Ph.D Thesis - Improvement of Conventional Leather Making Processes

IMPROVEMENT OF CONVENTIONAL LEATHER MAKING PROCESSES TO

REDUCE THE ENVIRONMENTAL IMPACT

Doctoral Thesis directed by

Dr. José Costa López Dr. Jaume Cot Cosp

Eduard Hernàndez Balada

Barcelona, December 2008

Programa de doctorat d’Enginyeria del Medi Ambient i del Producte

Bienni 2006-2008

El Dr. JOSÉ COSTA LÓPEZ, Catedràtic del Departament d’Enginyeria Química de la

Universitat de Barcelona,

CERTIFICA QUE:

El treball d’investigació titulat “IMPROVEMENT OF CONVENTIONAL

LEATHER MAKING PROCESSES TO REDUCE THE ENVIRONMENTAL

IMPACT” constitueix la memòria que presenta l’Enginyer Químic EDUARD

HERNÀNDEZ BALADA per a aspirar al grau de doctor per la Universitat de

Barcelona. Aquesta tesi doctoral ha estat realitzada dins del programa de doctorat

“Enginyeria del Medi Ambient i del Producte”, bienni 2006-2008, en el Departament

d’Enginyeria Química de la Universitat de Barcelona.

I per què així consti als efectes oportuns, signa el present certificat a Barcelona, a 8 de

Desembre de 2008.

Dr. José Costa López

Codirector de la tesi doctoral

El Dr. JAUME COT COSP, professor de recerca del Departament d’Ecotecnologies

de l’Institut d’Investigacions Químiques i Ambientals de Barcelona (IIQAB),

pertanyent al Consell Superior d’Investigacions Científiques (CSIC)

CERTIFICA QUE:

El treball d’investigació titulat “IMPROVEMENT OF CONVENTIONAL

LEATHER MAKING PROCESSES TO REDUCE THE ENVIRONMENTAL

IMPACT” constitueix la memòria que presenta l’Enginyer Químic EDUARD

HERNÀNDEZ BALADA per a aspirar al grau de doctor per la Universitat de

Barcelona. Aquesta tesi doctoral ha estat realitzada dins del programa de doctorat

“Enginyeria del Medi Ambient i del Producte”, bienni 2006-2008, en el Departament

d’Enginyeria Química de la Universitat de Barcelona.

I per què així consti als efectes oportuns, signa el present certificat a Barcelona, a 8 de

Desembre de 2008.

Dr. Jaume Cot Cosp

Codirector de la tesi doctoral

Luck is what happens when preparation meets opportunity

Seneca

Life is what happens to you while you are busy making other plans

John Lennon

Acknowledgments

ACKNOWLEDGMENTS

Hi ha moltes persones a les quals dec un enorme reconeixement en la realització de la

present tesi doctoral. El suport incondicional i a distància de la meva família ha estat

sens dubte cabdal. Voldria destacar particularment als meus pares, avis, germà i tiets,

que em van fer sentir tan aprop quan ens separava tot un oceà. A tota la resta de

família, els dec un sentit agraïment per tots els àpats de rebuda i comiat cada cop que

tornava a Catalunya.

Al Professor Jaume Cot (o Jaume a seques, com em va fer acostumar a dir-li) li dono

les gràcies per obrir-me les portes del Consell Superior d’Investigacions Científiques

(CSIC) i haver fet possible la meva estada al Departament d’Agricultura dels Estats

Units (USDA). La seva bondat, humiltat, sentit de l’humor i capacitat científica són un

exemple a seguir.

No em voldria oblidar dels companys del CSIC que em van donar suport i ànims quan

vaig decidir fer les Amèriques. Especial menció mereixen el Drs. Albert Manich, Agustí

Marsal, Fernando Fernández i Merche Catalina. A tota la resta d’amics i companys

del CSIC, que són massa nombrosos per enumerar-los tots, moltes gràcies per les

enriquidores estones passades junts.

També voldria agraïr al codirector de tesi Dr. Costa, així com al Prof. Mans i Dra.

González, el suport rebut durant els darrers anys i la generositat per ajudar-me a fer

tràmits a distància.

I would like to acknowledge a lot of amazing people I met during my stay at the United

States of America. I must start thanking my three overseas “aunts”: Maryann Taylor,

Ellie Brown and Lorelie Bumanlag. Their kindness, generosity and wisdom were

absolutely priceless. With them, I felt home.

Special mention goes to my first office mate at the USDA, Brian Coll, who took the

patience to teach me proper English and “show me around”. I would also like to thank

Joe Lee, not only for sharing his endless knowledge in leather, but for his true

friendship during all this time. To all of you, I will truly miss you.

Acnowledgements

I would also like to extend a BIG thank-you to the Fats, Oils and Animal Coproducts

Research Unit staff, and to Dr. William Marmer in particular. Thanks for believing in

me and giving me the opportunity to stay at the USDA two more years beyond the initial

appointment.

Visit of my parents to the USDA. Top – With Ms. Maryann Taylor Bottom – With Dr. Ellie Brown

Table of contents

Table of Contents SUMMARY .................................................................................................................. i 1. HIDES AND SKINS ................................................................................................ 1

1.1 History of leather processing ...................................................................... 2

1.2 Economics of hides and skins in the U.S. market ...................................... 3

1.3 Structure of hides and skins ........................................................................ 5

1.4 Conversion of hides and skins into leather ................................................. 9

2. PRESERVATION OF RAW HIDES AND SKINS ............................................... 14

2.1 Causes and signs of decay of hides and skins .......................................... 15

2.2 Preservation of raw hides and skins with common salt ............................ 18

2.2.1 Salt pack curing ......................................................................... 18

2.2.2 Brine raceways .......................................................................... 18

2.2.2.1 Brine curing in the industry ......................................... 20

2.2.2.2 Advantages and disadvantages of brine curing ........... 21

2.4 Alternatives to brine curing ...................................................................... 23

2.4.1 Alternative methods ................................................................... 24

2.4.2 Alternative chemicals ................................................................ 26

3. FILLERS IN THE LEATHER INDUSTRY .......................................................... 28

3.1 Looseness and veininess .......................................................................... 29

3.1.1 Veins ......................................................................................... 29

3.1.2 Grain break ................................................................................ 30

3.2 The upgrading of veiny or coarse break leather ……............................... 31

3.3 Whey and whey products ......................................................................... 32

3.3.1 Description and characterization ............................................... 32

3.3.2 Uses of whey ............................................................................. 35

3.4 Gelatin ...................................................................................................... 36

3.4.1 Description and characterization ............................................... 37

3.4.2 Uses of gelatin ........................................................................... 39

3.4.3 Gelatin in the leather industry ................................................... 39

3.5 Enzymes .................................................................................................... 40

Table of contents

3.5.1 Enzymes in the leather industry ................................................ 42

3.5.2 Microbial transglutaminase ……….......................................... 42

3.5.3 Reactivity of TGase with gelatin and whey proteins ................ 45

4. MATHEMATICAL MODEL OF RAW HIDE CURING WITH BRINE ............. 48

4.1 Letter of acceptance .................................................................................. 50

4.2 Abstract ..................................................................................................... 51

4.3 Resum ....................................................................................................... 52

4.4 Introduction .............................................................................................. 53

4.5 Theory ....................................................................................................... 53

4.6 Experimental ............................................................................................. 59

4.6.1 Materials .................................................................................... 59

4.6.2 Methods ..................................................................................... 59

4.6.3 Analyses ..................................................................................... 59

4.6.3.1 Chloride concentration determination ........................ 59

4.6.3.2 Fluorescence imaging ................................................. 59

4.7 Results and discussion .............................................................................. 60

4.7.1 Epifluorescence microscopy ...................................................... 60

4.7.2 Determination of diffusion coefficients …................................. 61

4.7.3 Determination of optimum brine curing conditions ….............. 63

4.8 Conclusions .............................................................................................. 64

4.9 Definition of terms ................................................................................... 65

4.10 References .............................................................................................. 66

4.11 Acknowledgments .................................................................................. 67

5. EVALUATION OF DEGREASERS AS BRINE CURING ADDITIVES ............... 68

5.1 Letter of acceptance ..................................................................................... 70

5.2 Abstract ........................................................................................................ 71

5.3 Resum .......................................................................................................... 72

5.4 Introduction ................................................................................................. 73

5.5 Experimental ................................................................................................ 74

5.5.1 Materials ....................................................................................... 74

5.5.2 Methods ........................................................................................ 74

5.5.2.1 Stratigraphic study ......................................................... 74

Table of contents

5.5.2.2 Degreaser study .......................................................... 75

5.5.3 Analyses ..................................................................................... 75

5.5.3.1 Determination of moisture and ash content ................ 75

5.5.3.2 Determination of fat content ....................................... 75

5.5.3.3 Determination of thermal stability .............................. 76

5.5.3.4 Back-scattered/Low Vacuum Scanning Electron

Microscopy (SEM-BSE) ......................................................... 76

5.5.3.5 Statistical analysis ....................................................... 77

5.6 Results and Discussion ............................................................................. 77

5.6.1 Stratigraphic study ..................................................................... 77

5.6.2 Degreasing study ....................................................................... 81

5.7 Conclusions .............................................................................................. 85

5.8 References ................................................................................................ 86

5.9 Acknowledgments .................................................................................... 87

6. PROPERTIES OF BIOPOLYMERS PRODUCED BY TRANSGLUTAMINASE

TREATMENT OF WHEY PROTEIN ISOLATE AND GELATIN ......................... 88

6.1 Letter of acceptance .................................................................................. 90

6.2 Abstract ..................................................................................................... 91

6.3 Resum …................................................................................................... 91

6.4 Introduction .............................................................................................. 92

6.5 Experimental ............................................................................................. 93

6.5.1 Materials .................................................................................... 93

6.5.2 Sample preparation .................................................................... 94

6.5.3 Analyses ..................................................................................... 95

6.5.3.1 Gel strength ................................................................. 95

6.5.3.2 Viscosity ..................................................................... 95

6.5.3.3 Rheology ..................................................................... 95

6.5.3.4 SDS-PAGE ................................................................. 96

6.5.3.5 Statistical modeling .................................................... 96

6.6 Results and discussion .............................................................................. 96

6.6.1 Gel strength ................................................................................ 96

6.6.2 Viscosity .................................................................................... 98

6.6.3 Rheological properties ............................................................. 100

Table of contents

6.6.4 SDS-PAGE .............................................................................. 101

6.7 Conclusions ............................................................................................ 103

6.8 References .............................................................................................. 104


7. WHEY PROTEIN ISOLATE: A POTENTIAL FILLER FOR THE LEATHER

INDUSTRY .............................................................................................................. 108

7.1 Letter of acceptance ................................................................................ 110

7.2 Abstract ................................................................................................... 111

7.3 Resum ..................................................................................................... 112

7.4 Introduction ............................................................................................ 113

7.5 Experimental ........................................................................................... 114

7.5.1 Materials .................................................................................. 114

7.5.2 Methods ................................................................................... 114

7.5.2.1 Preparation of WPI-Gelatin blends ........................... 114

7.5.2.2 Application of WPI-Gelatin blends to wet blue leather

................................................................................................ 115

7.5.2.3 Retan/Color/Fatliquor (RCF) .................................... 116

7.5.2.4 Drying ....................................................................... 116

7.5.3 Analyses ................................................................................... 116

7.5.3.1 Mechanical properties ............................................... 116

7.5.3.2 Subjective evaluation ................................................ 116

7.5.3.3 Protein concentration determination ......................... 117

7.6 Results and discussion ............................................................................ 117

7.6.1 Shoe upper wet blue ................................................................ 117

7.6.2 Upholstery wet blue ................................................................. 121

7.6.3 Mechanical properties .............................................................. 123

7.7 Conclusions ............................................................................................ 126

7.8 References .............................................................................................. 127


8. GENERAL CONCLUSIONS AND RECEOMMENDATION ........................... 129

8.1 Preservation of raw hides and skins with brine. Conclusions ................. 130

8.2 Preservation of raw hides and skins with brine. Recommendations ....... 131

Table of contents

8.3 Obtaining and characterization of potential fillers for leather.

Conclusions ................................................................................................... 132


Recommendations ......................................................................................... 133

9. REFERENCES ..................................................................................................... 135

10. NOTATION ........................................................................................................ 144

11. GLOSSARY OF TERMS ................................................................................... 148

12. RESUM EN CATALÀ ....................................................................................... 157

List of figures

List of Figures

FIGURES OF CHAPTER 1

Figure 1.1 – Leather articles …………………………………………....................... 3

Figure 1.2 – Typical distribution of a cow’s weight after the slaughtering .................. 5

Figure 1.3 – Composition of hide ................................................................................. 5

Figure 1.4 – Structure of collagen ................................................................................ 6

Figure 1.5 – Schematic cross-section of a bovine hide ................................................ 7

Figure 1.6 – Structural comparison of hides and skins ................................................. 8

Figure 1.7 – Unit processes and operations in leather processing ................................ 9

Figure 1.8 – Pile of wet blue leather ........................................................................... 11


Figure 2.1 – (a) Scratches on the grain of leather; (b) Leather damaged by bites of

insects ......................................................................................................................... 15

Figure 2.2 – Pinholes (open grain) in sheepskin ........................................................ 17

Figure 2.3 – (a) Process flow drawing of a typical brine curing system; (b) Raceway

brine cure tank ............................................................................................................ 19

Figure 2.4 – Red heat damage on salted skins ............................................................ 23


Figure 3.1 – Veininess in calfskin ......................................................................................... 29

Figure 3.2 – Scanning electron microscope images of blue stock without and with

visible veins ................................................................................................................ 30

Figure 3.3 – Samples of shoe upper leather showing coarse break and fine break .... 30

Figure 3.4 – Processing of whey protein isolate ......................................................... 32

Figure 3.5 – Snack fortified with whey protein isolate .............................................. 36

Figure 3.6 – Transformation of collagen into gelatin in the course of hydrolysis ..... 36

Figure 3.7 – Conversion of chrome shavings into gelatin .......................................... 40

Figure 3.8 – Role of an enzyme in a given reaction ................................................... 40

List of figures

Figure 3.9 – Lock and key analogy for enzymes and substrate .................................. 41

Figure 3.10 – Temperature and pH activity profiles of microbial transglutaminase .. 43

Figure 3.11 – Cross-linking of gelatin chains upon reaction with microbial

transglutaminase ......................................................................................................... 45

Figure 3.12 – Effect of mTGase concentration, incubation time, pH and incubation

temperature on breaking strength of a 10% (w/w) type A gelatin .............................. 46

Figure 3.13 – Reduction of a disulfide bond by two thiol-disulfide exchange reactions

involving DTT ............................................................................................................ 47


Figure 4.1 – Mathematical model of the curing process of a raw hide ..................... 54

Figure 4.2 – Dimensionless sodium chloride concentration field within the hide

during the curing process ............................................................................................ 56

Figure 4.3 – Dimensional sodium chloride concentration field within the hide during

the curing process for various soaking numbers ........................................................ 57

Figure 4.4 – Epifluorescent microscopic images of a cross section of a hide at

different stages of curing ............................................................................................ 60

Figure 4.5 – Determination of transport parameter � from experimental data. The

graph corresponds to c0p = 30% (w/v) and Na = 3 ..................................................... 62


Figure 5.1 – Stratigraphic distribution of water, ash, and hide salt saturation in a hide

treated for various intervals of time with a 500% float of an initial 95 °SAL brine .. 79

Figure 5.2 – Denaturation temperature (TD) of the grain, middle, and flesh layers

of a hide treated for various intervals of time with a 500% float of an initial 95 °SAL

brine ............................................................................................................................ 80

Figure 5.3 – Composite images of hide samples, collected at different curing times

by low vacuum, mixed signal SEM imaging .............................................................. 81

List of figures


Figure 6.1 – Gel strengths of gels formed from (a) bovine type B gelatin, 1 to 10%

(w/w), and (b) WPI-gelatin blends, 10% WPI with 0 to 3% added gelatin ............... 98 Figure 6.2 – Viscosities of solutions of (a) gelatin, (b) WPI and (c) WPI-gelatin

blends ....................................................................................................................... 100

Figure 6.3 – Time sweep analysis of 10% (w/w) WPI and WPI-gelatin blends ...... 101

Figure 6.4 – SDS-PAGE gels of WPI, gelatin, and WPI-gelatin blends .................. 102


Figure 7.1 – Flow diagram for retan, color and fatliquor formulation of upholstery

and shoe upper wet blue ........................................................................................... 116

Figure 7.2 – mTGase and protein uptake profiles by shoe upper wet blue pretreated

with a solution containing 0, 2.5 or 5% mTGase and treated with a solution of

5% WPI + 0.5% gelatin ............................................................................................ 118

Figure 7.3 – mTGase and protein uptake profiles by upholstery wet blue pretreated

with a solution containing 0 or 2.5% mTGase and treated with a solution of

2.5% WPI + 0.25% gelatin ……………………………………………………....... 122

Figure 7.4 – Subjective properties of upholstery crust leather ................................. 123

Figure 7.5 – Effect of the various treatments of leather with WPI and mTGase on

the tensile strength of upholstery and shoe upper crust leather ................................ 124


the Young’s modulus of upholstery and shoe upper crust leather ............................ 125


the tear strength of upholstery and shoe upper crust leather .................................... 125


Figure 12.1 – Model matemàtic del procés de curat d’una pell crua ........................ 160

Figure 12.2 – Mostres de cuir amb un toc de flor gruixut o fi .................................. 163

Figure 12.3 – Reacció de crosslinking entre molècules proteiques amb l’enzim

mTGase ..................................................................................................................... 165

List of figures

Figure 12.4 – Força de gel, viscositat, i mòdul elàstic (G’) d’una barreja proteica

composta per 10% (w/w) WPI i diferents quantitats de gelatina (de 0.5 a 3%),

en un ambient reductor ............................................................................................. 166

Figura 12.5 – Imatges de microsopia epifluorescent del wet blue després d’haver

Estat tractat amb una solució etiquetada de WPI i gelatina ...................................... 167

List of tables

List of Tables

TABLES OF CHAPTER 1

Table 1.1 – U.S. hides and skins production ........................................................................... 4

Table 1.2 – U.S. hide and skin exports by country .................................................................. 4

Table 1.3 – Typical reagents needed and wastes produced during the manufacturing

of leather .................................................................................................................................. 13

TABLES OF CHAPTER 2 Table 2.1 – Typical range of emission factors for conventional leather processing .................. 22

TABLES OF CHAPTER 3

Table 3.1 – Composition of different whey products .............................................................. 33

Table 3.2 – Summary of properties of the basic constituent proteins in whey ....................... 34

Table 3.3 – Composition of whey protein isolate .................................................................... 35

Table 3.4 – Physicochemical properties of types A and B of gelatin ...................................... 37

Table 3.5 – Dependence of molecular weight on the bloom strength of type B gelatin ......... 38

Table 3.6 – List of proteinaceous substrates reported to be reactive towards TGase ............. 44

TABLES OF CHAPTER 4

Table 4.1 – Transport coefficient � for various conditions of initial brine concentration (c0p)

and soaking Number (Na) ...................................................................................................... 62 TABLES OF CHAPTER 5 Table 5.1 – Effect of commercial degreasers on brine curing [0.5% w/w] ............................. 82

Table 5.2 – Effect of degreaser 1 on brine curing ................................................................... 83

Table 5.3 – Effect of sophorolipid on brine curing ................................................................. 84

TABLES OF CHAPTER 7 Table 7.1 – Uptake rate coefficient k for various treatments ................................................ 118

Table 7.2 – Subjective evaluation of shoe upper crust leather .............................................. 120

Summary

i

Summary The research reported in the present PhD dissertation was developed in its totality at the

United States Department of Agriculture (USDA), Eastern Regional Research Center

(ERRC) located in Wyndmoor, Pennsylvania.

During my stay at the ERRC from July 2005 to October 2008, I was assigned to two

CRIS (Current Research Information System) projects. The main goals of each project

are shown below. Further information about them can be found at the ERRC website

(http://cris.csrees.usda.gov/).

1. New and efficient processes for making quality leather

Project number: 1935-41440-013-00D

Lead Scientists: Dr. William N. Marmer and Dr. Cheng-Kung Liu

Objectives: Develop new technology for preparing hides for tanning. Establish drying

and finishing processes and develop in-line nondestructive tests for improving the

quality and durability of leather. Additional funding was obtained to expand the scope

of hide preparation research by investigating ways to impart efficiency to short-term

hide preservation (brine-curing).

2. Sustainable technologies for processing of hides, leather, wool and associated

byproducts

Project number: 1935-41440-014-00D

Lead Scientist: Dr. Eleanor M. Brown

Objectives: 1. Functional modification, leather and leather byproducts. Develop a

foundation for the use of new chemical and biochemical technologies (a) in the

production of high quality chrome-free leathers; (b) in expanding the range of high

Summary

ii

value biomaterial applications for solubilized proteins from leather byproducts. 2.

Functional modification, wool: modify wool to impart functionality for improved

performance and expanded uses of domestic wool.

Therefore, my assignments were divided into two different areas.

1. New and efficient processes for making quality leather

The preservation of raw hides and skins with a highly concentrated sodium chloride

solution (brine) is the most traditional and cost effective method. Nevertheless, it is a

lengthy process and detrimentally impacts the environment due to the disposal of salt

during the hide's conversion into leather. A mathematical model that described the

diffusion of sodium chloride in the hide during the curing process was developed in

order to search for the optimum brine curing conditions such as brine concentration and

float percentage. The diffusion of salt into the hide was characterized by the transport

coefficient �, which was found to be in the order of 10-5 s-1. From the model it was

found that the use of an initially saturated brine (35.9 g NaCl/100 ml water) as well as a

minimum float of 500% yielded an optimal diffusion rate. These findings corroborated

the generally accepted rule which states that about five kg of brine per one kg of hide is

required for a proper curing. The model also revealed that hides that were cured with

diluted brines (e.g. 20 or 25 g NaCl/100 ml water) would not receive a proper cure

regardless of the float percentage used. Importantly, the model put in evidence that the

results obtained were highly dependent on the hide salt saturation level required to reach

a proper cure, which was targeted at 85%. Finally, the proposed mathematical model

may be used to optimize the curing process under any given conditions and thus

rationalize the amount of salt and time employed to properly preserve raw hides and

skins.

Another goal of the project was to find ways to accelerate the uptake of sodium chloride

by the hide during the cure. By accomplishing this, the turn-around times in raceways

would be reduced and thus additional curing capacity created. By means of a

stratigraphic analysis of the hide at varying curing time intervals, it was found that salt

entered the hide mainly from the flesh side whereas water was withdrawn from both

sides of the hide, with the epidermis acting as a semipermeable membrane. These

findings were supported by epifluorescence microscopy and scanning electron

Summary

iii

microscopy in back-scattered electron mode. Bearing in mind that the adipose tissue

that adheres to the flesh side of the hide is believed to slow down the penetration of salt,

three commercial degreasers as well as a glycolipid-based surfactant (sophorolipid, SL)

were tested as brine curing additives. One of the three commercial degreasing agents

was proved to significantly enhance the uptake of salt by the hide during the cure,

simultaneously as it decreased the fat content. Trials with the SL also turned out

successfully; when the SL was used above its solubility limit, the degreasing ability was

comparable to that of commercial degreasers. The usage of SL in the leather

manufacturing has not yet been attempted on a commercial scale, but the promising

results here presented as well as their antimicrobial properties make them a very

promising candidate.

2. Sustainable technologies for processing of hides, leather, wool and associated

byproducts

The usage of leather byproducts in later stages of tanning is established practice,

although not well publicized in the literature. One of the products tanners frequently

use as a post tanning agent is called filler. Fillers minimize the veiny, loose and pipey

areas of the hide to obtain a uniform leather product. Back in the 1970’s, extracts of

vegetable tannins were used for that purpose. Recently, leather industry suppliers offer

filling agents such as polymers, resins and proteins. One of the proteins more

commonly used in U.S. tanneries was casein. Current casein prices in the American

market ($5.8-5.9/lb) encouraged the search for cheaper sources of protein.

Whey protein isolate (WPI), a byproduct of the cheese industry ($0.9-1.2/lb), and

gelatin, a byproduct of the leather industry ($2.4/lb), were selected for that purpose.

Biopolymers formed by the enzymatic crosslinking of dissimilar proteins have the

potential for generating novel products. Thus, small amount of gelatin was added to the

less expensive WPI and reacted with the enzyme microbial transglutaminase (mTGase)

under reducing conditions. The improvement in physical properties over either protein

component, given the same treatment, suggested the possibility of greater utilization and

new products from these coproducts.

Next, a biofiller composed of WPI and small amounts of gelatin was evaluated as a

filling agent for shoe upper and upholstery wet blue. Also, the effect of pretreating the

Summary

iv

wet blue with mTGase was examined. The outcome of the process was assessed by

subjective properties of the crust leather such as grain break, fullness, handle or color.

The general appearance of both shoe upper and upholstery leather was markedly

improved upon treatment with the biofiller, yielding fuller crust leather with a tighter

grain break and enhanced color. Importantly, the proteins contained in the filler were

not considerably removed by further processing. Furthermore, although treated samples

were a little stiffer and presented slight lower tear strength than the untreated samples,

the various treatments did not negatively affect the mechanical properties of the crust

leather. The pretreatment of the wet blue leather with mTGase affected the kinetics of

protein uptake and also contributed in the further improvement of the grain break in

samples with very bad original break. Noteworthy, it was proved that a 200% float

satisfactorily enabled the proteins to be taken up by the wet blue. If this technology is

to be transferred to the industry, use of a shorter float could be feasible due to a stronger

mechanical action. Further research that explores the possibility of using even cheaper

sources of protein as a raw material for bio-based leather products is an interesting

option currently being examined at the ERRC laboratories.

CHAPTER 1

Hides and skins

Chapter 1

2

1. HIDES AND SKINS

1.1 History of leather processing

From the earliest civilizations right to the present time, leather has been considered

nature’s product with inherent beauty and universal appeal. Prestige, durability,

physical properties and eye-appeal are some of the many natural qualities of this

product.

The process of leather making predates recorded history. The earliest record of the use

of leather dates from the Paleolithic period, when primitive man removed the hides and

skins from wild animals after having hunted them for food. They used those hides and

skins to make coats and footwear. Primitive man learned that skins rotted away after a

relatively short time. Throughout the following centuries, they discovered that if the

skins were stretched out and allowed to dry in the sun, they became stiff and hard but

also lasted much longer (Covington, 1997). Much more time had to pass until it was

eventually discovered that the bark of certain trees contained tannin or tannic acid

which could be used to convert raw skins into what we recognize today as leather.

Although it is hard to substantiate chronologically the exact time this tanning method

materialized, many claim that it had to be around 5,000 BC (Thomson, 1981).

Later on, wall paintings and artifacts in Egyptian tombs indicated that leather was used

for military equipment, sandals, clothes, gloves, buckets, bottles, etc. Also the Romans

used leather on a wide scale for footwear, clothes, and military equipment including

shields, saddles and harnesses. As a matter of fact, the manufacture of leather was

introduced to Britain by the Roman invaders.

Through the centuries, leather manufacture expanded steadily and by medieval times

most towns had a tannery located on the stream or river, which they used as a source of

water for processing and as a source of power for their water wheel driven machines.

During the middle age, leather was used for all kind or purposes: footwear, clothes,

bags, cases, upholstery of chairs and couches, book binding and military uses.

With the discovery and introduction of basic chemicals such as lime and sulfuric acid,

leather production slowly became a chemically based series of processes. Until the later

part of the 19th century, there were relatively few changes in the methods used to

Hides and skins

3

produce leather. However, the industrial revolution did not bypass tanning. A wider

range of dyestuffs, synthetic tanning agents and oils were introduced during that time.

Together with precision machinery, these changes and continued innovations to the

present day have combined to make tanning into a viable, modern manufacturing

industry.

Figure 1.1 – Leather articles. From left to right, a woman’s bag, leather seats in automobile and leather jacket.

1.2 Economics of hides and skins in the U.S. market

The hides and meatpacking industries may be regarded as a bridge between production

of the hide as a byproduct of the food industry and its manufacture into leather goods,

for which it provides a basic raw material.

Hides need some form of preservation after being removed from the animal. Once this

is accomplished, they can be shipped great distances (e.g. overseas) or stored until used.

Thus, the preserved hide becomes an article of international commerce. The supply of

hides is determined by the amount and type of meat in people’s diet. For instance, the

United States is a hide exporting nation since its appetite for beef exceeds the capacity

for tanning of the industry. On the contrary, Japan has a strong demand for leather

goods but a limited hide supply, which turns it into a hide importing nation

(Thorstensen, 1993).

Table 1.1 shows the estimate values of total slaughtered cattle in the Unites States over

the last years. On average, about 60% of the hides produced in the U.S. from 2001 to

2005 were exported, in their majority to Asia (Table 1.2). Latest figures available from

the United States Hide Skin & Leather Association (USHSLA) stated that an 80% of the

hides produced in the U.S. in 2008 were exported, half of which were shipped to China

Chapter 1

4

and Hong Kong (Reddington, 2008). This trend concludes that the United States’

exporting character is currently growing even further.

Table 1.1 – U.S. hides and skins production (1,000 hides)

Year Total slaughter Exports Imports Net exports

2001 35,530 23,471 1,721 21,750

2002 35,734 20,783 1,299 19,484

2003 35,647 19,330 1,153 18,177

2004 32,880 18,704 1,316 17,388

2005 32,535 19,200 1,355 17,845

Source: U.S. Leather Industry Statistics (2006 edition)

Table 1.2 – U.S. hide and skin exports by country (1,000 hides)

Destination 2005 2003 2001

China 8,191 5,434 5,417

Korea 4,089 4,860 7,602

Taiwan 1,718 1,941 2,751

Hong Kong 1,331 2,534 1,382

Mexico 1,287 1,348 1,647

Thailand 651 819 888

Italy 594 826 920

Japan 333 475 1,343

Vietnam 165 15 1

Brazil 141 160 76

India 107 11 41

Source: U.S. Leather Industry Statistics (2006 edition)

At the present time it is not uncommon for cattle hides to be produced in the United

States, converted into leather in China and shipped back to the U.S. to make garments.

As a matter of fact, hide sales to China are soaring. By the end of April 2006, U.S.

exports sales to China amounted to 47% of all exports worldwide which compares with

36% a year earlier (Leather International, 2006).

Hides and skins

5

1.3 Structure of hides and skins

Raw hides and skins are byproducts of the meat industry, and in turn are the raw

material of the leather industry. Figure 1.2 depicts that only about one third of the

animal live weight is manufactured as edible meat. Hide, hair, bones and organs

account for an approximate 23% of the animal’s weight.

Figure 1.2 – Typical distribution of a cow’s weight after the slaughtering.

Hides and skins are removed from the carcass of the animal during the slaughtering

process. By convention, the tanner employs the word hide to refer to the skin covering

large animals, such as cows, steers, horses, buffalos, etc., and the word skin is mostly

used to refer to smaller animals such as calves, sheep, goats, pigs, etc. The term hide is

never applied to the small animals.

The approximate composition of a freshly flayed hide is as follows (Figure 1.3).

Figure 1.3 – Composition of hide (Sharphouse, 1971).

Chapter 1

6

Collagen, with a content of 29%, is the main structural protein in the hide. Up to twenty

eight different types of collagen are described in literature. Among them, fibril-forming

collagens types I, II, III, V and XI are the majority components of skin, cartilage and

bone (van der Rest and Garrone, 1991). Collagen molecules consist of three

polypeptide chains, each coiled in a left-handed helix. The three chains are thrown into

a right-handed triple superhelix stabilized by periodic hydrogen bonds (Rich and Crick,

1955; Ramachandran and Kartha, 1955). The triple helices, also known as

tropocollagen, associate laterally and longitudinally to form microfibrils. These, in turn,

form fibrils, aggregates of which constitute various forms of connective tissue (Figure

1.4).

Figure 1.4 – Structure of collagen.

Other structural proteins present in the hide are elastin (0.3%) and keratin (2%). The

former helps skin to return to its original position when it is poked or pinched, whereas

the latter is the constituent protein of the hair. The figure for keratin varies widely

depending on the amount of hair present. Among the non-structural proteins, albumens

and globulins account for 1% and mucins and mucoids 0.7%.

An interesting approach of seeing the structure of a hide is to examine a cross section

(Figure 1.5).

Hides and skins

7

Figure 1.5 – Schematic cross-section of a bovine hide

(Sharphouse, 1971).

Starting from the hair side the following elements are found:

• Hair. It is composed by an actively growing root zone embedded in the skin and

a visible dead hair shaft above the skin. Hair is primarily keratin, a sulfur-

bearing protein. The structure of a hair follicle is quite complex and up to five

concentric layers can be differentiated (Wagner and Bailey, 1999).

• Epidermis. It is the outermost layer of skin. It consists of a variety of cell layers

produced in the geminative basal cell region located at the base of the dermis. It

is hard, quite inert chemically, and constantly in the state of flaking.

• Sweat glands. They release sweat and undesirable body wastes through the

pores of the skin.

• Sebaceous glands. They release oil into the hair and onto the surface of the skin

in order to maintain a proper body temperature in warm-blooded animals.

• Corium. It consists of a network of collagen fibers intimately woven. In the

upper part of the corium, the fibers are very thin and tightly woven and towards

the center they are coarser and stronger. The orientation of the fibers and

Chapter 1

8

whether they are loosely or tightly-woven will determine some characteristics of

the resultant leather.

• Flesh. It is composed of varying amounts of fatty adipose tissue, blood vessels,

nerves and voluntary muscle.

Hides and skins differ in their structure, depending upon the habits of life of the animal,

season of the year, age, sex and breeding. Figure 1.6 depicts the different structure of a

goatskin, sheepskin and cattle hide. The amount of hair and fat present as well as the

tightness of the structure are three important parameters used to differentiate them. For

instance, goatskin presents less hair and fat and a more firm structure than sheepskin

and cattle hide. Cattle hide presents fat in both sides of the hide and has a tighter

structure than that of sheepskin but more open than goatskin.

Figure 1.6 – Structural comparison of hides and skin (Thorstensen, 1993).

Hides and skins

9

1.4 Conversion of hides and skins into leather

The conversion of a raw hide or skin into leather requires numerous processing steps,

which may be grouped in the following five categories (Figure 1.7).

Figure 1.7 – Unit processes and operations in leather processing (Saravanabhavan et al., 2003).

1. Hide preservation. Treatment given to raw hides or skins just removed from

the carcass of the animal to minimize putrefaction and bacterial action, but

enabling the skins to be rehydrated conveniently in preparation for tanning.

Sometimes the preservation of hides is carried out after cutting away the

subcutaneous tissues or flesh, which accounts for about 20% of the weight of the

hide. This process is known as fleshing.

2. Beamhouse (Pretanning). It refers to the processes in the tannery between the

removal of the skins or hides from storage and their preparation for tanning.

Despite this definition, hide preservation is typically not considered a

beamhouse process. Beamhouse operations are of tremendous importance in the

ultimate quality of the leather. The following operations are considered

beamhouse:

a. Trimming. Cut away useless or unwanted material from the edges of raw

hides or skins to give them a better shape.

Preserved Hides and Skins

Pelt Wet Blue Leather

Crust Leather

Finished Leather

Finishing Operations Preservation Pretanning Tanning

Post tanning Operations

Trimming Soaking

Unhairing Liming

Deliming Bating

Scudding Pickling

Wringing Splitting

Retanning Dying

Fatliquoring Setting Drying

Conditioning Staking Toggling Buffing

Spraying Plating

Unit Operations

Outcome

Stages

Chapter 1

10

b. Soaking. Treat hides or skins with water, sometimes with the addition of

a disinfectant, to cleanse them, remove salt and other soluble matter, and

to rehydrate and soften them.

c. Unhairing. Removal of hair or wool from hides or skins. Various

chemicals may be used for this purpose: lime-sulfide, an oxidizing agent

in acid solution, or an enzyme preparation.

d. Liming. Treatment of hides or skins with lime intended to loosen hair,

fat, flesh, etc. Sometimes it is done simultaneously to the unhairing.

e. Deliming. Removal of alkali and pH adjustment for bating.

f. Bating. Enzymatic action for the removal of unwanted inter-fibrillary

proteins, as well as any remaining hair roots, epidermal structure and

fatty cells.

g. Scudding. Working over the grain surface of limed, or bated, pelt with a

blunt-bladed tool, by hand or machine, to eliminate hair fragments,

pigment granules, lime soaps and other impurities.

h. Pickling. Treatment of pelts with an acid liquor, such as a solution of

sulfuric acid and sodium chloride, to preserve them or to prepare them

for tanning, especially chrome tanning.

3. Tanning. Tanning is defined as a process by which putrescible biological

material is converted into a stable material which is resistant to microbial attack

and has enhanced resistance to wet and dry heat (Gustavson, 1956). Chrome

tanning is the most frequently used method to tan hides, mainly due to the short

time it takes (4 to 6 hours) and because it produces leather that combines both

the best chemical and physical properties sought after in the majority of leather

uses (Leather Facts, 1973). A hide or a skin that has been subjected to the usual

beamhouse processes, chrome-tanned and left wet receives the name of wet blue

(Figure 1.8). Wet blue may be stored or exported in this state.

Hides and skins

11

Figure 1.8 – Pile of wet blue leather.

Alternatives to chrome tanning include vegetable tanning, carried out by means

of tanning agents contained in the barks, woods, fruits, leaves, etc., of plants.

Although they have not been implemented in the industry, natural products like

genipin (Ding et al., 2007) or salts of metals such as aluminum (Brown and

Dudley, 2005) or titanium (Peng et al., 2007) were reported to be successfully

tan the skins and hides.

4. Post tanning operations. The order of processes varies considerably for

different leathers. The control and choice of these operations will determine the

characteristics of the leather made.

a. Wringing. Removal of excess moisture from the stock for a proper

splitting.

b. Splitting. Adjustment of the thickness of the hide to the specified

requirements. The cut off layer, named split, is still a valuable raw

material for making into sueded type of leathers. Some hides may need

further thickness adjustment in a process called shaving.

c. Retanning, Coloring and Fatliquoring (RCF). These three operations

have vastly different purposes, but they are considered as a unit because

one follows the other without interruption. To retan is to subject an

already tanned leather to a further tanning treatment to modify its

properties. Coloring, also called dyeing, gives the required color to the

Chapter 1

12

leather. The aim of fatliquoring is to soften the leather by the use of oils

that lubricate the fibers.

d. Setting out. Multiple purpose operation, which smoothes and stretches

the leather while squeezing out excess moisture from it.

e. Drying. Removal of all but equilibrium water from the stock. Moisture

content after this step should be around 10-12%.

5. Finishing operations. The act of making completely tanned leather more

attractive, serviceable and durable. It can include one or more of the following

processes:

a. Conditioning. Application of a fine mist of water to raise the water

content to about 25%.

b. Staking. Mechanical operation that softens and flexes the leather.

c. Toggling. The straining and fixing of leather onto frames with toggles.

The purpose is to dry leather keeping it under tension.

d. Buffing. Abrade or grind a leather surface, especially the grain surface,

by a moving band of abrasive paper or cloth.

e. Spraying. Apply a liquid in the form of very fine droplets designed to

enhance the appearance and/or give the grain or flesh surface special

properties.

f. Plating. Mechanical finishing process used to subject the surface finish

of leather to a high pressure from a heated, polished plate or cylinder to

obtain desired smoothness, flow-out, gloss and film formation.

Hides and skins

13

Table 1.3 summarizes the reagents typically used in the tannery for the conversion of

hides and skins into leather, as well as the different wastes generated at each one of the

processing stages.

Table 1.3 – Typical reagents needed and wastes produced during the manufacturing of leather

(Rao et al., 2003)

Reagents Liquid Waste Solid Waste Air pollutants

Beamhouse operations

Water Lime

Sulfide Enzymes

Salt & Acid

Salt Protein Lime

Dusted salt Trimming Fleshing

Hair

Sulfide

Tanning

Water Chrome

Vegetable tannins

Salt Chrome Tannins

Splits Shavings

Vegetable bark

Post tanning operations

Water Chrome Syntans

Dyes Fatliquors

Dyes Greases Syntans Chrome

Finishing

Binders Pigments

Organic solvents Formaldehyde

Lacquers

Tanned wastes Buffing dust

Organic solvents Buffing dust

Formaldehyde

CHAPTER 2

Preservation of raw hides and skins

Chapter 2

15

2. PRESERVATION OF RAW HIDES AND SKINS

2.1 Causes and signs of decay of hides and skins

The purpose of preserving hides and skins is to temporarily prevent deterioration from

the time they are removed from the animal until they are processed into a product that is

no longer susceptible to putrefaction or rotting. A hide that has not been properly cured

will yield a finished leather of poor quality. Therefore, this very first step plays an

essential role in the quality of the finished product.

Hide damage can be classified into two categories: physical damage and putrefaction.

• Physical damage. Occurs before the slaughter of the animal. It becomes an

important issue because these defects will have an adverse effect on the finished

leather even if the hides receive a proper cure. It includes tears, scratches, cuts,

hook marks, contamination with dirt (manure), insect attack, etc (Figure 2.1).

Figure 2.1 – (a) Scratches on the grain of leather; (b) Leather damaged by bites of insects (Tancous et al., 1959).

• Putrefaction. The main object of preservation is to fight the damage caused by

bacteria and the proteolytic enzymes produced by them. Bacteria are one-celled

microorganisms that multiply very rapidly when they feel comfortable in the

surrounding environment. These conditions are summarized in the following

list:

a) Food. Bacteria secrete a digestive juice that contains enzymes. The

function of these enzymes is to nourish bacteria by breaking down

molecules of the substrate (hide). Three different kinds of bacteria are

a b


16

involved in the degradation of skin proteins: anaerobic, aerobic and

facultative (which can grow with or without oxygen). Anaerobic

bacteria deteriorates the proteins into the stage of amino acids and

therefore it is one of the most dangerous. A species named Clostridium

histolyticum was the first to be reported to produce the enzyme

responsible for collagen degradation, collagenase (Mandl et al., 1958).

b) Water. Bacteria need water to live, grow, multiply and produce

enzymes. Nevertheless, there are some bacteria that have become

accustomed to dry conditions that form dormant spores which are

capable of reproducing when moisture is available again, sometimes

even after years. The critical moisture, below which level the skin is not

conducive for bacterial attack was found to be at around 50% (Stuart and

Frey, 1938).

c) pH. Bacteria require a near neutral or slightly alkaline environment to

survive. Generally, microorganisms can not survive in an environment

with a pH very much lower than 5 or higher than 8.

d) Temperature. Bacteria are more likely to reproduce in a warm

surrounding, leading to poor cure efficiency and loss of hide substance.

Literature reported that optimum temperature for bacteria was between

15 to 37 °C and that a better preservation was attained if curing was

carried out at a temperature between 10 to 18 °C (McLaughlin and

Rockwell, 1922).

e) Pre-curing period. This term defines the time comprised between the

slaughtering of the animal and the commencement of the curing process.

It was reported that a five hours period led to degenerative changes in the

cells lying around the sweat glands, and that another 6 hours led to

structural damage of the skin and the breaking down of the polypeptide

chains into dipeptides (Chattopadhyay, 1998).

Chapter 2

17

f) Presence of certain chemicals. Microorganisms can be killed by a

variety of chemical substances. These chemical poisons may be used as

short term preserving agents or along with salt in order to obtain hides

that will remain preserved in a long term period. A bacteriostat is a

chemical agent that stops or inhibits the activity of bacteria at whichever

state they are in. A bactericide is a chemical agent that kills bacteria

outright. They used to be based on mercury compounds or chlorinated

phenols, but they were ruled out with the enforcement of more strict

environmental laws.

Typically, preserved hides may be graded according to the following classification:

• Very good. No sign of putrefaction, raw skin smell, fresh appearance.

• Good. No sign of putrefaction, smells good, not so fresh as raw skin, appearance

good but not so fresh.

• Fair. Signs of putrefaction present, slight putrid smell, dull appearance.

• Poor. Several bacterial damage, strong putrid smell, slimy appearance.

Hair slippage, bad odor, the presence of mold and grain damage (e.g. discoloration) are

amongst the most common signs of a hide decay. Hair slippage has been used as an

indicator by packers and tanners that hides or skins have not been preserved to the

fullest degree. It is a sign that either autolytic or bacterial degeneration has occurred to

the extent that the hair and epidermis is loosened and can be easily removed. This

phenomenon may be accompanied by degradation of the hair follicle leaving a hole in

the grain (Didato et al., 1999) (Figure 2.2).

Figure 2.2 – Pinholes (open grain) in sheepskin.


18

2.2 Preservation of raw hides and skins with common salt

Preservation of hides with sodium chloride (NaCl) is the most frequent curing method

used currently. The hygroscopic nature of sodium chloride reduces the water content of

the hide as well as lowers the water activity of the remaining moisture (Bailey, 2003).

Sodium chloride takes the water away from the bacteria that inhabit the surface of the

hide. Yet an exception to this statement are the halophilic bacteria, which grow in a

concentrated salty environment (see Section 2.3.).

There are two distinct processes that use sodium chloride as a preserving agent: salt

pack curing and brine curing.

2.2.1 Salt pack curing

Also known as green salting, is a more antique method of salt preservation. It is not

common in the United States or Europe but it is extensively applied in warm countries

like India. It consists of sprinkling solid salt onto the flesh surface of the hide, usually

about one kilogram of salt per kilogram of hide. As the solid salt slowly diffuses into

the hide, the water inside migrates out. Successive layers of salt and hides are added to

the pack until a height of four to five feet. Because of the slow diffusion of salt into the

hide, a considerable time (approximately 30 days) is needed to insure a satisfactory and

uniform curing.

2.2.2 Brine raceways

This method is extensively used in American and European hide processing facilities. It

consists of a huge vat containing a very concentrated or saturated solution of sodium

chloride (brine) where hides are suspended for a minimum of 18 h. The brine is kept

close to saturation by circulating it through a salt-box or Lixator, or by having excess

salt in the brining vat. Brine curers also add bactericide in the brine to keep low the

presence of bacteria both in the hide and in the vat.

Chapter 2

19

Figure 2.3 – (a) process flow drawing of a typical brine curing system (Thorstensen, 1993) (b) raceway brine cure tank, provided with two small paddles to keep the solution agitated (courtesy of Diamond Crystal Salt Co.).

Tanners and meatpackers monitor the concentration of brine in the raceway with an

instrument called salometer. This hydrometer is a long, narrow, graduated stem

attached to a weighted bulb. It is more buoyant the higher the salinity of the liquid and

therefore sinks less deeply into the liquid. The salometer scale ranges from 0 °SAL

(pure water) to 100 °SAL (saturated brine).

There are other hydrometers that can be used for the same purpose, which measure the

specific gravity or the sodium chloride percentage by weight. The specific gravity is the

relation between the weight of a certain volume of any substance and the weight of the

same volume of water. This value rises from 1.000 (pure water) to 1.198 (saturated

brine at 25 °C).

At 25 °C, 100 ml of saturated brine hold 31.7 g of salt, or likewise 100 g of saturated

brine holds 26.5 g of sodium chloride. Saturation levels can also be referred as g of salt

per 100 ml of water. In this case, 35.9 g of salt saturates 100 ml of pure water, at 25 °C.

The following equation relates brine concentration (g NaCl/100 g brine) and salometer

degrees (°SAL), at 15 °C.

[ ] 0004.0789.3)( −⋅=° NaClSALSalometer

a

b


20

It is important to note than the solubility of sodium chloride is slightly dependent on

temperature, with values ranging from 35.7 to 39.8 g/100 g water, at 0°C and 100 °C

respectively.

Despite the low knowledge of technology involved, there are a few aspects that need to

be taken into consideration when curing hides and skins in a raceway.

• Brine needs to be kept always in fast motion. Otherwise the hides settle to the

bottom and will not receive a proper cure.

• Brine needs to be changed regularly due to the accumulation of dirt and manure

in the bottom of the vat. The removal of dirt, clay, manure and urine during

brining results in hides cleaner than those obtained in salt pack curing. If no

action is taken, the actual float decreases and the hides are more likely to receive

a poor cure. Float (or float percentage) stands for the ratio between the volume

of brine and the volume of hides.

• A large float and a high concentration of brine are essential to attain a proper

cure of the hides. A generally accepted rule stipulates a minimum 500% float

(about 5 m3 of brine per m3 of hide) in order to reach a good cure.

2.2.2.1 Brine curing in the industry

Brine curing is performed in conjunction with one of two hide processing methods:

green fleshing or cured fleshing.

1. Green fleshing + Brine curing. It consists of fleshing the hides first and then

brine cure. This is the option of choice for packers that flesh in operations

adjacent to their kill floors. There are three main reasons that justify this modus

operandi:

• Green fleshing has allows the removed flesh to be processed in a

rendering plant, since it is not contaminated with salt, thus preserving

some value in the flesh. In fact, production of biodiesel from solid

tannery waste is being currently considered (Kolomaznik, 2008).

Chapter 2

21

• With the adipose tissue mostly removed, salt penetrates more quickly

and to a greater extent into the hide. This fact also leads to an additional

weight increase in the hides fleshed before cure (Meat Industry, 1979).

• Hides fleshed before the cure show significantly lower bacterial levels

than hides that have not been prefleshed.

2. Brine curing + cured fleshing. In this case, the hide is cured first and then

fleshed. Packers who do not have adjacent hide processing facilities or hide

processors that are located at some distance from their clients, usually choose

this method. Similarly, this method presents two advantages.

• The 18 h curing time in the raceway gives some time to soften and

remove the feedlot mud that adheres to the hair of cattle (especially from

January through April).

• From an operational point of view, the hide has to be handled twice: put

it in the raceway, cured and fleshed. Conversely, green fleshing requires

the hide to be washed in a fresh water tank first, fleshed, returned to the

brine raceway, cured and then removed. Therefore, the hide needs to be

handled three times and more wash tank/raceway capacity is needed.

2.2.2.2 Advantages and disadvantages of brine curing

Brine curing of hides and skins presents the following advantages and disadvantages.

The most relevant advantages are the following:

• It is relatively inexpensive in comparison to the other preservation methods or

curing materials (current commodity prices for sodium chloride are around

$0.11/kg).

• A raceway can process a high volume of hides and skins (various thousands per

day).

• There is not high tech knowledge involved.

• It involves the usage of safe chemicals.

The most important disadvantages of the brine-curing method are detailed next.


22

• Water pollution. Production of leather entails a high consumption of water. In

fact, the conversion of 1 ton of raw hides into leather requires between 20 and

40 m3 of water (Ramasami and Prasad, 1991), which will be almost fully

discharged upon completion of the process.

Soaking of brine cured hides releases about 40-50% of the total dissolved solids

(TDS) of the whole leather making process (Table 2.1). Technology to treat a

stream heavily contaminated with TDS and chlorides (e.g. membrane processes)

is not cost effective. In addition, the presence of common salt in irrigation water

can be extremely adverse, leading to an increase in soil salinity and reduction of

crop yields (Daniels, 1998).

Table 2.1 – Typical range of emission factors for conventional leather processing

Parameters Soaking

Volume of effluent 6 - 9

Biological oxygen demand (BOD) 6 - 24

Chemical oxygen demand (COD) 18 - 60

Total solids 200 - 500

Dissolved solids 190 - 400

Suspended solids 15 - 60

Chloride as Cl- 90 - 250

Total chromium as Cr -

Note: all values expressed in kg/ton of hide or skin processed. They were obtained from the formula (concentration x volume of effluent)/ton of leather processed (Ramasami et al., 1998).

• The large amount of salt required. Most industrial curing operations try to

maximize salt concentration in the brine to 97 °SAL (25.6 g NaCl/100 g brine).

• Despite the historically low cost of common salt, economics of brine curing of

hides and skins has been affected recently by increasing commodity prices for

sodium chloride (a 10-15% increase over the past few years) (Godsalve, 2007).

• The enormous amount of hides being cured at once makes it difficult to ensure

that they remain in the vat a minimum of 18 hours. Brining in a raceway is a

batch process and hides are not tagged. It is probable that hides are removed

Chapter 2

23

from the vat in a different order that they were put in. These hides will be

undercured and are likely to present deterioration in long term storage.

• Overloading the vat with hides causes the float to fall below the recommended

500%. With a lower float, the concentration of salt in the brine decreases

rapidly, which also leads to a slower rate of diffusion of salt into the hide.

Under this scenario, it is likely that a hide removed from the raceway after 18

hours may not be fully cured.

• Red heat damage. Hides that have received a proper cure are more susceptible

to be attacked by bacteria that grow in a concentrated salt environment.

Figure 2.4 – Red heat damage on salted skins (Source: BLC Leather Technology).

Archaea, an extremely halophilic bacteria belonging to the family

Halobacteriaceae, was demonstrated to cause significant damage on brine cured

hides (Bailey and Birbir, 1993). A concentration of at least 1.5 to 2 M NaCl was

needed for growth and optimally most species required 2 – 4 M NaCl (Grant et

al., 2001). A study showed that 98% of brine cured American hides were

contaminated with those microorganisms (Bailey and Birbir, 1993).

Furthermore, a total of 332 extremely halophilic Archaeal strains were isolated

and 94% of these strains were protease positive (Bailey and Birbir, 1993).

2.4 Alternatives to brine curing

Issues associated to the preservation of raw hides and skins with common salt

encouraged the search for alternatives. Over the last 30 years, different substances were

assayed as preserving agents, in either salt-less or less salt methods. Unfortunately for


24

the majority of cases, they were not applied in the industry due to the high cost

involved. Also, new proposed techniques that efficiently preserved the hides were

studied, but again the high capital investment ruled them out. A quick summary of

these new methods and substances is listed next.

2.4.1 Alternative methods

• Drying. It is the earliest method of preservation of raw hides and skins. Even

though this method is still used by primitive men to cover huts or making tents,

it presents a few disadvantages:

a) If the drying is too slow, which is likely to happen in a cold and wet

climate, putrefaction may occur before the moisture is low enough to

prevent the bacterial attack.

b) If the drying is too fast and the temperature too high, it is likely that

some areas of the hide start to become gluey, thus preventing inner layers

from drying.

c) Hides are thicker than skins and therefore more difficult to dry out.

d) Increase of slipperiness of the pelt during fleshing.

e) The dried hides may be subject to insect attack, which is as bad as the

damage caused by bacteria.

• Cooling and chilling. Some countries use cooling systems to preserve skins.

The preservation time depends on the temperature, going from 3 weeks to 2 days

for temperatures of 0 and 15 °C, respectively. There are three methods to cool

raw hides and skins (Kanagaraj and Chandra Babu, 2002).

Chapter 2

25

a) Cooled air treatment. Just flayed hides are cooled at 3 to 5 °C for about

one hour, which preserves them in a sufficient extent to confront the

transport from the slaughterhouse to the tannery without decaying

(approximately 12 hours).

b) Addition of ice. The addition of ice cubes or flakes in a container that

encloses hides cause a drop of about 20 °C and then can be stored for 24

hours without further treatment. This technique was improved by the use

of a preservative solution to produce ice.

c) Use of dry ice. The use of dry ice cools hides to temperatures of -35 °C

and effectively preserves them for a minimum of 48 hours. The use of

carbon dioxide, however, entails risk of suffocation.

• Freezing. It is a medium term preservation technique that consists of storing

hides at temperatures around -10 to -20 °C. Freezing is rarely used nowadays

due to the high investment required and to the micro-distortion within the fiber

structure caused by the formation of ice crystals.

• Freeze drying. In this case, water is removed by means of applying low

pressure at low temperatures, which gives a flexible and porous cured hide. This

preservation technique cuts back money on shipping and makes the soaking

process easier. However, the operational costs are too high for common use.

• Gamma irradiation. The irradiation of hides with gamma rays along with the

application of a bactericide was reported as a successful salt-less preservation

technique. However, the high cost capital investment required and the difficulty

to control the minimum amount of radiation needed to attain a proper cure

impeded its commercial adoption (Bailey, 1999).

• Electron beam irradiation. The application of electron-beam-irradiation to

cattle hides, also known as EvergreenTM technique, yields a preservation level


26

comparable to that of brine. This high tech method, however, presented the

same disadvantages as the irradiation with gamma rays (Bailey et al., 2001).

2.4.2 Alternative chemicals

• Formaldehyde (H2CO). The use of low amounts of this aldehyde was proved to

efficiently preserve the hide but its usage was ruled out due to its toxicity

(Sharphouse and Kinweri, 1978).

• Soda ash (Na2CO3). Even though soda ash is not a true preservative, its high

alkalinity created an environment where bacteria and proteolytic enzyme activity

was inhibited. This method was only analyzed as a short term preservation

technique (storage time up to 8 days) (Rao and Henrickson, 1983).

• Potassium chloride (KCl). It could be effectively used as a curing agent.

Furthermore, this substance presented a few advantages over the traditional

brine curing:

a) Production of less excess salt.

b) Possible effective use of the water stream as a plant fertilizer.

c) More rapid kinetics on uptake of salt.

d) Elimination of halophilic bacteria.

However, preservation with potash did not take off due to the more expensive

cost of potash over brine. In addition, the low solubility of potassium chloride at

low temperatures made this technique unviable in cold climate tanneries (Bailey

and Gosselin, 1996).

• Boric acid (H3BO3). This substance, used either in a salt-less or less-salt

method, effectively cured skins for storage times up to two weeks. The main

Chapter 2

27

advantage is the reduction by more than 80% of the TDS and chlorides in the

effluents. However, the cost for that new susbtance was about 15-20% higher

than for the traditional brine curing (Kanagaraj et al., 2005).

• Silica gel. This powerful dehydrating agent was proved to be effective in short

and long term preservation of raw hides and skins (Kanagaraj et al., 2001;

Munz, 2007). The advantages of this method are various:

a) Reduction of TDS and chlorides content in soaking liquors.

Furthermore, these liquors can replace pure water for irrigation

purposes.

b) Stronger dewatering of the hide which does not affect the subsequent

rehydration in the soaking step.

c) No impact on the quality of the final product.

However, the production cost of silicate was double that of common salt, and

therefore this method has not been extensively used yet.

• Azardirachta Indica. This herbal preservation agent, commonly named Neem

tree, was demonstrated to be an efficient agent in the curing of goatskins. By

using it instead of sodium chloride, both TDS and solid waste discharge were

reduced, and the quality of the finished crust leather was not adversely affected

(Preethi et al., 2006).

CHAPTER 3

Fillers in the leather industry

Chapter 3

29

3. FILLERS IN THE LEATHER INDUSTRY

3.1 Looseness and veininess

The presence of veins and loose or pipey areas in finished leather figure amongst the

most important concerns that tanners are facing in today’s leather processing. Thus, the

upgrading of leather that presents these defects is one of the most value adding

opportunities for a tanner.

3.1.1 Veins

Although the term veiny is widely accepted among tanners, the term “prominent blood

vessels”, which includes veins, arteries and their branches is more appropriate. A vein

can be compared to a tube which is either empty or full of blood and which is located in

the deeper or very superficial layers of the dermis. Veininess was firstly believed to be

caused by an improper bleeding of the animal after death, followed by coagulation of

animal blood in the blood vessels of the skin (Orthmann and Higby, 1929). Later on,

many more causes were believed to exert an influence on the appearance of veins: the

age, diet and breed of the animal, the climate, the period of slaughter, the preservation

method, the manufacturing process, etc. Belly and neck areas of the hide are most

likely to suffer from veininess.

Veins are very easily recognizable; they are very apparent in stock that is inspected

visually (Figure 3.1).

Figure 3.1 – Veininess in calfskin.

A significant difference in the structure of veiny and non-veiny leather can also be

observed when looking them under a scanning electron microscopy (Figure 3.2).


30

Figure 3.2 – Scanning electron microscope images of blue stock (a) without and (b) with visible veins. The flesh side of the hide lies on the bottom of the picture, and the grain side on the top.

3.1.2 Grain break

The grain break, also known as break of leather, is characterized by the wrinkles formed

on the surface of leather when it is bent grain inward. A fine break, characterized by the

presence of many fine wrinkles per linear inch, is more pleasing to the eye and thus

more desirable than a coarse break (Figure 3.3). Because no objective standard has

been established to evaluate the break of hides, it remains a matter of subjective

personal inspection.

Figure 3.3 – Samples of shoe upper leather showing (a) coarse break, and (b) fine break.

The break is a naturally occurring characteristic of the skin or hide, although it can be

influenced by processing. Typically, the butt has a finer break than the shoulder and

belly areas.

Frequently, grain break, pipiness and looseness are used interchangeably. These three

conditions have in common that the grain separates to some degree from the corium.

a b

a b

Chapter 3

31

The grain break, pipiness and looseness can be caused by inherent condition of the hide

or conditions associated to tannery processing.

3.2 The upgrading of veiny or coarse break leather

One common way to address this issue is by introducing a substance into the voids that

exist between the fibers of the leather. This substance is called filler or filling agent.

Filler’s objective is to give more body and substance to the leather by reducing

looseness and diminishing the appearance of veininess.

The nature of fillers has changed over time. At first, tanners used extracts of vegetable

tanning agents, barium compounds, glucose, flour and gum as fillers (Harris, 1974).

More recently, filling agents were made from conventional petroleum feedstocks, which

are becoming increasingly expensive. Therefore, the utilization of products from

renewable resources, and particularly those from waste proteins, became highly

interesting. Gelatin chemically crosslinked with glutaraldehyde was demonstrated to be

an effective filling agent, entering the loose areas of the hide and remaining attached to

the leather upon further processing (Chen et al., 2001). However, the potential toxicity

of glutaraldehyde advised against this method.

Our laboratory at the Eastern Regional Research Center (ERRC) has been working over

the last several years in the modification of various waste proteins with the enzyme

microbial transglutaminase (mTGase), with the goal of obtaining a product that could be

used as a filler. Unlike glutaraldehyde, mTGase is a nontoxic and food grade protein

crosslinker (see section 3.5.2). Gelatin, either commercial or experimental obtained

from tannery waste, whey, whey protein isolate (WPI) and sodium caseinate were

demonstrated to be reactive substrates towards the enzyme mTGase, and their reacted

products could be effectively used as potential fillers for leather (Taylor et al., 2001;

2002; 2003; 2004; 2005; 2006a; 2006b; 2007). These proteins, used alone or in

combination, were effectively bound to the leather and not significantly removed upon

further processing. In addition, the mechanical properties of the finished leather were

not adversely affected by the filling treatment (Taylor et al., 2008). Current prices of

sodium caseinate ($5.8/lb) and gelatin ($2.6/lb) emphasized the need for further

research into cheaper sources of protein to generate fillers for leather, like the less

expensive whey ($0.31/lb) or WPI ($1.05/lb) (USDA Agricultural Marketing Service).


32

Other technologies to upgrade the quality of finished leather have also been

investigated. A recent publication reported the use of polymeric micro-spheres that

penetrate selectively into the loose areas of the leather to subsequently expand by the

application of saturated steam (Tegtmeyer et al., 2007).

Next, a brief description of the products used in the preparation of new fillers for leather

(whey protein isolate, gelatin and microbial transglutaminase) is given (sections 3.3 to

3.5).

3.3 Whey and whey products

3.3.1 Description and characterization

Whey is a liquid that separates from clotted milk during manufacture of cheese and after

coagulation of the caseins at pH 4.6 and 20 °C (Eigel et al., 1984).

The majority component in whey is lactose (70-72%), followed by minerals (12-15%)

and whey proteins (8-10%). The whey protein fraction can be selectively or totally

removed from raw whey and concentrated by using various membrane processes (e.g.

diafiltration, electrodialysis, nanofiltration) or ion exchange columns (Figure 3.4).

Membrane processing of whey is more cost effective than ion exchange technology, but

it also yields a whey concentrate with a higher content of fat and less heat stability than

that obtained using the ion exchange columns.

Figure 3.4 – Processing of whey protein isolate (U.S. Dairy Export Council).

Chapter 3

33

Depending on the removal of non-protein constituents, whey products may be classified

as whey protein concentrate (WPC) or whey protein isolate (WPI). The composition of

these products is shown in Table 3.1.

Table 3.1 – Composition of different whey products (Jelen, 2002)

Product type Total protein (%) Lactose (%) Minerals (%)

Whey powder 12.5 73.5 8.5

Whey protein concentrate (WPC) 65.0-80.0 4.0-21.0 3.0-5.0

Whey protein isolate (WPI) 88.0-92.0 <1 2.0-3.0

Next, a brief description of the main constituent proteins in whey and whey products is

given.

Beta-lactoglobulin (�-Lg) is the most abundant of the whey proteins. It comprises 10%

of the total milk protein or about 58% of the whey protein. It has about 15, 43 and 47%

�-helix, �-sheet, and unordered structure, respectively (Kinsella, 1984).

The structure of �-Lg is pH and temperature dependant. Above its isoelectric point (pH

= 5.2), the protein exists as a dimer with a molecular weight of 36.7 kDa. However,

above pH 7.5 and below pH 3.5, the dimer dissociates to its monomeric form. Between

pH 3.5 and 5.2 the dimer polymerizes to a 147 kDa octomer (Cayot and Lorient, 1997).

The conformation of �-Lg can also be modified by temperatures above 65 °C, at which

the protein undergoes denaturation accompanied by conformational transitions that

expose highly reactive �-NH2 groups (Kinsella, 1984).

Alpha-lactalbumin (�-La) is the second most abundant protein in whey. It comprises

2% of the total milk protein which is about 13% of the total whey protein. A single

polypeptide chain contains 123 amino acid residues, which include eight cysteines

covalently linked by four disulfide bonds. The protein is arranged in a spherical and

highly compact structure that undergoes thermal denaturation at approximately 62 to 65

°C, depending on the pH (Rüegg et al., 1977; de Wit and Swinkels, 1980). Due to an

unusually high apparent heat resistance, �-La is extensively renatured upon cooling.


34

Bovine serum albumin (BSA) carries insoluble fatty acids in the blood circulatory

system, which in turn further stabilizes the protein molecule against heat denaturation

(de Wit, 1998).

Immunoglobulins (Ig) refer to four classes of glycoproteins ranging in size from 15 to

1,000 kDa: IgG1 and IgG2, IgA, IgM and IgE. These proteins consist of two 20 kDa

polypeptide chains and two 50 to 70 kDa polypeptide chains which are crosslinked by

disulfide bonds.

Lactoferrin (LF) is a 78 kDa glycoprotein consisting of a single polypeptide chain

linked to two glycans by N-glycosidic linkages. Its average concentration in cow’s milk

is 10 mg/ml but LF is found in higher concentration in whey protein products. LF is not

only a source of amino acids but also a regulatory factor with broad biological roles.

Table 3.2 summarizes the most relevant physicochemical properties of the proteins

contained in whey.

Table 3.2 – Summary of properties of the basic constituent proteins in whey (Eigel et al., 1984)

Property �-Lg �-La BSA Ig

Isoelectric point 5.2 4.2-4.5 4.7-4.9 5.5-8.3

Concentration in whey, g/l 2-4 0.6-1.7 0.4 0.4-1.0

Concentration in whey, % (w/w) 56-60 18-24 6-12 6-12

Molecular weight, Da 18,400 14,000 66,000 �146,000

Total amino acid residues/mol 162 123 582 NA

Cysteine residues/mol 5 8 35 NA

Disulfide residues/mol 2 4 17 NA

Sulfhydryl residues/mol 1 0 1 NA

Lysine residues/mol 15 12 59 NA

Glutamic acid residues/mol 16 8 59 NA

Chapter 3

35

The composition of WPI is shown in Table 3.3.

Table 3.3 – Composition of whey protein isolate (Morr and Foegeding, 1990)

Parameter Range Mean ± S.D.

Moisture 2.40-5.57 3.75 ± 1.34

Protein 88.6-92.7 91.0 ± 1.73

�-Lg 67.6-74.8 70.2 ± 3.3

�-La 8.3-17.5 14.3 ± 4.3

BSA 7.2-10.9 8.6 ± 1.6

Ig 5.9-7.5 6.9 ± 0.7

Lactose 0.42-0.46 0.44 ± 0.02

Total lipids 0.39-0.67 0.57 ± 0.13

Ash 1.37-2.15 1.82 ± 0.33

n=2.

3.3.2 Uses of whey

The United States is the world’s largest whey exporter; latest available figures from the

U.S. Dairy Export Council (USDEC, 2006) show an increase in exportation by 104%

between 2001 and 2006. Whey used to be considered a bothersome byproduct of the

cheese manufacture because of the high value of biological oxygen demand (40,000

mg/kg). Therefore, it constituted a major ecological issue if disposed off as a waste

material. However, the image of whey is changing rapidly and becoming a highly

priced resource which may be used within the same dairy installation or destined

somewhere else for other applications (e.g. low-cost additive to animal feed).

Due to its balanced amino acid profile and nutritious value, the main use of whey and

whey products fall in the food industry. In fact, whey is one of the preferred sources of

protein for bodybuilders. Whey can also be used to fortify a wide variety of food in

order to enhance its nutritional quality and functional attributes. Researchers at the

ERRC developed texturized whey proteins that can be used to increase up to 35% the

protein content in foods such as breakfast cereals, corn puffs or cheese curls (Onwulata

et al., 2001). (Figure 3.5).


36

Figure 3.5 – Snack fortified with whey protein isolate.

Also, various non-food uses for whey have been explored, such as incorporation into

foamed insulation materials for the construction industry or use as a binder for iron ore

pellets (Chambers and Ferretti, 1979). Currently, the conversion of whey into valuable

materials such as biosorbents, biopolymers, fat substitute or bacteriocins is being

thoroughly examined.

3.4 Gelatin

Gelatin is obtained by the partial hydrolysis of collagen, which is the main protein in

skin, bones, hides and white connective tissues of the animal body. Gelatin does not

exist in nature and is classified as a derived protein because it is obtained from collagen

by a controlled partial hydrolysis. This hydrolytic process may be carried out with acid,

alkali or enzymes and in all cases entails the partial separation and rupture of the three

polypeptide chains of the triple-helix of the parent collagen (Figure 3.6). The degree of

hydrolysis attained leads to gelatin of various molecular weights, which may range from

20 to 250 kDa, with an average molecular weight between 50 and 70 kDa.

Figure 3.6 – Transformation of collagen into gelatin in the course of hydrolysis. Triple helix collagen molecules break off into random coiled structured gelatin.

Hydrolysis

Chapter 3

37

Gelatin is classified as type A or B. The former is produced by acid processing of

collagenous raw materials, typically pigskin. The latter is produced by means of an

alkaline or lime process, and cattle hides or bones are the most common starting

material.

3.4.1 Description and characterization

The physical and chemical properties of gelatin depend on factors such as the source of

collagen, the method of manufacture, the extraction conditions, the pH, and the

chemical nature of additives or impurities. Table 3.4 summarizes the most relevant

properties for these two types of gelatin.

Table 3.4 – Physicochemical properties of types A and B of gelatin (Glicksman, 1969)

Property Type A Gelatin Type B Gelatin

Moisture (%) 8-12 8-12

pH 3.8-5.5 5.0-7.5

Isoelectric point 7.0-9.0 4.7-5.1

Bloom strength (g) 50-300 50-275

Viscosity (mps) 20-70 20-75

Ash (%) 0.3 0.5-2.0

Cystine (%) 0.1 None-trace

Lysine (%) 11.3-11.7 11.1-11.4

Glutamic acid (%) 4.1-5.2 4.5-4.6

The capability to form heat-reversible gels is one of the most useful and unique

properties of gelatin solutions. The gelation process is believed to proceed through

three stages: first, the individual molecular chains are rearranged in a helical structure.

Next, crystallites are formed by the union of two or three ordered segments. Finally, the

formed structure is stabilized by lateral interchain hydrogen bonding within the helical

regions (Von Hippel, 1967).

The bloom strength gives an indication of the strength of a gel formed from a gelatin

solution of known concentration. The strength of the gel is function of the

concentration, the intrinsic strength of the gelatin sample, the pH, the temperature, and


38

the presence of additives. Also, the bloom strength is proportional to the average

molecular weight (Table 3.5).

Table 3.5 – Dependence of molecular weight on the bloom strength of type B gelatin

Bloom strength Average molecular weight (kDa)

50 – 125 (Low Bloom) 20 – 25

175 – 225 (Medium Bloom) 40 – 50

225 – 325 (High Bloom) 50 – 100

Source: Sigma Gelatin data sheet (1996).

Aside from gel strength, there are other relevant physical and chemical properties to

characterize gelatins.

• Viscosity. Gelatin exhibits Newtonian behavior above 40 °C, and non-

Newtonian at temperatures between 30 and 40 °C. Viscosity of aqueous

solution of gelatin increases with gelatin concentration and decreasing

temperatures. Viscosity is also affected by the same factors as those mentioned

for gel strength.

• Solubility. Gelatin is soluble in water and in most highly polar organic solvents,

and practically insoluble in less polar solvents. The ratio of exothermic solvent

absorption is characteristic of each particular gelatin.

• Amphoteric character. The hydrolysis of collagen creates terminal amino and

carboxyl groups that make gelatin positively charged in strong acidic solutions

and negatively charged in a strong alkaline environment. This important

characteristic allows gelatin to be reactive with a wide range of substances.

• Swelling. This property is important in the solvation capacity of gelatin. The

rate of swelling is affected by factors such as pH, temperature, time and the

presence of electrolytes. Swelling is an essential characteristic to consider if

gelatin is intended to be used in photographic film processing or as

pharmaceutical capsules coating.

• Stability. Even though dry gelatin has a shelf life of many years if stored at

room temperature, it decomposes above 100 °C. In addition, aqueous solutions

of gelatin are highly susceptible to bacterial activity and breakdown by

proteolytic enzymes.

Chapter 3

39

3.4.2 Uses of gelatin

Gelatin has a wide variety of applications, mainly in the food, pharmaceutical and

photographic industries. Food applications are centered on edible products such as

candy, marshmallow, pies, ice cream, wafers, etc. The addition of small amounts of

gelatin during the manufacture of these products contributes in preventing the

crystallization of sugar or ice, inhibiting water separation, increasing the viscosity or

stabilizing the foam. Most edible gelatin is type A, but type B is also used.

Type A and B gelatins are also formulated in the manufacture of soft and hard capsules

for the pharmaceutical company. In addition, the use of gelatin as coating agents for

tablets is becoming more predominant due to the extra protection that provides against

medication adulteration.

Gelatin also has a long tradition as a binder in light sensitive products, particularly in

the manufacture of photographic film. Over the last 40 years, the photographic industry

has progressively switched from hide-derived gelatin to that obtained from bones.

3.4.3 Gelatin in the leather industry

Interestingly, gelatin can also be isolated from chrome shavings. Shavings are small

pieces of leather shaved off when the thickness of wet or dry tanned leather is rendered

uniform by a bladed cylinder. Therefore, this treatment can add value by using a

cleaner production pathway to a valuable product extracted from what was previously

hazardous waste.

The conversion of this waste material into gelatin can be done with enzymes (Cabeza et

al., 1997) or by means of classic hydrolysis (Catalina et al., 2007) (Figure 3.7). Gelatin

obtained through this technology was reported to have a potential application in

microencapsulation (Cabeza et al., 1999) and finishing agents for leather (Catalina et

al., 2007).


40

Figure 3.7 – Conversion of chrome shavings into gelatin.

3.5 Enzymes

Enzymes are proteins capable of catalyzing (i.e. accelerating) a reaction in which

various substrates are converted to products through the formation of an intermediate

enzyme-substrate complex. The objective is accomplished by lowering the energy

required to reach the substrate transition state (Figure 3.8).

Figure 3.8 – Role of an enzyme in a given reaction.

Chapter 3

41

As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do

they alter the equilibrium of these reactions. However, enzymes do differ from most

other catalysts by being much more specific. The specific action of an enzyme with a

single substrate can be explained using a lock and key analogy. In this analogy, the

lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate)

fits into the key hole (active site) of the lock (enzyme). Smaller keys, larger keys, or

incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do

not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock

(Figure 3.9).

Figure 3.9 – Lock and key analogy for enzymes and substrate, first postulated in 1894 by Emil Fischer.

The activity of an enzyme depends on various factors: temperature, pH, concentration of

substrates and reactants, diffusion of the site of activity, and presence of certain

substances such as ions, inhibitors (molecules that decrease enzyme activity) or

activators (molecules that enhance enzyme activity).

It is important to point out the difference between enzyme activity and enzyme

concentration. The former may be measured as units of change in a specified substrate

over a period of time. The latter is defined as the quantity of enzyme present in the

sample. Therefore, doubling the concentration of enzyme does not entail doubling its

activity in any case.


42

3.5.1 Enzymes in the leather industry

Enzymes have been around in the leather industry for a long time and are used at

different stages of the beamhouse. Proteolytic enzymes are used in soaking, liming and

bating, with the mission of opening up the fibers, increasing area yield, improving the

cleanliness of hides and mitigate the problem of fine or short hair (Alexander, 1988).

Alkaline serine protease isolated from Aspergillus tamari was recently reported to be an

effective dehairing agent which eliminates the nuisance of sulfide in tannery effluent

disposal (Anandan et al., 2007). However, one has to bear in mind that the use of

excessive amounts of enzymes may lead to undesirible effects such as loose leather and

abraded grain.

Enzymes have been applied with success in gelatin isolation from chrome shavings also.

Pepsin and trypsin were reported to be the most promising enzymes on the isolation of

high quality products from tannery wastes (Cabeza et al., 1997). However, cost was a

major deterrent to using research grade enzymes. In 2000, Taylor et al. found a

commercial trypsin preparation that proved to be not only efficient in solubilizing the

shavings but also cost effective.

3.5.2 Microbial transglutaminase

Protein-glutamine: amine �-glutamyl-transferase (EC 2.3.2.13), commonly known as

transglutaminase (TGase) (Webb, 1992), is an enzyme capable of forming inter- or

intra-molecular crosslinks in many proteins. The enzyme catalyzes an acyl transfer

reaction between the �-carboxamide group of peptite-bound glutamine residues as acyl

donors and primary amines as acceptors. When the �-amino group of peptide-bound

lysine acts as acyl acceptor, an �-(�-glutamyl) lysine crosslink is formed.

In the absence of amine substrates, TGase can catalyze the deamidation of glutamine

residues during which water molecules are the acyl acceptors.

Chapter 3

43

Transglutaminase can also catalyze an acyl transfer reaction between the �-carboxamide

group of peptite-bound glutamine residues and various primary amines.

Transglutaminase’s activity depends strongly on pH and temperature, regardless of the

substrate. Optimum conditions of pH and temperature are 7 and 50 °C, respectively.

Figure 3.10 – (a) Temperature and (b) pH activity profiles of microbial transglutaminase (Source: Ajinomoto, Inc.).

Although the earliest papers that reported on TGase were released in the late 1950’s, it

was not until the 1980’s that research on this enzyme and particularly its reactivity

towards food proteins arose. By that time, TGase was isolated from mammalian

systems such as pig liver or plasma coagulation and its activity was found to be

dependent on the availability of calcium ions (Folk, 1980). The unfeasibility to produce

large amounts of TGase triggered off the research to find a more cost-effective way to

produce the enzyme. In 1989, Ando et al. were the first investigators to isolate TGase

from a microorganism, the Streptoverticillium mobaraense. The advantages of this new

microbial transglutaminase (mTGase) over its predecessor were tremendous:

inexpensive, readily available, broad specificity and an activity independent of calcium

ion concentration. Ajinomoto Co., Inc. and Amano Pharmaceutical Co. Ltd. jointly

granted a patent shortly

a b


44

after that discovery and began to produce the enzyme in an industrial scale. Later on,

the enzyme was also identified in Physarum polycephalum (Klein et al., 1992), fish

(Yasueda et al., 1994) and plants (Pallavicini et al., 1992).

After the enzyme became readily available and economical, the industry started to show

interest on the ability of the enzyme to modify proteins functionality. In the 1990’s, the

literature exploded with scientific papers reporting the effects of mTGase on food

proteins.

Table 3.6 lists examples of various food proteins that were successfully treated with

TGase or mTGase and the effect exerted on their functional properties.

Table 3.6 – List of proteinaceous substrates reported to be reactive towards TGase

Substrate Effect of transglutaminase Reference

Gelatin, caseinate, soy protein isolate, egg yolk • Increase in breaking strength of gels Sakamoto et al.,

1994

Caseinate • Increase of gel forming ability,

modification of breaking strength, strain and cohesiveness of the gels formed.

Nonaka et al., 1992

Whey protein + Soybean 11S globulin

• Modification of heat stability, emulsifying properties and foaming capacity and stability of formed biopolymers.

• Obtaining of films with better mechanical properties and more resistance to solubilization.

Yildirim et al., 1996, 1997, 1998

Soy protein

• Increase in solubility, decrease in surface hydrophobicity, improvement of emulsifying and foaming properties, reduction of bitterness.

Babiker et al., 1996a

Gluten • Improvement of solubility, foaming,

emulsifying and surface functional properties.

Babiker et al., 1996b

�-lactoglobulin • Enhancement of heat stability, increase of viscosity and gel forming ability

Tanimoto and Kinsella, 1988

Chapter 3

45

3.5.3. Reactivity of TGase with gelatin and whey proteins

The ultimate goal of enzymatic modification of proteins is to improve their

functionality. Protein functional properties can be defined as “physico-chemical

properties that influence the structure, appearance, texture, viscosity, mouthfeel, or

flavor retention of the product” (Morr and Ha, 1993).

Gelatin is a reactive substrate towards the enzymatic action of mTGase, meaning that

the reaction will take place under the right conditions of pH, temperature and substrate

and enzyme concentration (Figure 3.11).

Figure 3.11 – Cross-linking of gelatin chains upon reaction with mTGase.

Sakamoto et al. (1994) studied the optimum reaction conditions of mTGase with gelatin

and found that an incubation period of 4 hours with 30 units mTGase/g gelatin, at pH 6

and 50 °C yielded gels with higher breaking strengths (Figure 3.12).


46

Figure 3.12 – Effect of (a) mTGase concentration, (b) incubation time, (c) pH and (d) incubation temperature on breaking strength of a 10% (w/w) type A gelatin (Sakamoto et al., 1994).

All kind of gelatins are susceptible to undergo enzymatic modification: pigskin gelatin,

both types A and B (McDermott et al., 2004; de Carvalho and Grosso, 2004), and fish

gelatin, from skin and bones of cod, haddock, and pollock (Yi et al., 2006) or the skin

of bigeye snapper and brownstripe red snapper (Jongjareonrak et al., 2006).

There are proteins that need to be treated prior to undergo the enzymatic reaction with

mTGase. This is the case of the two most abundant proteins in WPI, �-Lg and �-La.

As opposed to gelatin, the globular structure of these two proteins causes the reactive

groups to be buried and therefore inaccessible to the action of the enzyme. A less

globular structure of these proteins can be achieved by undergoing a denaturation

process, which enhances their reactivity towards the enzyme (Færgemand et al., 1997).

Denaturation of whey proteins can be achieved using different methods. The use of a

reductant or the application of heat are among the two most common. DTT is generally

(a) (b)

(c) (d)

Chapter 3

47

the reductant of choice for cleaving disulfide bonds given that it is needed in much

lower concentration than other reductants (e.g., glutathione, cysteine) to complete the

reaction (Grazú et al., 2003). DTT breaks down the disulfide bond by two sequential

thiol-disulfide exchange reactions, resulting in DTT becoming a six-member ring

structure. (Figure 3.13).

Figure 3.13 – Reduction of a disulfide bond by two thiol-disulfide exchange reactions involving DTT.

Whey proteins can also be denatured by the action of heat. Upon heating, whey

proteins undergo a conformational transition that unfolds their structures making the

reactive groups available. As an example, sulfhydryl groups react with the disulfide

bonds in a thiol-disulfide exchange reaction. This reaction, along with interactions of

other nature (e.g. electrostatic, hydrophobic) are responsible for the aggregation of the

thermally denatured whey proteins, which may result in the formation of gels.

Even though the apparent order of denaturation of individual whey proteins is Ig > BSA

> �-Lg > �-La, their rates of denaturation are strongly influenced by factors such as pH,

ionic composition, or total solids concentration.

CHAPTER 4

Mathematical model of raw hide curing with brine

Chapter 4

49

4. MATHEMATICAL MODEL OF RAW HIDE CURING WITH BRINE

Authors: Eduard Hernàndez Balada1,2, William N. Marmer1, Karel Kolomazník3, Peter

H. Cooke1 and Robert L. Dudley1.

1U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional

Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038 USA

2Department of Chemical Engineering, University of Barcelona, Martí i Franquès 1,

08028 Barcelona, Spain

3Tomas Bata University, Mostni 5139, 760 01 Zlín, Czech Republic

Manuscript published at the Journal of the American Leather Chemists´Association

(JALCA), Vol. 103 (5), 167-204, 2008.


50

4.1 Letter of acceptance

THE JOURNAL OF THE AMERICAN LEATHER CHEMISTS

ASSOCIATION c/o The American Leather Chemists Association, 1314 50th Street, Suite 103,

Lubbock, Texas 79412 Mobile phone: (616) 540-2469 E-mail: [email protected] November 19, 2007 Dr. William N. Marmer USDA, Agricultural Research Service Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor, PA 19038 Dear Dr. Marmer, This will acknowledge receipt and acceptance for publication of the manuscript entitled “Mathematical Model of Raw Hide Curing with Brine”. It is tentatively scheduled for publication in the May 2008 issue of the Journal. The manuscript now lists you as the corresponding author. As instructed in your cover letter of November 14, 2007, I will change the manuscript to footnote Dr. Hernàndez as the corresponding author. Could you please sign the attached “Transfer of Copyright” form and return it to me. Thank you for this interesting contribution. Sincerely yours, Robert F. White Journal Editor cc: Dr. Eduard Hernàndez Balada

Chapter 4

51

4.2 Abstract

The most common method of preserving raw hides is brine curing with sodium

chloride. However, this process has three important disadvantages: first, the length of

time that it takes, which is a minimum of 18 hours; second, the insufficient degree of

curing reached in some hides due to an overload and possibly the low efficiency of the

brine raceway; and finally, the environmental impact associated with the discharge of

large quantities of electrolytes in the soaking step. Our long term goal is to address all

three issues. Initially, we have carried out a study of the salt uptake and its diffusion

mechanism in order to attempt a reduction in the curing time. A continuous reaction

mathematical model of a closed one dimensional system that describes the diffusion of

sodium chloride in the hide during the curing process was chosen in the search for the

optimum brine curing conditions such as the optimum brine concentration and percent

float. The effect of these two parameters on the values of transport coefficient � was

reported. Brine diffusion into the hide was tracked by measurement of the chloride

concentration of the residual brine solution. In addition, a piece of hide was cured with

a fluorescently labeled brine solution and analyzed by means of epifluorescent

microscopy for direct visualization of the sodium location within the hide.


52

4.3 Resum

El mètode més comú per a preservar pells crues és el curat amb salmorra. Tot i així,

aquest procés presenta tres desavantatges: en primer lloc, el temps que requereix, un

mínim de 18 hores; en segon lloc, l’insuficient nivell de curat que s’assoleix en algunes

pells a causa de la sobrecàrrega i baixa eficàcia del tanc de curat; i finalment, l’impacte

mediambiental associat a la descàrrega d’elevades quantitats d’electròlits en l’operació

de remullat. El nostre objectiu a llarg terme és resoldre els esmentats problemes.

Inicialment, estudiem l’absorció de sal i el seu mecanisme de difusió per tal de reduir el

temps de curat. Un model matemàtic de reacció contínua d’un sistema unidimensional

tancat fou seleccionat per descriure la difusió del clorur de sodi a la pell durant el procés

de curat per tal de trobar les condicions òptimes de curat, tals com la concentració

òptima de salmorra i el percentatge òptim de bany. L’efecte d’aquests dos paràmetres

en els valors del coeficient de transport � són presentats. La difusió de sal a la pell va

ser monitoritzada mitjançant la determinació de concentració de clorurs en la solució

residual de salmorra. A més, una mostra de pell fou curada amb una solució de

salmorra etiquetada amb una substància fluorescent i analitzada mitjançant microscopia

epifluorescent per a la visualització directa del sodi en la pell.

Chapter 4

53

4.4 Introduction

Raw hides and skins are ~ 60-70% water and ~ 25-30% protein. In this form the hide is

susceptible to bacterial activity within hours after being removed from the carcass. The

autolytic degradation of skins/hides is assumed to be due to a combined action of tissue

enzymes and bacteria, the latter requiring moisture to be viable.1 Curing is the process

that provides an environment in which bacteria cannot survive. Several curing agents

have been reported in the literature, e.g., potassium chloride,2 silica gel,3,4 boric acid,5

and herbal-based products.6 Common salt, in spite of its inherent impact on the

environment and the large amount required, is the most popular and inexpensive

material used to preserve hides and skins. A suitable improved method would yield

savings in salt, shipping and effluent treatment costs as well as a diminished

environmental impact. Mathematical modeling has been reported to be a powerful tool

in the optimization of processes such as soaking of salted cattle hides.7,8 Curing has

been modeled in substrates such as cheese9,10 and meat11, but has not been studied for

the particular case of raw hides.

The aim of this work is to develop and verify a mathematical model that describes the

diffusion of sodium chloride in the hide during the curing process. Upon its

verification, the model is applied in the search of the optimum values of process

variables such as brine concentration, float percentage and curing time.

4.5 Theory

We propose a continuous reaction model to describe the diffusion of sodium chloride

from the bath containing brine solution to the surface of the solid phase (hide). The

model assumes that salt will further diffuse into the hide’s inner volume where it will

form a non-stationary concentration field (Figure 4.1). It also assumes that diffusion

takes place only into the flesh side and that hide parameters such as thickness, surface

and properties of both hair and flesh sides will remain constant throughout the whole

process.


54

Figure 4.1 – Mathematical model of the curing process of a raw hide.

Curing can be considered as a counter diffusion in which sodium chloride soaks into the

skin as water simultaneously washes out. Equation (1) describes a non-stationary one

dimensional concentration field inside the inner volume of the solid phase, defined by

Fick’s second law. Boundary condition (1a) assumes that sodium chloride diffuses into

the hide from the flesh side only. Terms are defined at the end of the paper.

),(),(2

2

τττ

xxc

Dxc

∂∂⋅=

∂∂

bx <<0 0>τ (1)

0),0( =∂∂ τxc

(1a)

Equation (2) corresponds to a mass balance of a closed system in which salt flow at the

hide surface is equal to accumulation speed of sodium chloride in the bath. Equations

(2a) and (2b) are the initial boundary conditions (� = 0). They assume a null initial

content of sodium chloride in the fresh hide and a constant initial value of brine

concentration in the bath respectively.

)(),( 00 τ

ττ

∂∂⋅=

∂∂⋅⋅− c

Vbxc

DS (2)

0)0,( =xc (2a)

pcc 00 )0( = (2b)

Chapter 4

55

Equation (3) is valid under an ideal mass transfer from the bulk solution to the surface

of the solid phase.

)(),( 0 τετ cbc ⋅= (3)

The introduction of dimensionless parameters (equations 4a to 4e) has been

demonstrated to be a useful tool in the model development.

pc

cC

0⋅=

ε (4a)

pc

cC

0

00 = (4b)

bx

X = (4c)

2bD

Fo

τ⋅= (4d)

VV

Na 0= (4e)

The dimensionless time, also called Fourier’s number [F0], assesses the proximity of the

process to the equilibrium, i.e. equilibrium is reached when F0 � �. The dimensionless

soaking number [Na] expresses the ratio between the volumes of liquid and solid

phases. The replacement of the dimensionless parameters into the previous model leads

to a new dimensionless model (Eq. (5a) to (5f)).

),(),( 02

2

00

FXX

CFX

FC

∂∂=

∂∂

10 << X 00 >F (5a)

)(),1( 000 FCFC = (5b)

)(),1( 00

00 F

FCNa

FXC

∂∂

⋅−=∂∂

ε (5c)

0),0( 0 =∂∂

FXC

(5d)

0)0,( =XC (5e)


56

1)0(0 =C (5f)

the analytical solution of which can be obtained by means of Laplace’s transformation:

( ) �∞

=

⋅−

⋅⋅−⋅

−⋅

⋅⋅⋅+

+=

1 )sin()sin(

)cos(

)cos(2,

20

nnn

n

nn

gFn

o

gNagg

gg

egXNa

NaNa

FXCn

εεε (6)

Where gn are the roots of the transcendent equation (7).

ε

nn

gNagtg

⋅−=)( (gn > 0) (7)

The three dimensional concentration field graphic corresponding to Eq. (6) is shown in

Figure 4.2.

Figure 4.2 – Dimensionless sodium chloride concentration field within the hide during the curing process.

Chapter 4

57

Replacing Eq. (7) into Eq. (6) and rearranging terms (for X = 1), we obtain an equation

that illustrates the variation of brine concentration with time.

( ) �∞

=

⋅−

⋅++

⋅++

=1

220

20

2n n

gF

o gNaNa

eNa

NaNa

FCn

εεε

(8)

In addition, integration of Eq. (6) leads to Eq. (9) which calculates the optimal time in

order to reach a certain content of sodium chloride in the skin, C (integral average

concentration).

�∞

=

⋅−

⋅+⋅+⋅−

+=

1222

2

20

2)(n n

gF

o gNaNae

NaNa

NaFC

n

εεε (9)

Figure 4.3 shows the curves of the integral average concentration for various values of

soaking number.

Figure 4.3 – Dimensional sodium chloride concentration field within the hide during the curing process for various soaking numbers.


58

Determination of Diffusion Coefficients

The value of effective diffusion coefficient of sodium chloride in the hide can be

evaluated from experimental data. Crank12 suggested an equation for the diffusion

coefficients study at short times:

τλπ

ττ ⋅⋅+⋅=∞−

−=

NaNa

cccc

CP

P 12)()(

)(00

000 (10)

From the mass balance

VcVcVc p ⋅⋅+⋅=⋅ ∞∞ 00000 ε (11)

We get

ε+⋅=∞ NaNac

c P00 (12)

Transport parameter � is defined as a ratio of the effective diffusion coefficient D’ to

pore half length (a) square.

22

'aD

bD ==λ (13)

when the factor for the tortuosity of the pores:

ba=ξ (14)

� is an important value from an engineering point of view since it includes two

phenomena not considered in the presented model, which are the transport of water

from the hide to the bath and the interaction between sodium chloride and water counter

flows during the curing.

Chapter 4

59

4.6 Experimental

4.6.1 Materials

Fresh cow hides were purchased from a local abattoir. They were soaked for 2 h (with

surfactant) and then fleshed. Approximately 6 × 10 in (15 × 25 cm) pieces were cut and

stored at -20°C. They were thawed at 4°C just before use. Food grade sodium chloride

of purity minimum 99.82% was obtained from US Salt Corporation (Watkins Glen,

NY). All other chemicals were reagent grade and used as received.

4.6.2 Methods

Thawed hide was cut into square pieces of approximately 4 × 4 in (10 × 10 cm) with an

average weight of ~ 100 g. They were transferred to a Dose drum (Model PFI 300-34,

Dose Maschinenbau GmbH, Lichtenau, Germany), and tumbled at 6 rpm with brine

solution for varying time intervals after which they were pulled out of the drum, hand-

squeezed to wipe excess water, sealed in plastic bags and placed in the refrigerator. A

fraction of the residual brine solution was also collected at different time intervals. Two

sets of experiments were carried out: a constant 300% (v/v) float (volume of brine

solution/volume of hide) at different initial salt concentration levels (20%, 25%, 30%

and 35% (w/v), which correspond to 64, 80, 96 and 100 °SAL, respectively) and a

constant 30% (w/v) initial salt concentration (weight of NaCl/volume of solution) at

different float percentages (300%, 500%, 750% and 1000% v/v). Density of hide was

assumed to be ~ 1g/cm3.

4.6.3 Analyses

4.6.3.1 Chloride concentration determination

Chloride concentration was determined by classical Mohr titration.13 Residual brine

samples were diluted (1:100 v/v) in nano pure water prior to titration. All samples were

run in triplicate.

4.6.3.2 Fluorescence imaging

CoroNa ™ Green Sodium Indicator fluorescent dye (Invitrogen, Carlsbad, CA) was

used as a probe of sodium ions diffusing into raw hide from brine solution. A piece of

raw hide of approximately 1 × 1 in (2.5 × 2.5 cm) was immersed in a beaker containing

500% v/v float of a 30% w/v NaCl solution and 5µM of the fluorescent dye and gently


60

agitated. A 2-3 mm wide slice of the sample was excised manually with a stainless steel

razor blade (cutting from flesh surface toward the grain) at regular intervals of

incubation time, then mounted onto Petri dishes for imaging, using a Leica MZ FLIII

stereomicroscope (Leica Microsystems, Bannockburn, IL, USA) equipped for

epifluorescence and with a model DC200 color charge couple device camera system at

2.5x magnification. Samples were irradiated with blue (470/40 nm) and UV (360/40

nm) light, and images of the fluorescence were acquired at 0.1 (blue) and 0.44 (UV)

seconds of exposure time. Control samples, immersed in 500% v/v nano-pure water

and 5µM dye, were examined under the same conditions of concentration and time to

assess the penetration of the fluorescent dye in the absence of salt, and a blank sample

was also examined to evaluate possible autofluorescence of the untreated raw hide.

4.7 Results and discussion

4.7.1 Epifluorescence microscopy

The diffusion of labeled sodium ions into the cross section of hide samples was

followed by means of epi-fluorescent microscopy. In Figure 4.4, increased fluorescence

indicating diffusion of salt started on the flesh side and gradually moved toward the hair

side.

Figure 4.4 – Epifluorescent microscopic images of a cross section of a hide at different stages of curing. The hide was cured with 30% (w/v) labeled sodium chloride solution.

15min 30min 1h 2h 3h 4h 5h 28h 48h

Flesh

Hair

15min 30min 1h 2h 3h 4h 5h 28h 48h

Flesh

Hair

Flesh

Hair

Chapter 4

61

The lack of fluorescence development at the hair side validated the mathematical

assumption described by Eq. (1a); this can be attributed to the existence of a thin

protective barrier of sebaceous oil.14 The series of images demonstrate the advance of

fluorescence due to sodium ions into the cross sections of hide as well as increases in

fluorescence intensity throughout curing time. Signal saturation in the area near the

flesh side, denoted by a very bright fluorescence, was observed after 5 h of curing. The

apparent retrograde movement of the labeled sodium between 2 h and 5 h may be due to

the shrinking of the hide caused by dehydration. Surprisingly, sodium ions did not

seem to reach the hair side even after 48 h of curing. This could be due to many factors.

In order to determine if the penetrability of the dye is a technical factor, a sample of

hide incubated in an aqueous solution of dye for 24 hours was examined under the

microscope and then transferred to a beaker with concentrated brine solution for 24

more hours. The dye did not fluoresce in the uncured sample but showed a high

fluorescence after being cured for 24 hours (graphs not shown). However, fluorescence

was absent or undetectable in the upper part of the corium, leading to the possibility that

the dye may not penetrate into the tightly-woven and dense structure of the corium,

possibly due to its size (MW = 586 Da). The use of scanning electron microscopy with

energy dispersive X-Ray spectroscopy (SEM-EDS) and elemental mapping to measure

the amount and location of salt in a brine-cured hide is an alternative method to

fluorescence imaging and this approach is planned.

4.7.2 Determination of diffusion coefficients

The diffusion of salt in the hide was evaluated by means of the transport coefficient �,

which can be calculated from the slope of the straight line that results from plotting

C0(t) versus square root of time (Eq. 10). Taking into account early published results12

and the accuracy of our measurements, the linear dependence holds approximately as

far as to the value of C0(t) = 0.6. Figure 4.5 depicts this correlation for the particular

case of c0p = 30% (w/v) and Na = 3.


62

Figure 4.5 – Determination of transport parameter � from experimental data. The graph corresponds to c0p = 30% (w/v) and Na = 3.

As seen in Table 4.1, all � values, except from that of c0p=35% (w/v), are on the order of

10-5 s-1. These results are of the same order of magnitude as those reported in the

mathematical model of soaking15, which suggests that the diffusion of salt does not

significantly differ between curing and soaking. A numerical value of � for c0p=35%

(w/v) may not be reliable, because the brine was initially supersaturated and the model

was developed for homogeneous solutions solely. Note than a saturated brine solution

holds 31.7g of salt in 100 ml of solution, (c0n) at 25°C.16

TABLE 4.1 Transport coefficient � for various conditions of initial brine concentration (c0p) and

soaking number (Na) Na = 3 C0p = 30% (w/v)

C0p (% w/v) �·105 (s-1) R2 C0p (% w/v) �·105 (s-1) R2

20 4.2 0.835 3 5.3 0.949

25 3.8 0.921 5 9.0 0.887

30 5.3 0.949 7.5 4.1 0.905

35 10.7 0.776a 10 8.6 0.876 aR2 < critical value for � = 0.05.

A comparison of the individual values of � is not simple, since they may be affected by

some of the following factors: 1. The thickness of the hide, which may vary throughout

the process and exerts a strong effect on the value of �, as seen in Eq. (13). 2. The pore

length, which varies among the hides and is hardly measurable. 3. The dry matter

content of the hide, the variation of which may extensively modify �. In fact, � may

drop up to two orders of magnitude between a wet and a dry hide.15,17 4. The

Chapter 4

63

temperature, which affects the diffusion rate and was sometimes difficult to keep at

25°C during the process. 5. The influence of the error in the measurement, i.e. the

difficulty to measure a small chloride concentration diminution with the Mohr method

despite the very low coefficient of variation (CV) found for this technique (< 1%).

Even so, one can draw the conclusion on the effect that both c0p and Na exert on the

values of �. Increasing values of c0p yielded larger values of � as a consequence of an

increasing gradient concentration between the solid phase and the solution, which is in

accordance with Fick’s second law of diffusion. This fact corroborates a general

practice applied in curing raceways, where solid salt is periodically added to the brine

solution to keep it close to saturation (� 97 °SAL). The float percentage also exerts a

remarkable effect on the values of transport parameter �. Larger floats yielded faster

diffusion of salt into the hide, even though that effect became less significant for Na >

5. That experimental observation corroborates the common practice in tanneries, which

operate at Na ~ 4 even though the generally accepted rule requires a Na � 5 in order for

hides to receive a proper cure.18 In addition, a large float will help maintain an almost

constant salt concentration. The outstandingly low value of � obtained for Na = 7.5

may be due to factors inherent to the hide, e.g., poor fleshing, which slows down salt

penetration, agglutination of the fibers and content of dry matter.

4.7.3 Determination of optimum brine curing conditions

An 85% salt saturation of the water remaining in the hide was established as a minimum

standard in order to attain a proper degree of cure.19 One can calculate the theoretical

minimum soaking number needed to attain this saturation percentage in the equilibrium,

that is, at infinite time, and without further additions of salt into the solution. From Eq.

(8), if � � then Fo � �; thus the second summand can be neglected, giving:

Na

Nacc

Cop +

==ε

00 (15)

Replacing 0cc ⋅= ε and rearranging for Na,


64

cc

cNa

p −⋅⋅=

0εε

(16)

Using ncc 085.0 ⋅⋅= ε , a porosity of � = 0.5 and c0p = 30 % (w/v), and assuming that all

pores are filled up with brine solution, a soaking number of 4.4 is needed to reach 85%

saturation. Solving Eq. (16) for c0p = 20 and 25% (w/v), negative values of Na are

obtained, indicating the unfeasibility to attain 85% saturation. On the other hand, the

minimum soaking number would drop to 2.8 if the cure was started out with a saturated

brine solution (31.7 % (w/v), or 100 °SAL). Notice that these values depend on the

porosity of the hide, which varies from one to another and within itself.20 Therefore,

slightly different values of minimum Na’s would be obtained using another value of

porosity.

Working out the value of c0p from Eq. (16) and using Na = 3, we received a minimum

initial brine concentration of 30.8% (w/v) in order to achieve the target saturation level.

By means of Eq. (9), the plot of which is depicted in Figure 3, one is able to calculate

the curing time needed to reach an 85% salt saturation in the hide. As just mentioned

above, this level cannot be achieved for c0p = 20, 25 and 30% (w/v) and Na = 3.

However, a time of 4.2 h is obtained for a 35% (w/v) supersaturated brine and Na = 3.

The 85% saturation is also achieved in 4.9, 7.7 and 3.4 h if the hide is cured with a brine

of c0p = 30% (w/v) and Na = 5, 7.5 and 10, respectively. These values are substantially

lower than the 18 hours that usually are required for a full cure in a normal float (Na~5).

The calculations of those times contain the parameter �, and therefore are affected by

the same factors mentioned in the previous section. In spite of this, it is interesting to

note the decrease of curing time with increasing float percentages, except from the

erratic value obtained for Na = 7.5.

4.8 Conclusions

Over 20 millions brine-cured hides were exported by the U.S. in 2006 (U.S. Leather

Industry Statistics, 2007). Increasing commodity prices for sodium chloride over the

past few years together with issues associated with water pollution set the alarm off in

the leather and meatpacking industries. The purpose of research reported in this article

Chapter 4

65

was to optimize the brine curing of hides and skins under specific process conditions by

means of mathematical modeling. The diffusion of salt into the hide was characterized

by the transport coefficient �, which was found to be in the order of 10-5 s-1. The usage

of saturated brine as well as large floats (>500%) yielded higher values of �, therefore

higher diffusion rates. From the model it was also possible to determine the minimum

float and initial brine concentration needed to attain an 85% salt saturation in the hide.

This saturation level was not achieved employing brines of initial concentration of 20

and 25% (w/v) independently of the float percentage used. For 30 and 35% brines, a

minimum float of ~ 440% and ~280% was found, respectively. Using a 30% (w/v)

brine, the targeted 85% saturation is attained in shorter times as the % float increases,

and one may expect the same trend for any other initial brine concentration. The

established 85% salt saturation in the hide obviously plays a critical role in the search

for optimum conditions of curing, and the need to attain this saturation level for a

proper cure will be discussed in our next contribution.

4.9 Definition of terms

a: pore half length of the skin [m]

b: thickness of cured hide [m]

c: concentration of sodium chloride in the hide moisture, at a distance x from the

boundary ( > 0) [mol m-3]

c0: concentration of sodium chloride in the bath ( > 0) [mol m-3]

c0n: concentration of saturated sodium chloride solution at 25°C [mol m-3]

c0p: initial concentration of sodium chloride in the bath ( = 0) [mol m-3]

c0�: equilibrium concentration of sodium chloride in the bath [mol m-3]

C : dimensionless concentration integral average [1]

C, C0: dimensionless concentrations [1]

D: diffusion coefficient of sodium chloride in the hide [m2 s-1]

D’: effective diffusion coefficient of sodium chloride in the hide [m2 s-1]

F0: Fourier number/dimensionless time [1]

Na: soaking number [1]

S: outer surface of the solid phase (skin) [m2]

V: volume of skin [m3]

V0: volume of brine solution [m3]


66

X: dimensionless distance [1]

Greek symbols

�: porosity of solid state [1]

�: transport coefficient [s-1]

: time (s) 4.10 References

1. Rao, B.R., and Henrickson, L.; Short-term preservation of cattlehide. JALCA 78, 48-

53, 1983.

2. Bailey, D.G., and Gosselin, J.A.; The preservation of animal hides and skins with

potassium chloride. JALCA 91, 317-333, 1996.

3. Kanagaraj, J., Chandra Babu, N.K., Sadulla, S., Suseela Rajkumar, G., Visalakshi

V., and Chandra Kumar, N.; Cleaner techniques for the preservation of raw goat

skins. J. Clean Prod. 9, 261-268, 2001.

4. Munz, K.H.; Silicates for raw hide curing. JALCA 102, 16-21, 2007.

5. Kanagaraj, J., John Sundar, V., Muralidharan, C., and Sadulla, S.; Alternatives to

sodium chloride in prevention of skin protein degradation–a case study. J. Clean

Prod. 13, 825-831, 2005.

6. Preethi, V., Rathinasamy, V., Kannan, N., Babu, C., and Sehgal, P.K.; Azardirachta

Indica: a green material for curing of hides and skins in leather processing. JALCA

101, 266-273, 2006.

7. O’Brien, D.J.; A mathematical model for unsteady state salt diffusion from brine-

cured cattlehides. JALCA 78, 286-299, 1983.

8. Kolomazník, K., Janacova, D., Vasek, V., and Blaha, A.; Chemical engineering and

automatics control in leather technology. Advanced Technologies: Research-

Development-Application (Lalic, B. ed.) Verlag Robert Mayer – Scholz, Germany,

pp. 475-516, 2006.

9. Guinee, T.P., and Fox, P.F.; Sodium chloride and moisture changes in Romano-type

cheese during salting. J. Dairy Res. 50, 511-518, 1983.

10. Turhan, M., and Kaletunç, G.; Modeling of salt diffusion in white cheese during

long term brining. J Food Sci. 57, 1082-1085, 1992.

Chapter 4

67

11. Bertram, H.C., Holdsworth, S.J., Whittaker, A.K., and Andersen, H.J.; Salt diffusion

and distribution in meat studied by 23Na nuclear magnetic resonance imaging and

relaxometry. J. Agric. Food Chem. 53, 7814-7818, 2005.

12. Crank, J.; The mathematics of diffusion, 2nd ed. Clarendon Press, Oxford, London.

1975.

13. Quantitative Analysis, 4th ed. (Pierce, Haenisch and Sawyer eds.) John Wiley &

Sons Inc., New York, 1958.

14. Sharphouse, J.C.; Types of hides and skins and principal uses. In Leather

Technician’s Handbook (Leather Producer’s Association eds.) Northampton, UK,

pp. 20-36, 1971.

15. Blaha, A., and Kolomazník, K.; Mathematical model of soaking. Part I. J. Soc.

Leather Tech. Chem. 73, 136-140, 1988.

16. Kallenberger, W.E.; Heat, humidity and cure quality. JALCA 82, 365-371, 1987.

17. Blaha, A., Kolomazník, K., and Dederle, T.; Mathematical model of the soaking

process. Part II. J. Soc. Leather Tech. Chem. 73, 172-174, 1989.

18. Bailey, D.G.; The preservation of hides and skins. JALCA 98, 308-319, 2003.

19. Trade Practices for Proper Packer Cattlehide Delivery, 3rd ed., Leather Industries of

America and U.S. Hide, Skin & Leather Association, pp. 12-19, 1993.

20. Allsop, T.F., and Passman, A.; Porosity measurement as a means of determining the

degree of processing of lamb pelts. J. Soc. Leather Tech. Chem. 87, 49-54, 2003.

4.11 Acknowledgments

The authors would like to thank Eleanor Brown, Laurelie Bumanlag, Gary Di Maio,

Rafael García, Michael Kurantz, Joseph Lee, John Phillips, Maryann Taylor and

Michaela Uhlíová for their technical support and assistance.

CHAPTER 5

Evaluation of degreasers as brine curing additives

Chapter 5

69

5. EVALUATION OF DEGREASERS AS BRINE CURING ADDITIVES Authors: Eduard Hernàndez Balada1,2, William N. Marmer1, Peter H. Cooke1 and John

G. Phillips1.


Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038 USA

2Department of Chemical Engineering, University of Barcelona, Martí i Franquès 1,


Manuscript accepted for publication at the Journal of the American Leather

Chemists´Association (JALCA). Tentatively scheduled for publication in the May 2009

issue.


70


THE JOURNAL OF THE AMERICAN LEATHER CHEMISTS ASSOCIATION

c/o The American Leather Chemists Association, 1314 50th Street, Suite 103, Lubbock, Texas 79412

Mobile phone: (616) 540-2469 E-mail: [email protected] November 12, 2008 Dr. William N. Marmer c/o Eastern Regional Research Center, USDA 600 East Mermaid Lane Wyndmoor, PA 19038-8598 Dear Dr. Marmer, Your manuscript "Evaluation of Degreasers as Brine Cure Additives" has been accepted for publication in JALCA. It is tentatively scheduled for the May 2009 issue. Please have the appropriate research leader sign the attached “Transfer of Copyright” form and return it to me. Thank you for this interesting contribution to JALCA. Sincerely yours, Robert F. White Journal Editor

Chapter 5

71

5.2 Abstract

The length of time needed for brine curing of raw hides and skins, a minimum of 18 h,

is a time consuming process. In this paper we initially report the results of an

investigation of the stratigraphic distribution of sodium chloride and water in fleshed

hides cured for varying intervals of time. We demonstrated that salt entered the hide

mainly from the flesh side. Water, on the other hand, was withdrawn from both sides of

the hide; the epidermis acted as a semipermeable membrane. Three commercial

degreasers as well as a glycolipid surfactant (sophorolipid) were tested as brine curing

additives and their efficiency evaluated according to the moisture, salt, salt saturation

and fat content levels in the brine cured hide. One of the commercial degreasers, when

used at 0.5% w/w, significantly removed fat from the hide as well as enhanced the

uptake of salt. The sophorolipid also was an effective degreasing agent, decreasing the

fat content of the brine-cured hide and, if used in excess, significantly increasing the

uptake of salt. The data presented here confirmed that the usage of an appropriate

degreasing agent in the brine is a suitable option for reducing the turn-around times in

raceways and thus creating additional curing capacity.


72

5.3 Resum

El període de temps que comporta la preservació de pells crues amb salmorra, que és

d’un mínim de 18 hores, és un important inconvenient del procés. En el present article

mostrem els resultats d’un estudi estratigràfic que descriu la distribució del clorur de

sodi i aigua en pells descarnades curades durant distints intervals de temps. Demostrem

que la sal penetra la pell majoritàriament pel costat de la carn. L’aigua, pel seu cantó,

migra cap a fora per les dues cares de la pell, amb l’epidermis actuant com una

membrana semipermeable. Tres desengreixants disponibles comercialment i un

tensioactiu glucolipídic (soforolípid) varen ser provats com a additius en la preservació

de pells amb salmorra, i la seva eficàcia fou mesurada d’acord amb els nivells d’aigua,

sal, saturació de sal i greix de la pell curada. Un dels desengreixants, emprat al 0.5%

w/w, rebaixà de forma significativa el contingut de greix a la pell, augmentant també

l’absorció de sal. El soforolípid demostrà també ser un agent desengreixant efectiu,

disminuint el contingut de greix de la pell curada, i en cas de ser emprat en excés,

augmentant també significativament l’absorció de sal. Les dades presentades en aquest

article confirmen que l’ús d’un apropiat agent desengreixador amb la salmorra és una

opció viable per tal de reduir el temps de processat de pells i per tant crear una major

capacitat de producció.

Chapter 5

73

5.4 Introduction

Curing of raw hides and skins with common salt (sodium chloride) is the most popular

preservation method used currently. Sprinkling of solid salt on the flesh surface of the

hide, known as green-salting, is extensively used in warm countries like India.

Conversely, most American and European hide processing facilities typically treat their

hides in raceways filled with a highly concentrated solution of sodium chloride (brine).

The economics of brine curing of hides and skins have been affected recently by

increasing commodity prices for sodium chloride.1 Furthermore, the large amount of

salt needed creates environmental issues when it is soaked out, contributing to 40% of

the total dissolved solids generated in the entire process of leather manufacturing.2

In our previous paper3 we developed a mathematical model that described the diffusion

of sodium chloride into the hide during the curing process. We concluded that the

usage of saturated brine as well as a minimum float of 500% yielded an optimal

diffusion rate. We also proved by means of epifluorescence microscopy that salt

diffused into the hide mainly from the flesh side, which presents a less compact

structure and greater porosity than the grain and upper corium. However, the fatty or

adipose tissue present on the flesh side was reported to retard the diffusion of salt into

the hide.4 Thus, a poor fleshing of the hide prior to the cure may further aggravate this

phenomenon.

The use of degreasing agents in the leather manufacturing process is quite widespread

and they are mainly utilized during soaking, liming and deliming to decrease the fat

content of the wet blue below the norm value.5 However, there is no literature

regarding the usage of these degreasers in the cure. If the use of a degreasing agent

along with the brine could accelerate the penetration of salt into the hide, then turn-

around times in the raceways would be reduced and thus additional curing capacity

would be created.

Sophorolipds (SL) are glycolipid surfactants produced in large quantities by the yeast

Candida bombicola. Among their desirable properties, they are biodegradable, non-

ecotoxic, and non-foaming.6 Sophorolipids are currently used in the cosmetic industry

and as an active ingredient of dishwashing detergents.7 The potential for their use in the

leather industry as a degreasing agent is therefore worthy of consideration.


74

In the present paper we first carried out a stratigraphic study to monitor the distribution

of sodium chloride and water in a hide throughout the curing process. The effect of the

cure on the collagen denaturation temperature was also examined. Furthermore, we

used scanning electron microscopy in backscattering electron mode to establish changes

in the distribution of sodium chloride within the hide. Next, we evaluated the effect that

commercial degreasers exerted on the penetration of salt into the hide, as well as their

fat removal efficiency. Finally, the possibility of using SL as brine curing enhancers

was investigated.

5.5 Experimental

5.5.1 Materials

Two fresh cow hides were purchased from a local abattoir. They were soaked in 200%

water with 0.15% Boron TS (Rohm & Haas Co., Spring House, PA) and 0.10% Proxel

(Chemtan Co. Inc., Exeter, NH) for 2 h and then fleshed. Approximately 6 × 10 in (15

× 25 cm) pieces were cut and stored at -20 °C. They were thawed at 4 °C just before

use. Food grade sodium chloride of minimum purity 99.82% (US Salt Corporation,

Watkins Glen, NY) was used in the brine preparation. Three commercial degreasing

agents marketed to the leather industry and identified as degreasers 1, 2 and 3, as well as

the experimental sophorolipid (SL) developed in our research facility, were used

without further purification. All other chemicals were reagent grade and used as

received.

5.5.2 Methods

5.5.2.1 Stratigraphic study

Thawed hide was cut into square pieces of approximately 4 × 4 in (10 × 10 cm) with an

average weight of approximately 100 g. They were transferred to a Dose drum (PFI

300-34; Dose 131 Maschinenbau GmbH, Lichtenau, Germany) and tumbled at 6 rpm in

a 500% float of 95 °SAL brine solution (25.1 g NaCl/100 g brine) for varying time

intervals, after which they were removed from the drum, hand-squeezed to wipe off

excess water, split into three layers (flesh, middle and grain), sealed in plastic bags and

stored at 4 °C until analysis.

Chapter 5

75

5.5.2.2 Degreaser study

Pieces of hides of approximately 100 g were tumbled at 6 rpm in a 500% float of

initially saturated brine (100 °SAL, 26.4 g NaCl/100 g brine) for 16 h. To the test

samples, the required volume of degreasers 1, 2 or 3 was added to the brine held in the

drum and tumbled for five minutes to ensure a uniform distribution of the product

within the brine. Due to its low solubility, the SL solution was prepared in the

laboratory by adding the required amount of SL powder to the saturated brine solution

and stirring for at least 24 h, after which the solution was dumped into the drum. An SL

solution that had been filtered through a sintered glass funnel immediately after 24 h of

stirring was also tested. Control samples to which no degreaser was added were also

run for all experiments. The concentration of degreasers or SL was always expressed

with respect to the combined weight of hide and brine. No further salt or brine was

added to the drums during the experiments. Brine density was determined by

gravimetric analysis. Hide density was assumed to be 1g/cm3.

5.5.3 Analyses

5.5.3.1 Determination of moisture and ash content

An oval arch punch (C.S. Osborne & Co., Harrison, NJ), 7/8 inches (2.2 cm) in

diameter, was used to sample the pieces of hide. Samples were clipped to minimize

variability due to long hair. Next, they were weighed on an analytical balance into dry

tared crucibles and dried in a vacuum oven at 80 °C for 18 h. After cooling in a

desiccator they were weighed and percentage moisture was calculated. Dry samples

were placed in a muffle furnace and ashed at 600 °C for 2 h. After cooling in a

dessicator, they were weighed and percentage ash was calculated. All samples were run

at least in triplicate.

5.5.3.2 Determination of fat content

In order to accurately compare results among different samples, fat content8 is

expressed in terms of moisture and ash free basis (MAFB).9 All samples were run in

triplicate.


76

5.5.3.3 Determination of thermal stability

Differential Scanning Calorimetry (DSC) analysis was performed on a Multi-Cell

Differential Scanning Calorimeter CSC-4100 (Calorimetry Sciences Corporation,

Lindon, UT). An aliquot of raw or cured hide of approximately 100 mg was weighed

into a stainless steel pan and placed in the calorimeter. Another pan containing

approximately the same weight of a 5% NaCl solution was used as a reference. When

raw hide samples were analyzed, nanopure water was used as a reference. Both sample

and reference capsules were weighed before and after the DSC run in order to determine

any possible weight loss by leakage. Samples were heated from 10 ºC to 120 ºC at

1.5 ºC/min. The peak maximum temperature was taken as the hide’s denaturation

temperature (TD). All samples were run in duplicate.

5.5.3.4 Back-scattered/Low Vacuum Scanning Electron Microscopy (SEM-BSE)

Backscattered electrons consist of high-energy electrons originating in the electron

beam that are reflected or backscattered out of the specimen interaction volume. The

brightness of the SEM-BSE image tends to increase with the atomic number. Two

major advantages over conventional SEM in the secondary electron image mode are the

high-quality atomic number contrast images produced and the simplicity of the

technique, which does not require a conductive coating.10

A piece of raw hide of approximately 1 × 1 in (2.5 × 2.5 cm) was immersed in a beaker

containing 500% v/v float of a saturated brine solution and gently agitated for varying

intervals of incubation time. A 2-3 mm wide slice of the sample was excised manually

with a stainless steel razor blade (cutting from flesh surface toward the grain), mounted

on aluminum stubs, and then irradiated at an accelerating voltage of 25 kV in low

vacuum mode (spot size 3.0, pressure 0.98 Torr) using a Quanta 200 FEG

environmental scanning electron microscope (FEI Company, Hillsboro, OR). Samples

were imaged as montages at a magnification of 50×. Individual montages of images at

each curing time were processed and analyzed using Fovea 3.0 plug-ins (Reindeer

Graphics, Asheville, NC) for PhotoShop, v. 7 (Adobe Systems, Inc., San Jose, CA).

The montages were spatially filtered with a Gaussian blur (3 pixels) to reduce local

noise and a line profile was drawn in each image extending from a point at the grain

surface to a point at the flesh surface. The line profiles were then plotted as gray level

Chapter 5

77

versus distance (data not shown), and the ranges of gray levels for different curing times

were plotted.

5.5.3.5 Statistical Analysis

Data were analyzed by analysis of variance followed by a Dunnett’s test to compare

each treatment (degreasing agent) with the control. Further comparisons were made

using orthogonal polynomial contrasts to test the linear and quadratic trends due to

degreaser 1 concentration. An orthogonal contrast was also used in the sophorolipid

study to determine the significance of the filtering effect.


5.6.1 Stratigraphic study

The layer-wise distribution of water and salt (ash) in a hide cured for various intervals

of time was monitored (Figures 5.1a and 5.1b). Typically, the flesh layer included the

flesh itself and the attached adipose tissue; the middle layer represented the majority of

the corium or true skin; and the grain layer included the junction of grain and corium

and the epidermis. The initial moisture content was highest in the region of the

epidermis and decreased toward the center of the corium. Whereas the diminishing

moisture content of the flesh layer leveled off at 54% after 2 h of cure, the moisture

content of the middle and grain layers continued to diminish over a 24-h period to less

than 45% (Figure 5.1a). The salt content of a raw hide was found to be less than a

0.5%. As curing progressed, salt content increased in all three strata: the flesh layer

contained the highest amount of salt at all curing times (Figure 5.1b). Noteworthy, the

salt content of the flesh layer after 30 min and 1 h of cure was 6.7 and 7.1%,

respectively, but only 1.6 and 1.9%, respectively, in the grain layer.

These figures demonstrated that salt entered the hide mainly from the flesh side, which

is in agreement with the findings of McLaughlin and Theis.11 It was also confirmed that

the epidermis acted as a semipermeable membrane, which allowed the withdrawal of

water by osmosis but did not allow salt to penetrate through it.12 Thus, whereas water

was extracted from the hide by both sides, only the flesh side was susceptible to

extensive absorption of salt, which then diffused into the hide and towards the grain.


78

The porous nature of the hide may have also contributed to the diffusion of salt through

the hide by capillarity.

By means of the equation 100359.01

%%

% ×��

��

�⋅��

��

�=WaterSalt

Saturation , the hide salt

saturation levels at various curing times were calculated (Figure 5.1c). O’Flaherty13

claimed that a 70% salt saturation of the water remaining in the hide with a maximum

of 50% moisture was needed for a proper cure. Later on, a new minimum standard of

85% salt saturation was established by the U.S. Hide, Skin & Leather Association.14

The salt saturation of the flesh layer was the highest of all three layers at all curing

times. The reason was found in the rapid dehydration experienced in the early stages of

the cure along with the almost exclusive penetration of salt from the flesh side of the

hide. The differences of hide salt saturation levels among the three layers become less

pronounced after 16 h of cure. Although we did not sample the hides beyond the 24 h

curing time frame, one could assume that the hide and surrounding brine reached

equilibrium after about one day of brining. In fact, it was previously reported that a

hide continuously brined for 48 h took up moisture as well as salt;4,15 brining a hide

beyond the equilibrium did not necessarily entail a more proper cure.

Chapter 5

79

Figure 5.1 – Stratigraphic distribution of (a) water (b) ash and (c) hide salt saturation in a hide treated for various intervals of time with a 500% float of an initial 95 °SAL. Each point is the mean of three values.

The denaturation or melting temperature (TD) of the grain, middle and flesh layers of a

hide cured for various time intervals was monitored (Figure 5.2). The hide’s collagen,

upon heating, undergoes a transition from the triple helix to a randomly coiled form in

which the three chains are separated.16 The flesh layer was the first to reach a constant

TD of 78 °C after only 2 h of cure. On the other hand, about 8 h of cure were needed to

level off the temperature curve of the middle and grain layers (TD = 82 °C). The

increase of the thermal stability of the hide upon brining could be ascribed to two

factors. The first and most important factor is the dehydration of the hide. Kopp et al.17

developed a mathematical function that showed an exponential decrease of TD with

increasing moisture content. The second factor is protein salting out and aggregation.

The brine, despite the disruption of some hydrogen bonds due to the withdrawal of

water, induces this phenomenon, which was reported to further stabilize the collagen

molecules, thus increasing the values of TD.18.

35 40 45 50 55 60 65 70 75

0 4 8 12 16 20 24

Flesh layer Middle layer Grain layer

Time (h)

0

5

10

15

20

0 4 8 12 16 20 24


Time (h)

0

20

40

60

80

100

0 4 8 12 16 20 24


Time (h)

35 40 45 50 55 60 65 70 75

0 4 8 12 16 20 24


Time (h)

35 40 45 50 55 60 65 70 75

0 4 8 12 16 20 24

Flesh layer Middle layer

35 40 45 50 55 60 65 70 75

0 4 8 12 16 20 24


Time (h)

0

5

10

15

20

0 4 8 12 16 20 24


Time (h)

0

5

10

15

20

0 4 8 12 16 20 24


Time (h)

0

20

40

60

80

100

0 4 8 12 16 20 24


Time (h)

0

20

40

60

80

100

0 4 8 12 16 20 24


Time (h)

1a

1c

1b


80

60

65

70

75

80

85

0 4 8 12 16 20 24

Flesh layerMiddle layerGrain layer

Time (h)

Figure 5.2 – Denaturation temperature (TD) of the grain, middle, and flesh layers of a hide treated for various intervals of time with a 500% float of an initial 95 °SAL brine. Each point is the mean of two values. Cross-sectioned samples of hides, cured up to 24 h duration, were examined in a

scanning electron microscope using a backscatter detector at low vacuum. Although the

backscatter signal is not specific for sodium chloride, results of backscattered intensity

variations were expected to reveal general trends related to diffusion of the dissolved

salt that could be further explored and mapped by energy dispersive x-ray

microanalysis.

The range of intensity in backscattered images was adjusted to maximize the dynamic

range of the detector by adjusting contrast and brightness values with the waveform

monitor using the 24-h cured sample. Then images of all the cured samples were

collected under the same conditions. Line profiles drawn on images from the grain side

to flesh side of an uncured hide (0 h) had a narrow range of low brightness values (69-

77) relative to the values set for the 24-h sample. At 0 h, higher values were located in

the lower half or flesh-side of the hide. Similar line profiles drawn on backscatter

images of cured hides indicated that the range of intensities increased monotonically

with longer curing times, with brightness values extending from a low around 20 to a

high above 200 (16 h), and the higher values were located within the flesh side of the

cross-section, with a boundary slowly moving toward the grain side of the cross-section

as the hours of curing increased (Figure 5.3).

Chapter 5

81

Figure 5.3 – Composite images of hide samples, collected at different curing times by low vacuum, mixed signal SEM imaging. Image heights were adjusted to an average value in order to facilitate comparisons. At 0 hours (control), the superficial brightness was low and variable in the lower, flesh side and uniformly brighter in the upper, grain side; the range of gray levels was narrow, between 69 and 77. At 1-24 hours of curing, the average values of brightness were increased almost monotonically, mainly from the flesh side, but not uniformly; some bright and dark patches were present through 1, 2, 4, 8 and 16 hours. At 24 hours, the brightness was the most uniform, suggesting that the concentrations of Na and Cl were evenly distributed in the hide.

5.6.2 Degreasing study

The effect of three commercial degreasers on the content of water, salt and extractable

fat of the hide after 16 h of brine curing was evaluated (Table 5.1). A degreaser

concentration of 0.5% (w/w) with respect to the combined weight of hide and brine was


82

used. Samples that had been brine-cured in the presence of degreaser 1 showed a

significantly higher salt content than the control, to which no degreaser had been added.

Furthermore, degreaser 1 was shown to be the most efficient as far as its fat-removal

capacity, significantly decreasing the extractable fat content with respect to the control.

Samples that had been treated with degreasers 2 and 3, despite tending to increase salt

content and decrease extractable fat, were not statistically more efficient than the

control. The composition of degreasers 2 and 3 was likely the cause of a lower

efficiency than degreaser 1. Although the specific compositions of these commercial

products are not disclosed, they are typically composed of a blend of nonionic

surfactants, modifiers and adjuvants. The hydrophilic-lipophilic balance (HLB) is a

widely used scale in the technology of surfactants; HLB values run from 0 to 20 and

indicate whether a surfactant has greater wetting (low end of the scale) or emulsifying

properties (high end of the scale). It is likely that degreaser 1 had a slightly higher HLB

than the other two products and hence a higher tendency toward forming oil-in-water

emulsions. As a matter of fact, the emulsification of fat in water was reported to be a

crucial step for the efficiency of the degreasing process.19

TABLE 5.1

Effect of commercial degreasers on brine curing [0.5% w/w]

Treatment Moisture

(%)

Salt

(%)

Hide salt

saturation (%)

Fat

(%, MAFB)

Control 46.5 ± 1.5 11.8 ± 0.9 70.9 ± 7.0 11.8 ± 2.6

Degreaser 1 47.9 ± 3.0 12.9 ± 0.9* 75.2 ± 1.1 7.3 ± 0.6*

Degreaser 2 46.5 ± 1.0 12.1 ± 0.8 72.1 ± 4.1 8.8 ± 3.6

Degreaser 3 46.7 ± 1.7 12.2 ± 0.4 73.1 ± 4.1 8.8 ± 2.2

Average of 8 values Values with * are significantly different from the control (Dunnett’s test, p<0.05)

Next, degreaser 1 was selected to study the effect of its concentration on the content of

water, salt and extractable fat of a hide after 16 h of curing with brine (Table 5.2). All

three tested concentrations of degreaser (0.25, 0.5 and 1% w/w) significantly defatted

the hide. Furthermore, a concentration of 0.5% also effected significantly higher salt

and hide salt saturation values than did the control. The statistical analysis showed

Chapter 5

83

evidence of a significant quadratic trend with increasing concentration of degreaser 1,

with a maximum occurring at a critical concentration between 0.5% and 1%. The usage

of a concentration larger than this critical point neither further increased the uptake of

salt nor additionally decreased the fat content. That could be ascribed to the fact that

the concentration of surfactants in degreaser 1 was well above the critical micelle

concentration.

TABLE 5.2

Effect of degreaser 1 on brine curing

Treatment Moisture

(%)

Salt

(%)

Hide salt

saturation (%)

Fat

(%, MAFB)

Control 53.4 ± 2.0 15.2 ± 0.6 79.5 ± 1.3 16.8 ± 2.2

Degreaser 1 [0.25%] 50.4 ± 2.0 13.8 ± 1.1 76.3 ± 3.5 7.4 ± 2.4*

Degreaser 1 [0.5%] 57.2 ± 4.2 17.1 ± 1.7* 83.5 ± 2.7* 3.8 ± 0.9*

Degreaser 1 [1%] 56.4 ± 2.9 16.5 ± 0.7 81.7 ± 1.9 9.7 ± 2.9*


The feasibility of using a sophorolipid (SL; a fermentation product, under investigation

at this location) as a degreasing agent for raw hides was examined. Due to the low

solubility of SL in the brine, the effect of filtering the SL solution prior to its use was

also examined. By doing this, we ensured that the brine solution contained the

maximum amount of soluble SL. Hide samples cured with the unfiltered brine-SL

solution displayed significantly higher values of salt and salt saturation than the control

(Table 5.3). The extractable fat was also significantly lower than the control. When a

filtered brine-SL was used, the values for moisture, salt, and extractable fat content were

significantly different from those of the control. Hence, SLs were demonstrated to have

a degreasing activity on the hide. The percentage of fat removed from the hide was 40.5

and 27.5% for the unfiltered and filtered SL-brine solution, respectively. These figures

are similar to those obtained for the commercial degreasers (from 25.4% for degreasers

2 and 3 to an average of 53.4% for degreaser 1). The undissolved SL likely provided a

reservoir of active surfactant as the initial concentration of solubilized SL suffered from

dilution or inactivation.


84

TABLE 5.3

Effect of sophorolipid on brine curing

Treatment Moisture

(%)

Salt

(%)

Hide salt

saturation (%)

Fat

(%, MAFB)

Control 45.0 ± 1.4 11.8 ± 0.6 72.8 ± 1.5 15.3 ± 0.5

SL Non-filtered 46.6 ± 0.8 12.5 ± 0.3* 74.9 ± 0.8* 9.1 ± 2.3*

SL filtered 49.7 ± 1.6* 13.0 ± 0.5* 73.0 ± 1.1 11.1 ± 1.9*


From Tables 5.1, 5.2 and 5.3 it can be seen that test samples with a significantly higher

amount of salt than the controls also exhibited a significantly lower fat content.

However, this statement was not verified in the other direction. It was also observed

that a significant effect of the degreaser on the content of salt and fat did not necessarily

entail a significant increase in the salt saturation levels. That could be attributed to the

wetting properties of the degreasers, which did not significantly alter the amount of

remaining moisture in the hide after the 16 h curing time frame (Tables 5.1 and 5.2).

It is important to bear in mind that the results presented in this study are based on the

analysis run on different areas of two cow hides. Variance in the results can be

attributed to some extent to the variability always found in biological material. In

addition, although the hides were washed, fleshed, cut up and stored in the freezer on

the same day that the animal was slaughtered, some damage to the hide due to a delayed

cure might have taken place. A delayed cure of only a few hours was reported to slow

down the diffusion of salt during the early stages of the cure.11 Furthermore, and for the

particular case of fat content, some variability could be attributed to the horizontal

distribution of grease, which is more abundant in the looser and more flexible areas

(e.g., belly;20). Despite all these facts, the coefficients of variation (CV) obtained for

the determination of moisture, salt and salt saturation were 3.97, 5.76 and 3.54%,

respectively, which indicate good precision.21 In the particular case of the

determination of extractable fat, a CV of 21.3% was relatively precise, considering the

low analytical values of that parameter.

Chapter 5

85

5.7 Conclusions

The purpose of research reported in this article was to evaluate the possibility of using

degreasers as brine curing additives, with the aim of enhancing the uptake of salt by the

hide. In the stratigraphic study carried out in order to get a better understanding of the

curing process, we found that the epidermis acted as a semipermeable membrane,

enabling the diffusion of water from the hide and into the surrounding float as well as

hindering the diffusion of salt into the hide, which occurred from the flesh side

overwhelmingly. These findings were corroborated by the pictures taken with a

scanning electron microscope run in the back-scattered electron mode. Furthermore, the

thermal stability of the hide increased upon curing, mainly due to the simultaneous

dehydration and salting out processes of the constituent collagen. If 0.5% (w/w) of a

commercial degreaser, made of a blend of nonionic surfactants, had been added to the

brine, the fat content of that hide was significantly decreased and the uptake of salt was

also significantly enhanced. Since another two commercial degreasers exhibited less

stimulation of salt uptake, we concluded that the composition of the degreaser was a

critical parameter for this specific purpose. The sophorolipid tested showed remarkable

degreasing properties and enhanced the uptake of salt by the hide if it was used above

the solubility limit. These facts along with its low-foam properties and low cost (from

$1 to 3/kg;22) make it an attractive choice of surfactant. One may hypothesize that the

addition of small amounts of a proteolytic enzyme along with a degreasing agent would

enhance the fat removal action, since it would facilitate the breakdown of the

proteinaceous membrane of the fat-containing sac.23 Nevertheless, the detrimental

effect that this treatment could have on the grain is an important drawback.

In this paper, we gave an overview of the brine curing process of raw hides and also

suggested the use of an additive (e.g., commercial degreasers, sophorolipid) to enhance

the uptake of salt and remove a major amount of the fat. It is important to note that

actual raceways or vats are operated continuously. Thus, it will be essential to find a

way to remove the fat that builds up in the vat.


86

5.8 References

1. Personal communication, Ed Godsalve, WE CO., 1991, Inc., 2006.

2. Rajamani, S. Cleaner tanning technologies in the beam house operation. UNIDO

report, pp. 9-18, 1998.

3. Hernàndez Balada, E.; Marmer, W.N.; Kolomazník, K.; Cooke, P.H.; Dudley, R.L.

Mathematical model of raw hide curing with brine. JALCA 103, 128-134, 2008.

4. Stuart, L.S.; Frey, R.W. Effect of adipose tissue fat on the green-salting of heavy

hides. JALCA 35, 414-418, 1940.

5. Stockman, G.B.; Rangarajan, R.; Didato, D.T. The replacement of

nonylphenolethoxylates (NPEs) as degreasing agents in wet blue manufacture.

World Leather, October 2005.

6. Garcia, R.A.; Solaiman, D.K.Y. Government scientists find new uses for rendered

products. Render, June 2008.

7. Solaiman, D.K.Y. Applications of microbial biosurfactants. Inform 16, 408-410,

2005.

8. Taylor, M.M.; Diefendorf, E.J.; Phillips, J.G.; Feairheller, S.H.; Bailey, D.G. Wet

process technology I. Determination of precision for various analytical procedures.

JALCA 81, 4-18, 1986.

9. Donmez, K.; Kallenberger, W.E. Total crude fat determination in hides. JALCA 89,

369-390, 1994.

10. Robinson, B.R.; Nickel, E.H. A useful new technique for mineralogy: the

backscattered-electron/low vacuum mode of SEM operation. Am. Mineral. 64,

1322-1328, 1979.

11. McLaughlin, G.D.; Theis, E.R. Science of hide curing. JALCA 17, 376-399, 1922.

12. Tancous, J.J. A study of a drawn condition, caused through osmosis, encountered in

some brine-cured hides. JALCA 58, 143-154, 1963.

13. O’Flaherty, F. Curing study part 1: Moisture to ash relationship. Leather and Shoes

138, 31-34, 1959.

14. Trade Practices for Proper Packer Cattlehide Delivery, 3rd ed., Leather Industries of


15. Strandine, E.J.; DeBeukelaer, F.L.; Werner, G.A. Stratigraphic distribution of

sodium chloride and water in fresh and brined steer hide. JALCA 46, 19-34, 1951.

16. Miles, C.A.; Bailey, A.J. Thermal denaturation of collagen revisited. Proc. Indian

Acad. Sci. (Chem. Sci.) 111, 71-80, 1999.

Chapter 5

87

17. Kopp, J.; Bonnet, M.; Renou, J.P. Effect of collagen crosslinking on collagen-water

interactions (a DSC investigation). Matrix 9, 443-450, 1989.

18. Komsa-Penkova, R.; Koynova, R.; Kostov, G.; Tenchov, B.G. Thermal stability of

calf skin collagen type I in salt solutions. Biochim. Biophys. Acta Protein Struct.

Mol. Enzymol. 1297, 171-181, 1996.

19. Thanikaivelan, P.; Rao, J.R.; Nair, B.U.; Ramasami, T. Progress and recent trends

in biotechnological methods for leather processing. Trends Biotechnol. 22, 181-

188, 2004.

20. Balderston, L. The distribution of grease in leather. JALCA 17, 405-407, 1922.

21. Taylor, M.M.; Diefendorf, E.J.; Artymyshyn, B.; Hannigan, M.V.; Phillips, J.G.;

Feairheller, S.H.; Bailey, D.G. The chemical and physical analysis of contemporary

thru-blue tanning technology, 1984. Animal Biomaterials Laboratory, ERRC,

Philadelphia, PA 19038. Available from senior author at

[email protected].

22. Sun, X.X.; Choi, J.K.; Kim, E.K. A preliminary study on the mechanism of harmful

algal bloom mitigation by use of sophorolipid treatment. J. Exp. Mar. Biol. Ecol.

304, 35-49, 2004.

23. Langridge, D.A.; Long, A.J.; Addy, V.L. The impact of sebaceous grease on the

leather industry. JALCA 101, 45-50, 2006.

5.9 Acknowledgments

The authors appreciate the assistance of the following: Dr. Richard Ashby, Guoping

Bao, Dr. Eleanor Brown, Nicholas Latona, Joe Lee, Dr. Justin Martin, Paul Pierlott, Dr.

Daniel Solaiman, Maryann Taylor and Amanda Tiscavitch.

CHAPTER 6

Properties of biopolymers produced by transglutaminase treatment of whey protein isolate and gelatin

Chapter 6

89

6.PROPERTIES OF BIOPOLYMERS PRODUCED BY TRANSGLUTAMINASE

TREATMENT OF WHEY PROTEIN ISOLATE AND GELATIN

Authors: Eduard Hernàndez Balada1,2, Maryann M. Taylor1, John G. Phillips1, William

N. Marmer1 and Eleanor M. Brown1.


Research Center, 600 E Mermaid Lane, Wyndmoor, PA 19038, USA 2Department of Chemical Engineering, University of Barcelona, c/ Martí i Franquès 1,


Manuscript accepted for publication at the Bioresource Technology journal. Tentatively

scheduled for publication around March 2009.

Properties of biopolymers produced by transglutaminase treatment of WPI and gelatin

90


Chapter 6

91

6.2 Abstract

Byproduct utilization is an important consideration in the development of sustainable

processes. Whey protein isolate (WPI), a byproduct of the cheese industry, and gelatin,

a byproduct of the leather industry, were reacted individually and in blends with

microbial transglutaminase (mTGase) at pH 7.5 and 45 °C. When a WPI (10% w/w)

solution was treated with mTGase (10 U/g) under reducing conditions, the viscosity

increased four-fold and the storage modulus (G') from 0 to 300 Pa over 20 hours.

Similar treatment of dilute gelatin solutions (0.5 to 3%) had little effect. Addition of

gelatin to 10% WPI caused a synergistic increase in both viscosity and G', with the

formation of gels at concentrations greater than 1.5% added gelatin. These results

suggest that new biopolymers, with improved functionality, could be developed by

mTGase treatment of protein blends containing small amounts of gelatin with the less

expensive whey protein.

6.3 Resum

La utilització de subproductes és important en el desenvolupament de processos

sostenibles. El concentrat de sèrum de proteïna (WPI, per les seves sigles en anglès), un

subproducte de la indústria del formatge, i la gelatina, un subproducte de la indústria

pelletera, van ser reaccionats per separat i conjuntament amb la transglutaminassa

microbiana (mTGase) a pH 7.5 y 45 ºC. Quan una solució (10% w/w) de WPI era

tractada amb mTGase (10 U/g) sota condicions reductores, la viscositat es multiplicava

per quatre i el mòdul elàstic (G’) passava de 0 a 300 Pa en 20 hores. Un tractament

similar de solucions diluïdes de gelatina (0.5 a 3%) va sorgir poc efecte. L’addició de

gelatina a un 10% de WPI va provocar un efecte sinèrgic en ambdós viscositat i G’, amb

la formació de gels a concentracions de gelatina superiors a 1.5%. Aquests resultats

suggereixen que nous biopolímers, amb funcionalitat millorada, poden ser

desenvolupats mitjançant el tractament de mescles de proteïnes amb mTGase, contenint

petites quantitats de gelatina juntament al més econòmic sèrum de proteïna.


92

6.4 Introduction

Whey and gelatin are both proteinaceous agricultural byproducts. Whey is an abundant,

inexpensive and readily available byproduct of the cheese industry. The United States

is the world’s largest whey exporter; latest available figures from the U.S. Dairy Export

Council (USDEC, 2006) show an increase in exportation by 104% between 2001 and

2006. Despite the exports, a considerable amount of whey is wasted through disposal,

and would be available for potential new uses. These uses could be for food or nonfood

products.

Animal hides, skins and bones, major byproducts of the meat industry, are rich sources

of collagen, the structural protein of connective tissue. Gelatin, produced by partial

hydrolysis of collagen, is a polydisperse mixture of varying sized collagen fragments.

Gelatins are classified by type, edibility, and gel strength. Type A gelatin is obtained by

acid treatment usually of pigskins, while type B is obtained by alkaline treatment of

cattle hides or bones (Rose, 1992). An early step in leather manufacturing is the liming

of the hide to assist in hair removal and opening up of the collagen fiber bundle for

access by tanning chemicals. The hide may then be split; the upper layer would be

tanned to make leather, and the lower layer would provide a rich source for the

extraction of type B gelatin. Gelatin extracted from limed hides is edible, and is used in

sausage casings, as an ingredient in a variety of food products, as well as in

pharmaceuticals. Inedible gelatin may be surplus lower grade material from production

of edible gelatins or may be from an inedible source such as leather waste (Taylor et al.,

1994). The highest value market for inedible gelatin traditionally was the photographic

industry, where the demand for gelatin has declined markedly in recent years. The

Bloom value, a measure of gel strength of a 6.67% (w/w) gelatin, is often used to

predict the behavior of a gelatin in a particular application. Gelatin suppliers typically

give a nominal Bloom value for the gelatins they market, in this case 75 g. High Bloom

(greater than 225 g) gelatins are taken from the highest molecular weight fraction of

crude gelatin and are relatively uniform. High and low molecular weight gelatin

fractions are blended to produce low Bloom (less than 125 g) gelatins, and variation

between lots may be expected.

Transglutaminase (TGase, EC 2.3.2.13) is a calcium-dependent enzyme that catalyzes

an acyl transfer reaction between the γ-carboxyamide of a protein or peptide bound

Chapter 6

93

glutamine and a primary amine (Folk and Chung, 1973). An ε( γ-glutamyl)lysine bond

is formed when the primary amine is the ε-amino group of a lysine residue. In 1989,

Ando et al. used Streptoverticillium mobaraense to produce a calcium-independent

form of the enzyme, namely microbial transglutaminase, mTGase.

A variety of individual food proteins, including gelatin, casein and whey proteins have

been polymerized by the formation of mTGase-mediated intermolecular crosslinks

(Sakamoto et al., 1994; Rodríguez-Nogales, 2005). Biopolymers formed by the

enzymatic crosslinking of dissimilar proteins have the potential for generating novel

products (Motoki and Nio, 1983; Yildirim and Hettiarachchy, 1997; Oh et al., 2004).

The most abundant proteins in whey, β-lactoglobulin and α-lactalbumin, are globular

and contain two and four disulfide bonds, respectively (Farrell et al., 2004). The

reduction of disulfide bonds in globular proteins, prior to mTGase treatment, has been

shown (Færgemand et al., 1997) to increase access to primary amino groups.

Dithiothreitol (DTT) is generally the reductant of choice for cleaving disulfide bonds,

given that it is needed in much lower concentration than other reductants to complete

the reaction (Grazú et al., 2003).

In this study, biopolymers produced by treating gelatin or whey protein isolate (WPI)

and blends of WPI and gelatin with mTGase under reducing or nonreducing conditions

are characterized. In blends, WPI, the less expensive of the protein sources, is the

primary component and the focus is on the effect of including small amounts of gelatin

with the WPI in these biopolymers.

6.5 Experimental

6.5.1 Materials

mTGase, Activa TG-TI (Ajinomoto USA Inc., Paramus, NJ) with an active range of pH

4.0 to 9.0 at 0 to 70 °C was used without further purification. The mTGase activity, in

the presence of the maltodextrose carrier, under the assay conditions of Folk and Chung

(1985) was approximately 100 U/g. Type B gelatin, alkaline extracted from bovine

skin, was obtained from Sigma (St. Louis, MO), and characterized in this laboratory as

115 g Bloom. WPI, Alacen 895, containing 93.2% protein (manufacturer’s data), was

generously supplied by NZMP (formerly New Zealand Milk Products; Lemoyne, PA).


94

DTT was obtained from Calbiochem (San Diego, CA). All other chemicals were

reagent grade and used as received.

6.5.2 Sample preparation

Solutions of gelatin or WPI ranging from 1% to 10% (w/w) were prepared by

suspending protein powder in the required weight of deionized water. WPI-gelatin

solutions (10% WPI with 0.5 to 3% gelatin) were prepared by suspending the required

amounts of WPI and gelatin powders in deionized water and stirring for about 30 min to

ensure a uniform suspension. To test the effect of a reducing environment, a 10% DTT

(w/v) solution was prepared and the volume necessary to give a concentration of 10 mg

DTT per g protein was included in the preparation of selected samples. Samples were

allowed to swell at room temperature for four hours, and then adjusted to pH 7.5 with 1

N NaOH or 1 N HCl. Samples were then heated at 38 ºC for one hour, cooled to room

temperature and stored overnight at 4 ºC.

Typical conditions for mTGase-mediated crosslinking of gelatin are pH 6.5 and 50 °C

for 4 h (Taylor et al., 2001). For whey proteins, the optimum conditions are pH 7.5 and

40 °C for 8 h (Truong et al., 2004). To provide a basis for comparison, all reactions in

this study were performed at pH 7.5 and 45 °C for 5 h, considering that WPI was the

major protein component in the mixtures.

For crosslinking experiments, protein samples were prepared in 10 ml less than the

required volume. The appropriate concentration of mTGase was then prepared in 10 ml

of water and this solution added with stirring to gelatin, WPI, or WPI-gelatin solutions

to give the desired final protein concentration. Samples were readjusted to pH 7.5 and

incubated for 5 h at 45 ºC in a shaker bath under mild agitation. Reaction products were

then heated at 90 ºC for 10 min to deactivate the mTGase. Control samples, without

mTGase, were subjected to the same thermal treatment.

Gelatin and WPI solutions (1 to 10%) were treated with mTGase at 10 U/g of protein.

The effect of enzyme concentration (0 to 10 U/g of protein) was also investigated for

gelatin and WPI solutions at 10%. Finally, the effect of gelatin (0.5 to 3%) was tested

by adding gelatin to a 10% WPI sample containing mTGase at either 0 or 2 U/g of total

protein.

Chapter 6

95

6.5.3 Analyses

6.5.3.1 Gel strength

The gelatin used for this study was characterized by the standard Bloom test (AOAC

Method 948.21). The strength of a gel formed in a standard 59 mm Bloom jar from a

6.67% (w/w) solution of gelatin at pH 6.5 was measured on a TA.XT2 Texture

Analyzer (Stable Micro Systems, Surrey, UK). Gel strengths of experimental samples

were determined by the modified small sample Bloom test (Wainewright, 1977).

Samples were transferred to 39 mm weighing bottles, to a height of 40 mm, cooled to

room temperature and then chilled for 17 h at 10 ºC in a constant temperature bath.

Each sample was then placed under a 0.5 in. diameter analytical probe, which then was

driven into the sample to a depth of 4 mm at 1mm/s. The measured force, using the

correction factor (1.389) previously determined for small samples (Taylor et al., 1994),

was expressed in grams. After the determination of gel strength, samples were melted

at 60 °C for viscosity measurements.

6.5.3.2 Viscosity

Viscosity was determined using a Model LV 2000 Rotary Viscometer (Cannon, State

College, PA) equipped with a low Centipoise Adapter and a jacketed sample chamber

connected to a refrigerated bath circulator, Model RTE-8 (Neslab, Portsmouth, NH).

An 18 ml aliquot of each sample was added to the sample chamber and equilibrated for

at least 10 min. Viscosity was measured at a spindle speed of 60 rpm, corresponding to

a shear rate of 73.42 s-1. After the viscometer had been stabilized for one minute,

readings were taken at 60 °C for gelatin as recommended by Rose (1992) and at 25 ºC

for WPI and blended biopolymer solutions.

6.5.3.3 Rheology

Dynamic oscillatory rheometric measurements were carried out in a controlled stress

AR-2000 Rheometer (TA Instruments, New Castle, DE) at room temperature (21 ± 2

ºC) after enzyme deactivation and equilibration of samples for one hour. The sample

was placed between parallel 25 mm diameter plates and the gap between them was set

to 2.5 mm. Excess sample was trimmed off and a thin layer of mineral oil applied to the

exposed free edges of the sample to prevent moisture loss. Time sweep measurements

were used to study the evolution of the storage modulus G' as a function of time. A

strain sweep test was performed at a constant frequency of 0.1 Hz to find an oscillation


96

stress value that was within the linear viscoelastic range. This value, 0.5 Pa, was then

used to perform time sweep measurements at a constant 0.1 Hz frequency. Storage

modulus G' was recorded and analyzed over 20 h, at a rate of two points per hour.

6.5.3.4 SDS-PAGE

Inter-protein crosslinking was evaluated by polyacrylamide gel electrophoresis in

sodium dodecyl sulfate (SDS-PAGE) (Laemmli, 1970) using precast gradient gels (4 to

15%). Gels were calibrated using the broad range (BRM) SDS calibration standard

(Bio-Rad, Hercules, CA) that contains a mixture of nine proteins ranging in size from

6.5 to 200 kDa. Samples (approximately 0.5 mg) of lyophilized protein dissolved in

sample buffer (10 mM Tris-HCl at pH 8.0 containing 1 mM EDTA, 25 mg/ml SDS, 50

µl/ml β-mercaptoethanol and 0.1 µl/ml bromphenol blue) were heated at 40 ºC for 4 h.

Separation was achieved using a Phast System (Pharmacia Biotech Inc., Piscataway,

NJ). Gels were stained with Coomassie Blue.

6.5.3.5 Statistical modeling

Because x,y plots of the data points always showed curvature, second order models in

%gelatin or %WPI were generated by regression analysis of viscosity and gel strength

data for products from the treatment of combinations of gelatin, WPI, and blends with

mTGase and DTT. These models are of the form: Response = a + b*Gelatin +

c*Gelatin*Gelatin, where a, b, and c are regression coefficients. Analysis of covariance

(ancova) was performed on the models to determine whether the coefficients were

significantly different between treatments. Comparisons between treatments of the

slopes and second order coefficients were performed using orthogonal contrasts (Littell

et al., 2002). All tests of significance were performed at the P<0.05 level.


6.6.1 Gel strength

The effects on gel strength of increasing concentrations of gelatin (1 to 10%) in the

presence or absence of mTGase and DTT were investigated. The second order model

showed that gel strengths of all samples significantly increased (P<0.05) with increasing

gelatin concentration and were greatest at each concentration for samples containing

gelatin alone (Figure 6.1a). Because type I collagen, the primary source of gelatin, has

no disulfide bonds, the addition of DTT to gelatin was not expected to have a noticeable

Chapter 6

97

effect on gel strength, and at concentrations below 6% gelatin, the addition of DTT did

not impact the gel strength. At higher concentrations of gelatin (6 to 10%), however,

gel strengths were significantly (P<0.05) reduced by up to 15%. One possible

explanation is the presence of type III collagen, which is generally co-located with type

I collagen in connective tissue (van der Rest et al., 1990). The individual chains of type

III collagen are disulfide-linked (Boudko and Engel, 2004), and separation of these

chains may have interfered with the partial renaturation that normally accompanies

gelation. Inclusion of mTGase in dilute (1 to 5%) gelatin solutions, either with or

without DTT, resulted in gel strengths significantly (P<0.05) lower than for gelatin

alone (Figure 6.1a). The formation of intramolecular crosslinks, which would inhibit

the attainment of collagen-like structure, is favored over intermolecular ones in dilute

solutions (Sakamoto et al., 1994). At higher (6 to 10%) gelatin concentrations where

mTGase-mediated intermolecular crosslinking is more favorable, the resulting gel

strengths were about 90% of those for gelatin alone.

When the effect of mTGase (1 to 10 U/g gelatin) was determined, no trend could be

observed in the gel strength of a 10% gelatin (data not shown). The range of values was

between 188 g and 225 g, with an average gel strength of 207 ± 12 g, significantly

(P<0.05) lower than the 254 ± 1 g determined for 10% gelatin without enzyme. In the

presence of the reducing agent, DTT, the activity of mTGase was enhanced, as reported

by Kolodziejska et al. (2006), and gel strengths (263 ± 14 g) were comparable to those

for gelatin alone.

The formation of mTGase-mediated WPI gels has been previously reported at WPI

concentrations greater than 10% (Færgemand et al., 1997). Under the conditions of

these experiments, no gel formation was observed for WPI alone at concentrations up to

10%, with or without mTGase, under reducing or nonreducing conditions. Gels did

form when small amounts of gelatin (0.5 to 3%) were included in 10% WPI solutions

(Figure 6.1b). Gelatin alone at these concentrations forms only very weak gels, and so

long as the reaction conditions were not reducing, the gel strength was not significantly

altered whether the gelatin was alone or in a 10% WPI solution, with or without

mTGase at 2 U/g WPI-gelatin. Under reducing conditions, a dramatic increase in gel

strength was seen for mTGase-treated WPI-gelatin blends, the significance of which

was confirmed by the second order statistical model (P<0.05). Because reducing


98

conditions and gelatin were essential for enhancing the gel strength, it is likely that

crosslinking was between whey proteins and gelatin chains. The reduction of disulfide

bonds in the globular whey proteins by the action of DTT would expose reactive groups

to the action of the mTGase. In the absence of DTT, the WPI proteins were not

sufficiently unfolded to serve as effective substrates for mTGase.

Figure 6.1 – Gel strengths of gels formed from (a) bovine type B gelatin, 1 to 10% (w/w), and (b) WPI-gelatin blends, 10% WPI with 0 to 3% added gelatin. Gelatin gels were prepared in the presence or absence of mTGase (10 U/g protein) and DTT (10 mg/g protein), WPI-gelatin gels in the presence or absence of mTGase (2 U/g protein) and DTT (10 mg/g protein). Each data point represents a separate measurement. Abbreviations are: G - gelatin, W - WPI, R - reducing conditions (DTT), E - enzyme (mTGase).

6.6.2 Viscosity

The viscosities at 60 ºC, of gelatin solutions (1 to 10%), with and without DTT and

mTGase, were determined (Figure 6.2a). Neither DTT nor mTGase alone had a

significant effect on gelatin viscosity at this temperature. Similar to the case for gel

strength, the effect of mTGase under reducing conditions was not significant at gelatin

concentrations in the 1 to 6% range. Above 7% gelatin, the effect was a significantly

dramatic increase (P<0.001) in viscosity to values greater than 1800 mPa·s, beyond the

limit of the viscometer. These results are consistent with the formation of

intramolecular crosslinks at low gelatin concentrations and both intra- and

intermolecular crosslinks at higher concentrations (Clark and Courts, 1977; Yi et al.,

2006).

The viscosities at 25 °C of solutions of WPI (1 to 10%) with and without DTT and

mTGase were determined (Figure 6.2b). At WPI concentrations less than 7%, neither

Chapter 6

99

DTT nor mTGase, alone or together, had any significant effect on the viscosity of the

solution.

Between 7% and 10% WPI, mTGase alone did not affect the viscosity (data not shown),

and by inference had little ability to crosslink the proteins. In this concentration range,

the viscosity was decreased slightly when disulfide bonds were reduced and the protein

conformation was less well defined. A highly significant effect on the viscosity

(P<0.001) was observed as a result of the treatment that combined the action of DTT

and mTGase on a 10% WPI solution; a four-fold increase in viscosity was attained.

Viscosities measured at 25 °C for blends of gelatin (0.5 to 3%) with 10% WPI were

always higher than those for gelatin alone (Figure 6.2c). When mTGase was included

in blends of 10% WPI with gelatin, the increase in observed viscosity was not

significant. When both DTT and mTGase were included in the blend, their effect on

viscosity became more significant (P<0.001). In fact, viscosities for blends of 10%

WPI with more than 1.5% gelatin could not be measured because the solution formed a

gel that did not melt below 60 °C.


100

6.6.3 Rheological properties

The storage modulus (G') was determined for 10% WPI, blends of 10% WPI with 3%

gelatin, and samples treated with mTGase under reducing or nonreducing conditions

(Figure 6.3). A 10% WPI solution, which remained liquid at 20 °C, exhibited a constant

value less than 1 for G'. In contrast, for a 10% WPI solution treated with mTGase under

reducing conditions, G' remained nearly constant and low for the first 10 h and then

increased to 300 Pa between 10 and 20 h, suggesting the formation of a stable and

permanent gel structure (Comfort and Howell, 2002). Because G' can be related to the

amount of crosslinking, the similar and flat response of the WPI sample in a reducing

environment and the WPI sample with mTGase under nonreducing conditions suggest

that no significant crosslinking occurred under these conditions. WPI-gelatin blends

under reducing conditions without mTGase or with mTGase in a nonreducing

environment showed a small increase in G' between 10 and 20 hours, possibly due to

noncovalent interactions between gelatin and whey protein molecules. Chen and

Dickinson (1999) suggested that electrostatic interactions, hydrogen bonding or

Figure 6.2 – Viscosities of solutions of (a) gelatin, (b) WPI and (c) WPI-gelatin blends prepared as described for Fig. 6.1. Viscosities were measured at 60 °C for gelatin solutions and at 25 °C for WPI or WPI-gelatin blends. Abbreviations are as reported for Figure 6.1.

Chapter 6

101

hydrophobic interactions that have a physical nature and are temperature-dependent

might contribute to the appearance of weak gel-like behavior. A similar study of 3%

gelatin samples with or without mTGase (2 U/g gelatin) produced a constant G' value

near zero (data not shown). Blends of 10% WPI with 3% gelatin after incubation with

mTGase (2 U/g protein) under reducing conditions showed an exponential increase of

G' from approximately 70 Pa to approximately 5,000 Pa within the first ten hours. This

remarkable gain suggests the existence of an initial gelled network that became more

reticulated and stable with time (Eissa et al., 2004). Similar enhancements in storage

modulus were reported for blends of gelatin with small amounts of chitosan, a larger

polymer, and mTGase (Chen et al., 2003).

Figure 6.3 – Time sweep analysis of 10% (w/w) WPI and WPI-gelatin blends. Blends contain 10% (w/w) WPI and 3% (w/w) gelatin, mTGase was 10 U/g protein for WPI and 2 U/g protein for WPI-gelatin blends, DTT was 10 mg/g protein. Abbreviations are as designated for Figure 6.1.

6.6.4 SDS PAGE

Gelatin, WPI, and blended biopolymers were analyzed by SDS PAGE. Ten percent

gelatin (Figure 6.4, lane 2) reflects a polydisperse mixture of different sized collagen

fragments with some aggregated material in the stacking gel at the top, and faint bands

visible through the general smear in the separating gel. Although more distinct bands

can be seen in SDS PAGE patterns for gelatins isolated from a specific source (Taylor

et al., 1994) or high Bloom (250 g) commercial gelatins (Tosh et al., 2003), the pattern

seen here is typical of low Bloom (115g) commercial gelatins (Taylor et al., 2004),


102

which are sold on the basis of gel strength, and are blends of gelatins from different

preparations. The pattern for 10% gelatin treated with mTGase under reducing

conditions (Figure 6.4, lane 3) shows only a faint smear, representing small collagen

fragments that most likely lack the lysine or glutamine sidechains necessary for

mTGase-mediated crosslinking. Although samples prepared for lanes 2 and 3 were of

the same weight, the faintness of the pattern in lane 3 and the lack of material in the

stacking gel and the upper range of the separating gel suggests the formation of

aggregates so large they could not penetrate the stacking gel (Taylor et al., 2004). A

similar observation (Sharma et al., 2002) was interpreted to suggest that the protein had

become so highly polymerized that it could not penetrate the stacking gel; they also

demonstrated that although the highly polymerized products did not appear on the gels,

their presence could be confirmed by chromatography.

The SDS PAGE patterns for 10% WPI alone (Figure 6.4, lane 4) or after incubation

with mTGase 5 U /g under nonreducing conditions (Figure 6.4, lane 5) show the major

whey proteins, β-lactoglobulin (MW approximately 18 kDa) and α-lactalbumin (MW

approximately 14 kDa) as well as faint bands for bovine serum albumin (MW

approximately 66 kDa) and lactoferrin (MW approximately 86 kDa). Incubation of

10% WPI with mTGase 5 U /g under reducing conditions (Figure 6.4, lane 6) resulted in

bands, above both the separating gel and the stacking gel, showing formation of high

molecular weight polymers in addition to the typical WPI bands. An earlier study

(Færgemand et al., 1997) produced similar results.

1 2 3 4 5 6 7 8 9

Figure 6.4 – SDS-PAGE gels: lane 1, molecular weight markers, ranging in size from 200 kDa at the top to 6.5 kDa at the bottom; lane 2, gelatin 10% (w/w); lane 3, 10% gelatin after treatment with mTGase (10 U/g) under reducing conditions; lane 4, WPI 10% (w/w); lane 5, 10% WPI after treatment with mTGase (5 U/g); lane 6, 10% WPI after treatment with mTGase (5 U/g) under reducing conditions; lane 7, WPI 10% (w/w) with gelatin 1.5% (w/w); lane 8, WPI 10% (w/w) with gelatin 1.5% after treatment with mTGase (2 U/g); and lane 9 WPI 10% (w/w) with gelatin 1.5% after treatment with mTGase (2 U/g) under reducing conditions. This is a composite figure produced from two gels that were run simultaneously.

Chapter 6

103

The addition of 1.5% gelatin to 10% WPI had little effect on the gel pattern (Figure 6.4,

lane 7). In an earlier study, when small amounts of WPI were added to 10% gelatin

samples (Taylor et al., 2006), the whey component was clearly visible in the gel

patterns, and its participation in the formation of crosslinked biopolymers could easily

be monitored. Here, the lack of defined molecular weight bands in patterns for gelatin

and the low concentration of gelatin in the blends contribute to its invisibility.

Incubation of the WPI-gelatin blend with mTGase under nonreducing conditions

resulted in the appearance of a high molecular weight band, possibly representing

crosslinked gelatin fragments, but had little effect on the WPI pattern (Figure 6.4, lane

8). The gel pattern (Figure 6.4, lane 9) for a similar sample incubated under reducing

conditions shows a decrease in intensity of the WPI bands, an increase in high

molecular weight aggregates and a decrease in total protein. The pattern is consistent

with the formation of WPI-gelatin biopolymers in addition to aggregates of the

individual biopolymers. Although it is clear from the gel strengths and rheological data

that the inclusion of 1.5% gelatin in 10% WPI induces gel formation and enhances

physical properties, the biopolymers formed range in size from those formed by WPI

when treated with mTGase under reducing conditions to the much larger aggregates that

do not penetrate the gel.

6.7 Conclusions

Both gelatin (ICIS, 2006) and whey (FASS, 2006) are relatively low value byproducts

of the American food industry. While the actual prices vary over time the ratio is

relatively constant at approximately 5:1 (gelatin:whey). The addition of minor amounts

of relatively low quality gelatin to whey protein improves the strength and stability of

gels formed by the action of mTGase in a reducing environment. As a byproduct of the

meat industry, and a breakdown product of collagen, gelatin comes in a range of

qualities. The higher quality gelatins find a variety of uses in food, pharmaceutical and

industrial products and are heavily traded. Whey is a lower value byproduct that can be

recovered from the waste stream of the cheese industry. The use of mTGase to catalyze

the formation of crosslinks in and between protein molecules is well established, as is

the efficacy of its usefulness with either gelatin or WPI. When a small amount of

gelatin was added to WPI, before mTGase treatment under reducing conditions, a

dramatic rise in viscosity, higher gel strengths, and the appearance of high molecular

weight bands due to inter-protein crosslinking in SDS-PAGE gel patterns than for either


104

gelatin or WPI treated separately were observed. These results suggest that the reducing

environment partially unfolds the whey proteins, increasing access to glutamine and

lysine side chains, and that the gelatin chains crosslink the whey proteins to form a

network. The improvement in physical properties over either protein component, given

the same treatment, suggests the possibility of greater utilization and new products from

these byproducts.

6.8 References

Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H.,

Motoki, M., 1989. Purification and characteristics of a novel transglutaminase

derived from microorganisms. Agric. Biol. Chem. 53, 2613-2617.

Association of Official Analytical Chemists, 1990. Jelly strength of gelatine, final

action (948.21). In: Helrich, K. (Eds.), Official Methods of Analysis of the

Association of Official Analytical Chemists (15th ed.), AOAC, Arlington, VA,

pp. 929.

Boudko, S.P., Engel, J., 2004. Structure formation in the C terminus of type III collagen

guides disulfide cross-linking. J. Mol. Biol. 335, 1289-1297.

Chen, J., Dickinson, E., 1999. Interfacial ageing effect on the rheology of a heat-set

protein emulsion gel. Food Hydrocoll. 13, 363-369.

Chen, T., Embree, H.D., Brown, E.M., Taylor, M.M., Payne, F.P., 2003. Enzyme-

catalyzed gel formation of gelatin and chitosan: Potential for in situ applications.

Biomaterials 24, 2831-2841.

Clark, R.C., Courts, A., 1977. The chemical reactivity of gelatin. In: Ward, A.G.,

Courts, A. (Eds.), The Science and Technology of Gelatin, Academic Press,

New York, pp. 209-247.

Comfort, S., Howell, N.K., 2002. Gelation properties of soya and whey protein isolate

mixtures. Food Hydrocoll. 16, 661-672.

Eissa, A.S., Bisram, S., Khan, S.A., 2004. Polymerization and gelation of whey protein

isolates at low pH using transglutaminase enzyme. J. Agric. Food Chem. 52,

4456-4464.

Færgemand, M., Otte, J., Qvist, K.B., 1997. Enzymatic cross-linking of whey proteins

by Ca2+-independent microbial transglutaminase from Streptomyces lydicus.

Food Hydrocoll. 11, 19-25.

Chapter 6

105

Farrell, H.M., Jr, Jiménez-Flores, R., Bleck, G.T., Brown, E.M., Butler, J.E., Creamer, L.K.,

Hicks, C.L., Hollar, C.M., Ng-Kwai-Hang, K.F., Swaisgood, H.E., 2004. Nomenclature

of the proteins of cows' milk--sixth revision. J. Dairy Sci. 87, 1641-1674.

Folk, J.E., Chung SI, 1973. Molecular and catalytic properties of transglutaminases.

Adv. Enzymol. Relat. Areas Mol. Biol. 38, 109-191.

Folk, J.E., Chung, S.I., 1985. Transglutaminases. Methods Enzymol. 113, 358-375.

Grazú, V., Ovsejevi, K., Cuadra, K., Betancor, L., Manta, C., Batista-Viera, F., 2003.

Solid-phase reducing agents as alternative for reducing disulfide bonds in

proteins. Applied Biochem. Biotech. 110, 23-32.

ICIS News, Chemical Industry News, Chemical Prices & Chemical Suppliers from

ICIS, 2006 at http://www.icis.com/Articles/2006/08/28/2015785/Chemical-

Prices-F-J.html

Kolodziejska, I., Piotrowska, B., Bulge, M., Tylingo R, 2006. Effect of transglutaminase

and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide on the solubility of fish

gelatin-chitosan films. Carbohydr. Polym.. 65, 404-409.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head

of bacteriophage T4. Nature 227, 680-685.

Littell, R.C., Stroup, W.W., Freund, R.J. 2002. SAS for Linear Models, Fourth Edition.

Cary, NC: SAS Institute Inc.

Motoki, M., Nio, N., 1983. Crosslinking between different food proteins by

transglutaminase. J. Food Sci. 48, 561-566.

Oh, J.H., Wang, B., Field, P.D., Aglan, H.A., 2004. Characteristics of edible films made

from dairy proteins and zein hydrolysate cross-linked with transglutaminase. Int.

J. Food Sci. Tech. 39, 287-294.

NASS, National Agricultural Statistics Service, 2006, Dairy product prices. June 30,

2006.http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?docume

ntID=1450

Rodríguez-Nogales, J.M., 2006. Effect of preheat treatment on the transglutaminase-

catalyzed cross-linking of goat milk proteins. Process Biochem. 41, 430-437.

Rose, P.I., 1992. Inedible gelatin and glue. In: Pearson, A.M., Dutson, T.R. (Eds.),

Inedible Meat By-Products. Advances in Meat Research, Elsevier Applied

Science, New York, pp. 217-263.


106

Sakamoto, H., Kumazawa, Y., Motoki, M., 1994. Strength of protein gels prepared with

microbial transglutaminase as related to reaction conditions. J. Food Sci. 59,

866-871.

Sharma, R., Zakora, M., Qvist, K.B., 2002. Susceptibility of an industrial α-lactalbumin

concentrate to cross-linking by microbial transglutaminase. Int. Dairy J. 12,

1005-1012.

Taylor, M.M., Diefendorf, E.J., Marmer, W.N., Brown, E.M., 1994. Effect of various

alkalinity-inducing agents on chemical and physical properties of protein

products isolated from chromium-containing leather waste. J. Amer. Leather

Chem. Assoc. 89, 221-228.

Taylor, M.M., Cabeza, L.F., Marmer, W.N., Brown, E.M., 2001. Enzymatic

modification of hydrolysis products from collagen using a microbial

transglutaminase. I. Physical properties. J. Amer. Leather Chem. Assoc. 96, 319-

332.

Taylor, M.M., Marmer, W.N., Brown, E.M., 2004. Molecular weight distribution and

functional properties of enzymatically modified commercial and experimental

gelatins. J. Amer. Leather Chem. Assoc. 99, 129-141.

Taylor, M.M., Marmer, W.N., Brown, E.M., 2006. Preparation and characterization of

biopolymers derived from enzymatically modified gelatin and whey. J. Amer.

Leather Chem. Assoc. 101, 235-248.

Tosh, S.M., Marangoni, A.G., Hallett, F.R., Britt, I.J., 2003. Aging dynamics in gelatin

gel microstructure. Food Hydrocoll. 17, 503-513.

Truong, V.D., Clare, D.A., Catignani, G.L., Swaisgood, H.E., 2004. Cross-linking and

rheological changes of whey proteins treated with microbial transglutaminase.

J. Agric. Food Chem. 52, 1170-1176.

U.S. Dairy Export Council's 2006 Annual Report at http://usdec.org (accessed August

31, 2007)

van der Rest, M., Dublet, B., Champliaud, M-F., 1990. Fibril-associated collagens.

Biomaterials 11, 28 – 31.

Wainewright, F.W., 1977. Physical tests for gelatin and gelatin products. In: Ward,

G.A., Courts, A. (Eds.), The Science and Technology of Gelatin, Academic

Press, New York, pp. 507-534.

Chapter 6

107

Yi, J.B., Kim, Y.T., Bae, H.J., Whiteside, W.S., Park, H.J., 2006. Influence of

transglutaminase-induced cross-linking on properties of fish gelatin films. J.

Food Sci. 71, E376-E383.

Yildirim, M., Hettiarachchy, N.S., 1997. Biopolymers produced by cross-linking

soybean 11S globulin with whey proteins using transglutaminase. J. Food Sci.

62, 270-274.

6.9 Acknowledgments

The authors would like to thank Lorelie Bumanlag, Paul Pierlott, Michael Tunick and

Carlos Carvalho for their technical support in this work.

CHAPTER 7

Whey protein isolate: a potential filler for the leather industry

Chapter 7

109

7. WHEY PROTEIN ISOLATE: A POTENTIAL FILLER FOR THE LEATHER

INDUSTRY

Authors: Eduard Hernàndez Balada1,2, Maryann M. Taylor1, Eleanor M. Brown1,

Cheng-Kung Liu1 and Jaume Cot3.


Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038 USA 2Department of Chemical Engineering, University of Barcelona, Martí i Franquès 1,

08028 Barcelona, Spain 3 Consejo Superior de Investigaciones Científicas (CSIC), Research and Development

Center, Ecotechnologies Department, Jordi Girona 18-26, 08034 Barcelona, Spain

Manuscript accepted for publication at the Journal of the American Leather

Chemists´Association. Tentatively scheduled for publication in the March 2009 issue.


110


THE JOURNAL OF THE AMERICAN LEATHER CHEMISTS ASSOCIATION

c/o The American Leather Chemists Association, 1314 50th Street, Suite 103, Lubbock, Texas 79412

Mobile phone: (616) 540-2469 E-mail: [email protected] September 11, 2008 Ms. Maryann M. Taylor USDA, Agricultural Research Service Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor, PA 19038 Dear Ms. Taylor, This will acknowledge receipt and acceptance for publication of the manuscript entitled “Whey Protein Isolate: A Potential Filler for the Leather Industry”. It is tentatively scheduled for publication in the March 2009 issue of the Journal. Could you please sign the attached “Transfer of Copyright” form and return it to me. Thank you for this interesting contribution. Sincerely yours, Robert F. White Journal Editor cc: Dr. Eduard Hernandez Balada

Chapter 7

111

7.2 Abstract

The upgrading of leather that presents loose areas and poor grain break is one of the

most value adding opportunities for a tanner. Typically, petroleum-based products are

used to improve the final appearance and feel of crust leather. In this study, we

demonstrate that blends composed of whey protein isolate (WPI), a byproduct of the

cheese industry, and small amounts of gelatin, a byproduct of the leather industry, could

be effectively used as filling agents for both shoe upper and upholstery leather. Wet

blue leather from three different areas in the hide (butt, belly and neck) was treated with

the WPI-gelatin blend, retanned, colored and fatliquored, and their subjective and

mechanical properties evaluated. The effect of pretreatment of the wet blue samples

with various concentrations of the enzyme microbial transglutaminase (mTGase) was

also examined. It was found that the rate of uptake of the WPI-gelatin blend by

upholstery wet blue increased four-fold when it was pretreated with a 2.5% mTGase

solution. Conversely, this rate was decreased when shoe upper was pretreated with

increasing amounts of mTGase. The subjective properties (e.g. handle, fullness, color

and grain break) of both shoe upper and upholstery leather that were treated with the

WPI-gelatin blend were significantly improved over the controls. Importantly, the grain

break of the belly area of samples that were pretreated with enzyme (both upholstery

and shoe upper) was remarkably improved. Hence, fillers mainly composed by the less

expensive WPI were demonstrated to be effective filling agents for both upholstery and

shoe upper leather.


112

7.3 Resum

La millora de cuirs que presenten àrees buides i poca fermesa de flor és un dels majors

desafiaments que té l’adobador per afegir-los-hi valor. Típicament, matèries primeres

derivades del petroli són emprades en la millora de l’aspecte final i tacte del cuir. En el

present estudi, demostrem que barreges formades per sèrum concentrat de proteïna

(WPI, per les seves sigles en anglès), un subproducte de la indústria del formatge, junt

amb petites quantitats de gelatina, un subproducte de la indústria pelletera, es poden

emprar de forma efectiva com agents de rebliment en cuirs per tapisseria i calçat.

Diferents zones de pells adobades amb crom (flancs, coll i part posterior de l’animal)

van ser tractades amb una barreja de WPI-gelatina, tornades a adobar, colorejades i

engreixades, i les mostres avaluades respecte a les propietats subjectives i mecàniques.

L’efecte del pretractament de cuir amb vàries concentracions de l’enzim

transglutaminassa microbiana (mTGase) fou també examinat. Es va trobar que la

velocitat d’absorció de la mescla de WPI-gelatina per part del cuir per tapisseria

s’incrementava per quatre si era pretractat amb una solució de 2.5% mTGase. En canvi,

aquesta velocitat disminuïa quan el cuir per calçat era pretractat amb quantitats creixents

de mTGase. Les propietats subjectives (tacte, cos, color i fermesa de flor) de les pells

per tapisseria i calçat que van ser tractades amb la mescla WPI-gelatina van millorar

sensiblement en comparació amb els controls. En particular, la fermesa de flor de les

mostres dels flancs que van ser pretractades amb l’enzim (tant en cuirs per tapisseria

com per calçat) va ser millorada notablement.

Chapter 7

113

7.4 Introduction

The presence of loose areas with poor grain break in finished leather is one of many

concerns that tanners are facing in today’s leather processing. This problem becomes

particularly significant in the neck and belly areas of the hide with the belly area

exhibiting a looser break.1 Fillers are materials used to fill the interstices of the leather

and make the looseness less pronounced which in turn should improve cutting yields.

Over the last several years, this laboratory has explored alternatives to petroleum-based

fillers. Chen et al.2 demonstrated that collagen hydrolysate crosslinked with

glutaraldehyde could be suitable for filling low quality leather. More recent research has

focused on the use of gelatin polymerized by the action of microbial transglutaminase

(mTGase) (EC 2.3.2.13), an enzyme capable of forming crosslinks in a wide variety of

proteins. Both commercial and experimental alkali-extracted gelatins were effectively

crosslinked with mTGase, yielding products with improved functional properties.3-6

This enzyme also proved to be effective in the crosslinking of gelatin with sodium

caseinate, a dairy industry byproduct.7 Enzymatically modified gelatin and casein were

successfully applied as fillers in wet blue leather. It was later found that these modified

proteins were not removed during the washing process.8 Nevertheless, the relatively

elevated cost of gelatin and casein encouraged the search for a cheaper source of

renewable proteins. Whey and whey protein isolate (WPI), byproducts of the cheese

manufacturing industry, fulfill that condition and were also effectively reacted with

mTGase yielding viable products for use as filling agents.9,10

We recently demonstrated that the addition of small amounts of gelatin to whey protein

isolate (WPI) in the presence of mTGase and the reducing agent dithiothreitol (DTT)

yielded novel products with improved physical properties (e.g., viscosity, gel strength,

degree of polymerization) over either protein component.11 The main goal of that study

was to obtain biopolymers with unique properties at low cost, hence using WPI as the

majority component of the WPI-gelatin blend.

In the present study, we examine the suitability of biopolymers produced by combining

WPI with small amounts of commercial low Bloom gelatin as a filling agent for shoe

upper or upholstery leather. The effectiveness of the various treatments was assessed by


114

measuring the physical, mechanical and subjective properties of crust leather from three

different areas of the hide (butt, belly and neck).

7.5 Experimental

7.5.1 Materials

Microbial transglutaminase, Activa TG-TI (approximately 100 units/g), a commercial

mTGase formulation containing 99% maltodextrose as a carrier, with an active range of

pH 4.0 to 9.0 at 0 to 70°C, was obtained from Ajinomoto USA Inc. (Paramus, NJ) and

used without further purification. Type B gelatin, alkaline extracted from bovine skin,

and characterized in this laboratory as 115 g Bloom, was obtained from Sigma (St.

Louis, MO). WPI, Alacen 895, containing 93.2% protein (manufacturer’s data), was

generously supplied by NZMP (formerly New Zealand Milk Products; Lemoyne, PA).

Dithiothreitol (DTT) was obtained from Calbiochem (San Diego, CA). A Bicinchoninic

Acid (BCA) Kit for protein determination was purchased from Sigma (St. Louis, MO).

Trutan PA-65 and Trutan PRP-77 were obtained from the former Pilar River Plate Corp.

(Newark, NJ); Havana Dye (Derma Havana R Powder) was obtained from Clariant

Corporation (Charlotte, NC).; Altasol-CAM, Altasol 310-L and Eureka 400R were

obtained from Atlas Refinery, Inc. (Newark, NJ). Basyntan NNOL and Basyntan DLE

were obtained from BASF Corporation (Charlotte, NC). Upholstery and shoe upper wet

blue leather was obtained from commercial tanneries. All other chemicals were reagent

grade and used as received.

7.5.2 Methods

7.5.2.1 Preparation of WPI-Gelatin blends

One day prior to the treatment, the required amounts of WPI and gelatin powders were

suspended in water (200% float), mixed well and allowed to sit at room temperature for

at least two h. The amount of protein powder was calculated on the basis of the weight

of wet blue. Next, a 10% DTT (w/v) solution was prepared and the volume necessary

to give a concentration of 10 mg DTT per g of WPI was added. The pH was then

adjusted to 7.5 with 1 N NaOH or 1 N HCl and heated at 38 ºC for one h, cooled to

room temperature and stored overnight at 4 ºC. It is worth noting that all the

proteinaceous blends discussed in the present paper were prepared without adding

Chapter 7

115

mTGase to the mixture. By doing this, the WPI-gelatin blend can be stored for an

extended period of time without danger of it becoming a permanent gel.

7.5.2.2 Application of WPI-Gelatin blends to wet blue leather

Wet blue samples from the butt, belly, and neck were tumbled with water in a Dose

drum (Model PFI 300-34, Dose Maschinenbau GmbH, Lichtenau, Germany) for 30 min

at 50°C, drained and refloated (200% float, 50°C) in 4% sodium bicarbonate solution,

based on the wet blue weight. Upon pH stabilization, the float was drained, mTGase

solution (2.5% or 5% mTGase) with a 200% float was added and samples were

drummed for one h at room temperature (22 ± 4°C). Note that the mTGase

concentrations stated throughout the manuscript are mTGase + carrier concentration.

The float was drained and a WPI-gelatin solution was added (200% float). Control

pieces to which no enzyme or protein mixture was added were also run with a 200%

float of water. The samples were tumbled for one h at RT and then for five h at 45°C.

The floats were then drained and the samples washed twice for 10 min at 50°C (200%

float), drained, patted dried and stored at 4°C. Previous work carried out in our

laboratory typically employed a 400% float in all the above mentioned processing

steps.8-10,12 By cutting back the float to a half we reduce the consumption of water and

also increase the concentration of protein in the solution, which ultimately leads to a

higher concentration gradient between the solution and the leather.

Aliquots of 3 ml were extracted from the drum after varying time intervals throughout

the treatment of the samples with the mTGase or the WPI-gelatin solutions. Aliquots of

water after each wash were also collected to estimate the amount of protein that was

removed from the wet blue. One drop of 5% sodium azide solution was added to each

aliquot and they were stored at 4°C until needed to run the protein determination assay.

The following day, both treated and untreated samples were weighed and the amount of

reagents needed for the Retan-Color-Fatliquor (RCF) calculated.


116

7.5.2.3 Retan/Color/Fatliquor (RCF)

Control and test samples were retanned, colored and fatliquored in separate drums.

Shoe upper and upholstery samples followed slightly different procedures (Figure 7.1a

and 7.1b, respectively).

Retan/Color

• 150% float @ 30°C

• Add 2% Trutan PA-65 (20 min @ 30°C)

• Add 4% PRP-77 (30 min @ 30°C)

• Add 4% 400R (5 min @ 60°C)

• Add 6% Havana Dye (60 min @ 60°C)

• Add 1% formic acid and tumble until dye is exhausted, @ 60°C

• Batch wash x 3 (200% float @ 60°C, 10 min)

Fatliquor

• Refloat in 150% float @ 60°C

• Add 10% Altasol CAM and 2% Eureka 400R (60 min @ 60°C)

• Add 1.5% formic acid (target pH 3.0-3.5)

• Drain and wash for 5 min

Toggle dry, mill for 24 h, store for 48 h @ constant temp. & RH

Mechanical Properties & Subjective Evaluation

Retan/Color

• 150% float @ 30°C

• Add 2% Trutan PA-65 (20 min @ 30°C)

• Add 4% PRP-77 (30 min @ 30°C)

• Add 4% 400R (5 min @ 60°C)

• Add 6% Havana Dye (60 min @ 60°C)

• Add 1% formic acid and tumble until dye is exhausted, @ 60°C


Fatliquor


• Add 10% Altasol CAM and 2% Eureka 400R (60 min @ 60°C)

• Add 1.5% formic acid (target pH 3.0-3.5)

• Drain and wash for 5 min

Toggle dry, mill for 24 h, store for 48 h @ constant temp. & RH


Retan/Color

• 75% float @ 43°C

• Add 10% DLE Syntan, 2% Basyntan NNOL (5 min @ 43°C)

• Add 1% Havana dye in 25% float @ 60°C (45 min @ 60°C)

• Add 0.5% formic acid and tumble until dye is exhausted, @ 60°C


Fatliquor


• Add 5% Altasol CAM, 1% Eureka 400R

• Run 30 min @ 55°C

• Add Altasol 310L in 10% water @ 55°C

• Run 10 min @ 55°C

Haul, horse overnight, set out, toggle dry, store for 48 h @ constant temp. & RH


Retan/Color

• 75% float @ 43°C

• Add 10% DLE Syntan, 2% Basyntan NNOL (5 min @ 43°C)

• Add 1% Havana dye in 25% float @ 60°C (45 min @ 60°C)

• Add 0.5% formic acid and tumble until dye is exhausted, @ 60°C


Fatliquor


• Add 5% Altasol CAM, 1% Eureka 400R

• Run 30 min @ 55°C

• Add Altasol 310L in 10% water @ 55°C

• Run 10 min @ 55°C

Haul, horse overnight, set out, toggle dry, store for 48 h @ constant temp. & RH

Mechanical Properties & Subjective Evaluation Figure 7.1 – Flow diagram for retan, color and fatliquor formulation of (a) upholstery and (b) shoe upper wet blue.

7.5.2.4 Drying

After RCF, samples were removed from the drum and allowed to dry. Shoe upper crust

leather was allowed to hang freely, whereas stretching (toggling) was applied to

upholstery crust leather. Once dry, the leathers were conditioned, put into plastic bags

for one day and then staked twice. Shoe upper crust samples were not milled;

upholstery crust samples were milled for about 24 h. All samples were then kept on a

shelf in the conditioning room at 20°C and 65% relative humidity for at least three days.

7.5.3 Analyses

7.5.3.1 Mechanical properties

Tensile strength, Young modulus and tear strength were determined as described in a

previous publication.12

7.5.3.2 Subjective evaluation

Experimental and control crust leathers were assessed for handle, fullness, grain

tightness (break), color and general appearance by hand and visual examination.

a b

Chapter 7

117

Handle is defined as the sensation or feeling of certain physical properties of leather,

such as flexibility and smoothness, which can be perceived by touch with fingers and

hands��Fullness refers to the way a loop of leather feels in the palm of the hand when

compressed. A full leather fills the palm while a flat leather has more of a cardboard

effect. Grain break is the pattern of tiny wrinkles formed when the leather is bent grain

inward. Leather was rated on a scale of 1 to 5 for each functional property by two

experienced tanners, where higher numbers indicate a better property.

7.5.3.3 Protein concentration determination

Protein concentrations in the float, at different stages of the treatment, were determined

using the bicinchoninic acid (BCA) assay13 according to the directions supplied with the

kit. Samples were centrifuged at 13,400 rpm for 30 min in a microcentrifuge

(Eppendorf MiniSpin plus, Westbury, NY). One ml of protein supernatant was

removed and typically a 1:25 (v/v) dilution was prepared in order to fall within the

linear concentration range for the assay (200 to 1,000 µg/ml protein). A 50 µl aliquot of

the diluted solution was mixed with 1.0 ml of BCA reagent and incubated at 37°C for

30 minutes. The absorbance of a sample solution at 562 nm minus a reagent blank was

compared with a standard curve using known concentrations of bovine serum albumin.


7.6.1 Shoe upper wet blue

We first investigated the uptake of mTGase and WPI-gelatin blends by shoe upper wet

blue at both 2.5% and 5% mTGase concentration levels. Similar trends for the uptake

of mTGase were obtained for samples that were pretreated with 2.5 or 5% mTGase. In

both cases, the curve leveled off after only 20 minutes and the bath was not exhausted

after one hour of drumming (Figure 7.2a).

After draining the mTGase solution, a blend of 5% WPI and 0.5% gelatin, with respect

to weight of wet blue, was added and drummed one hour at RT followed by 5 h at 45°C.

A protein uptake of 98% was achieved with wet blue that was not treated with mTGase,

whereas wet blue pretreated with 2.5% and 5% mTGase reduced the percentage to 86%

and 83%, respectively (Figure 7.2b).


118

0

10

20

30

40

50

60

0 10 20 30 40 50 60

5% mTGase2.5% mTGase

Time (min)

0

20

40

60

80

100

0 1 2 3 4 5 6

5% mTGase2.5% mTGase0% mTGase

Time (h) Figure 7.2 – (a) mTGase and (b) protein uptake profiles by shoe upper wet blue pretreated with a solution containing 0, 2.5 or 5% mTGase and treated with a solution of 5% WPI + 0.5% gelatin. All percentages were calculated with respect to the weight of wet blue and added in a 200% float.

The absorption of the protein by the wet blue follows first order reaction kinetics. Hence,

[ ][ ] tAA ⋅−=��

�

��

�κ

0

ln

where [A] and [A]0 are the protein concentration remaining in the drum at time t and t =

0, respectively and k is the uptake rate coefficient. Table 7.1 shows k and the

correlation coefficient values for the uptake of WPI-gelatin by shoe upper wet blue and

shoe upper wet blue pretreated with 2.5% or 5% mTGase. The most rapid absorption,

reflected by the highest value of k, was obtained for the wet blue that was not pretreated

with enzyme (k = 0.608 h-1), followed by the one pretreated with 2.5% (k = 0.413 h-1)

and 5% mTGase (k = 0.323 h-1), respectively.

TABLE 7.1

Uptake rate coefficient k for various treatments

Treatment Wet blue % mTGasea % WPIa Gelatina k (h-1) R2

A Shoe upper 0 5 0.5 0.608 0.994

B Shoe upper 2.5 5 0.5 0.413 0.851

C Shoe upper 5 5 0.5 0.323 0.950

D Upholstery 0 2.5 0.25 0.365 0.830

E Upholstery 2.5 2.5 0.25 1.377 0.956 aPercentages calculated with respect to weight of wet blue.

a b

Chapter 7

119

The wet blue was washed twice immediately after draining the proteinaceous solution.

No detectable level of protein was found in washes of wet blue pretreated with 5%

mTGase. At 2.5% and 0% mTGase there was a protein removal of approximately 6%

and 8%, respectively, and approximately 75% of that protein was washed out in the first

wash. Part of that washed out protein could be due to the unbound protein adhered to

the hide or to the insufficient draining of the drum before the addition of water.

All crust samples were evaluated with respect to handle, fullness, grain tightness (break)

and color. The samples were rated on a scale of 1 to 5, with 1 being the worst and 5

being the best. From these ratings, an overall rating in which the grain break was

weighted more than the other ratings was also presented. Table 7.2 reports the results

on the above mentioned subjective properties of shoe upper wet blue subjected to

treatments A, B or C. Values that were equal to or better than controls are underlined.


120

TABLE 7.2

Subjective evaluationa,b,c

Treatment A Treatment B Treatment C

Hide

area Property Control Test Control Test Control Test

Handle 4 4 4 3 3.5 4.5

Fullness 4 5 4 5 4 4.5

Break 5 5 5 5 1.5 3.5

Color 3 5 3 5 4 4

Butt

Overall 4 5 4 5 3 4

Handle 3 4 3 2 2 4.5

Fullness 3 5 4 5 2 4.5

Break 4 5 5 5 1 4

Color 3 5 3 5 2.5 3.5

Belly

Overall 3 5 4 5 1.5 4.5

Handle 3 4 3 2 3.5 4.5

Fullness 4 5 4 5 4 4.5

Break 5 5 5 5 2.5 4

Color 2 5 2 5 3 3.5

Neck

Overall 3 4 1.5 4.5 2.5 4.5 aScale 1-5, 1=worst, 5=best bn=2 cA, B and C stand for treatments of shoe upper with a solution containing 5% WPI + 0.5% gelatin and pretreated with a solution of 0, 2.5 or 5% mTGase, respectively. All percentages were calculated with respect to the weight of wet blue and added in a 200% float

In all treatments, the test pieces were found to be equal to or superior to the control

pieces. Only the handle of the leather pretreated with 2.5% mTGase was rated slightly

lower than the control. Focusing on the grain break, it is important to note that the wet

blue samples used for the 0% and 2.5% mTGase batches had a good break before the

treatment while the 5% mTGase wet blue samples exhibited a poor break. The samples

that already exhibited a good break showed neither a significant improvement in break

Chapter 7

121

nor any detrimental effect. The 5% mTGase wet blue sample clearly showed significant

improvement in leather from all areas of the hide when comparing the break to the

control.

Next, we examined the effect of reducing the WPI to 2.5% and gelatin to 0.25% and

mTGase to 2.5%, for samples that exhibited a poor break. Approximately 80% of the

protein was taken up by the wet blue, with a rate coefficient rate of k = 0.262 h-1.

During the wash procedure, approximately 6% of the protein was removed, all of it in

the first wash. Although the break of the crust leather fared better than the control, the

improvement was not as dramatic as when a 5% mTGase treatment followed by 5%WPI

+ 0.5% gelatin was used (data not shown). These results suggest that 5% WPI + 0.5%

gelatin filled the leather better than 2.5% WPI + 0.5% gelatin, particularly in the belly

area.

7.6.2 Upholstery wet blue

The ability of WPI and gelatin to fill and improve upholstery wet blue was examined.

Given the smaller thickness of upholstery (1.0-1.2 mm) compared to shoe upper (2.0-

2.4 mm), a lower concentration of WPI and gelatin was selected to make up the

proteinaceous blend (2.5% WPI + 0.25% gelatin). The effect of an enzymatic

pretreatment of the samples with 2.5% mTGase prior to the addition of the

proteinaceous blend was also evaluated. About half the amount of the enzyme was

picked up by the wet blue within the first 30 minutes, and the curve leveled off

thereafter (Figure 7.3a). A complete uptake of protein was reached within the first 3 h

of tumbling for wet blue that was pretreated with 2.5% mTGase. Conversely, the

protein uptake trend for samples that were not pretreated with the enzymatic leveled off

at approximately 90% after 4 h of drumming (Figure 7.3b).


122

0

10

20

30

40

50

60

0 10 20 30 40 50 60Time (min)

0

20

40

60

80

100

120

0 1 2 3 4 5 6

0% mTGase2.5% mTGase

Time (h) Figure 7.3 – (a) mTGase and (b) protein uptake profiles by upholstery wet blue pretreated with a solution containing 0 or 2.5% mTGase and treated with a solution of 2.5% WPI + 0.25%. All percentages were calculated with respect to the weight of wet blue and added in a 200% float.

A remarkably faster uptake of protein was observed for samples that were pretreated

with mTGase, as can be seen from the uptake coefficient values (Table 7.1).

Approximately 5% of the protein was removed in the first wash regardless of the

enzymatic pretreatment. A non detectable amount of protein was removed during

subsequent washes.

The treatment of upholstery wet blue with 2.5% WPI + 0.25% gelatin considerably

improved the handle, fullness, and color of the resulting crust leather. Most

importantly, the break of the belly and butt areas was significantly improved when the

wet blue was pretreated with 2.5% mTGase prior to the addition of the proteinaceous

blend (Figure 7.4).

a b

Chapter 7

123

Figure 7.4 – Subjective properties of upholstery crust leather. D and E stand for treatments of upholstery with a solution containing 2.5% WPI + 0.25% gelatin and pretreated with a solution of 0 or 2.5 mTGase, respectively. All percentages were calculated with respect to the weight of wet blue and added in a 200% float.

7.6.3 Mechanical properties

Leathers from three different areas of the hide, neck, belly and butt, were tested for

mechanical properties. This report will present the test results from belly area only, the

primary area of concern. The three areas demonstrated the same tendency towards the

change of two major variables: percent WPI and percent mTGase. A 3-D regression

plot of the resultant tensile strength as a function of percent WPI and percent mTGase

simultaneously for both upholstery and shoe upper leather samples was developed

(Figure 7.5). The tensile strength decreases slightly with increasing percent WPI for

both upholstery and shoe upper leather. However, for upholstery leather, the tensile

strength increases significantly with percent mTGase, whereas for shoe upper leather,

Butt

0

1

2

3

4

5

Control Treatment D Treatment E

Belly

0

1

2

3

4

5


Neck

0

1

2

3

4

5


Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

Butt

0

1

2

3

4

5


Belly

0

1

2

3

4

5


Neck

0

1

2

3

4

5


Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

Handle

BreakFullness

ColorOverall

BreakFullness

ColorOverall


124

the tensile strength shows little change with percent mTGase. It is worthy to note that

as demonstrated in Figure 7.5, shoe upper leather has greater tensile strength than

upholstery leather and this is ascribable to the fact that shoe upper leather is thicker and

has more fiber network to resist the fracture.

Figure 7.5 – Effect of the various treatments of leather with WPI and mTGase on the tensile strength of (a) upholstery and (b) shoe upper crust leather. The regression graphic corresponds to samples from the belly area.

Young’s modulus is a value indicating the stiffness of leather. Young’s modulus of

upholstery leather increases significantly with both percent WPI and percent mTGase

(Figure 7.6a). On the other hand, for shoe upper leather Young’s modulus also

increases significantly with percent mTGase, but changes very little with percent WPI

(Figure 7.6b). Looking closely at Figure 7.6, one can notice that the range of Young’s

modulus values are higher for shoe upper leather than upholstery leather. Besides the

fact that shoe leather is thicker, this variation could be due to the use of different types

of fatliquors.

a b

Chapter 7

125

Figure 7.6 – Effect of the various treatments of leather with WPI and mTGase on the Young’s modulus of (a) upholstery and (b) shoe upper crust leather. The regression graphic corresponds to samples from the belly area.

Tear strength decreases with both percent WPI and percent mTGase for upholstery

leather (Figure 7.7a). However, for shoe upper leather the tear strength responds quite

differently to the changes of percent mTGase and percent WPI. As demonstrated in

Figure 7.7b, tear strength of shoe upper leather decreases pronouncedly with percent

mTGase but is relatively unchanged with percent WPI.

Figure 7.7 – Effect of the various treatments of leather with WPI and mTGase on the tear strength of (a) upholstery and (b) shoe upper crust leather. The regression graphic corresponds to samples from the belly area.

a b

a b


126

7.7 Conclusions

Current prices of sodium caseinate ($5.8/lb)14 and gelatin ($2.6/lb)15 emphasized the

need for research into cheaper sources of protein to generate fillers for leather. The less

expensive whey protein isolate ($1.05/lb)14 along with small amounts of gelatin were

successfully applied as a filling agent for upholstery and shoe upper leather. Subjective

properties such as fullness, handle and color of the resulting crust leather were

significantly improved. Furthermore, grain break for upholstery and shoe upper leather

fared markedly better when samples were pretreated with mTGase. The enzymatic

pretreatment of wet blue leather with mTGase also affected the protein uptake ratio.

The uptake coefficient for upholstery leather pretreated with 2.5% mTGase increased

four-fold over samples that were not enzyme pretreated. The trend was reversed for

shoe upper leather with a drop in the uptake coefficient value from 0.608 h-1 to 0.323 h-1

for samples that were not pretreated with mTGase or pretreated with 5% mTGase,

respectively. We further demonstrated that a 200% float satisfactorily enabled the

proteins to be taken up by the wet blue. If this technology is to be transferred to the

industry, use of a shorter float could be feasible due to a stronger mechanical action.

Importantly, the proteins are not considerably removed by washing, regardless of the

enzymatic pretreatment. Another advantage presented herein is that the proteinaceous

blend was added to the drum without any enzyme pretreatment, thus its preparation

becomes more convenient and feasible. Even though the various treatments did not

negatively affect the mechanical properties of the crust leather, filled samples were a

little stiffer and presented slight lower tear strength than the controls. Further research

that explores the possibility of using even cheaper sources of protein as a raw material

for bio-based leather products is an interesting option currently being examined in our

laboratory.

Chapter 7

127

7.8 References

1. Tancous, J. J., Roddy, W. T., and O’Flaherty, F.; Defects due to natural

characteristics of the skin or hide. In: Skin, Hide and Leather Defects (The Western

Hills Publishing Company), pp. 2-18, 1959.

2. Chen, W., Cooke, P. H., DiMaio, G. L., Taylor, M. M., and Brown, E. M.; Modified

collagen hydrolysate, potential for use as a filler for leather. JALCA 96, 262-267,

2001.

3. Taylor, M. M., Cabeza, L. F., Marmer, W. N., and Brown, E. M.; Enzymatic


transglutaminase. I. Physical properties. JALCA 96, 319-332, 2001.

4. Taylor, M. M., Liu, C. K., Latona, N. P., Marmer, W. N., and Brown, E. M.;

Enzymatic modification of hydrolysis products from collagen using a microbial

transglutaminase. II. Preparation of films. JALCA 97, 225-234, 2002.

5. Taylor, M. M., Liu, C. K., Marmer, W. N., and Brown, E. M.; Enzymatic


transglutaminase. III. Preparation of films with improved mechanical properties.

JALCA 98, 435-444, 2003.

6. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Molecular weight distribution

and functional properties of enzymatically modified commercial and experimental

gelatins. JALCA 99, 129-141, 2004.

7. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Characterization of biopolymers

prepared from gelatin and sodium caseinate for potential use in leather processing.

JALCA 100, 149-159, 2005.

8. Taylor, M. M., Bumanlag, L., Marmer, W. N., and Brown, E. M.; Use of

enzymatically modified gelatin and casein as fillers in leather processing. JALCA

101, 169-178, 2006.

9. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Preparation and characterization

of biopolymers derived from enzymatically modified gelatin and whey. JALCA 101,

235-248, 2006.

10. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Evaluation of polymers prepared

from gelatin and casein or whey as potential fillers. JALCA 102, 111-120, 2007.

11. Hernàndez Balada, E., Taylor, M. M., Phillips, J. G., Marmer, W. N., and Brown, E.

M.; Properties of biopolymers produced by transglutaminase treatment of whey

protein isolate and gelatin. Bioresour. Technol. In press.


128

12. Taylor, M. M., Marmer, W. N., and Brown, E. M.; Effect of fillers prepared from

enzymatically modified proteins on mechanical properties of leather. JALCA 103,

128-137, 2008.

13. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H.,

Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C.;

Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85, 1985.

14. http://www.ams.usda.gov/AMSv1.0/.

15. http://www.icis.com/Articles/2006/08/28/2015785/Chemical-Prices-F-J.

7.9 Acknowledgments

The authors would like to thank Lorelie Bumanlag, Gary Di Maio, Rafael García,

Nicholas Latona, Renée Latona and Joe Lee for their technical support and assistance.

CHAPTER 8

General conclusions and recommendations

Chapter 8

130

8. GENERAL CONCLUSIONS AND RECOMMENDATIONS

The main conclusions extracted from this work are compiled and discussed in this

section. Given that the research reported in the thesis was divided into two different

projects, the conclusions are also presented in two sections.

8.1 Preservation of raw hides and skins with brine. Conclusions

� A continuous reaction mathematical model was verified to describe the diffusion

of sodium chloride in the hide during the curing process.

� It was demonstrated that the infusion of sodium chloride into the hide occurred

from the flesh side overwhelmingly. That was ascribed to the semipermeability

of the epidermis, which enabled the diffusion of water from the hide and into the

surrounding float but also hindered the diffusion of salt into the hide.

� The thermal stability of the hide increased upon curing, mainly due to the

simultaneous dehydration and salting out processes of the constituent collagen.

The denaturation temperature of the hide increased gradually from the flesh to

the grain during the cure, corroborating that the absorption of salt took place

mainly from the flesh side.

� The diffusion of sodium chloride into the hide was characterized by the transport

coefficient �, which was found to be in the order of 10-5 s-1. By comparing the

individual values of � obtained for various values of initial brine concentration

and float percentage.

� The concentration of brine was found to play a critical role on the effectiveness

of the curing process. A proper cure of the hides could not be achieved by using

diluted brines (20 and 25% w/v, or 64 and 80 ºSAL, respectively) regardless of

the float percentage used.

� If employing an initially saturated brine (100 ºSAL), the usage of a minimum

float of 280% was found to be necessary in order to properly cure hides. A float

of 440% was needed if a less concentrated brine of 96 ºSAL was selected.

� It was concluded that the usage of a saturated brine (35% w/v or 100 ºSAL) as

well as a minimum float of 500% yielded higher values of �, therefore higher

diffusion rates. These results corroborated a general practice applied in curing

raceways, where a 97 ºSAL is typically employed in a 500% float.

� It was proven that the amount of fat adhered to the flesh side retarded the

diffusion of salt into the hide. The addition of a commercial degreasing agent,


131

made of a blend of nonionic surfactants, along with the brine significantly

decreased the fat content of the hide and significantly enhanced the uptake of

sodium chloride. In addition, the composition of the degreaser was found to be

a critical parameter for this specific purpose.

� A concentration of degreaser between 0.5 and 1% w/w (with respect to the

combined weight of hide and brine) was proven to be sufficient to significantly

defat the hide and facilitate the uptake of salt.

� A glycolipid surfactant produced by the yeast Candida bombicola, sophorolipid,

was effectively tested as brine curing enhancer agent. When used at a

concentration above its solubility limit of 0.5% w/w (with respect to the

combined weight of hide and brine), the sophorolipid showed remarkable

degreasing properties and enhanced the uptake of salt by the hide.

� By using an appropriate degreasing agent in the brine, turn-around times in

raceways could be reduced and thus additional curing capacity could be created.

8.2 Preservation of raw hides and skins with brine. Recommendations

For further investigation, the following recommendations are proposed.

� In the presented model, it was assumed that the thickness of the hide, b, was

constant throughout the curing. Nevertheless, it is well known that a hide

shrinks to a certain extent during the first stages of the cure. It would be

interesting to remake the mathematical model in which the thickness of the hide

would be a variable of the process instead of a parameter.

� More research needs to be done in order to establish a minimum salt saturation

level that ensures a proper preservation of the hides, currently set at 85%. A

comprehensive study that studies the relationship between the quality and

characteristics of the cured hide, the degree of saturation and the storage

conditions (e.g. temperature, time, humidity) needs to be carried out.

� More research needs to be done to increase the solubility of sophorolipids. By

accomplishing this, their field of applications would be greatly widened and

hide dealers would be more receptive towards its usage in an industrial scale.

� It is essential to find a way to remove the fat that builds up in the curing vats or

raceways, which are operated continuously.

� Hide dealers do not have a reliable method to assess the degree of curing of a

hide. Typically, they evaluate the completeness of curing by making a cut

Chapter 8

132

through the hide and looking for a uniform blue color on the cut surface that

indicates complete curing. Therefore, a non-destructive rapid test method for

cure validation is needed.

8.3 Obtaining and characterization of potential fillers for leather. Conclusions

� Both gelatin and whey protein isolate (WPI), which are relatively low value

byproducts of the American food industry, were proved to be reactive substrates

towards the enzyme microbial transglutaminase (mTGase).

� A reducing environment was needed in order to partially unfold the globular

whey proteins and thus expose the reactive glutamine and lysine groups to the

action of the mTGase.

� A dramatic rise in gel strength, viscosity and elastic modulus was observed

when a small amount of gelatin (ranging from 0.5 to 3% w/w) was added to 10

% w/w WPI and reacted with mTGase under reducing conditions. Also, the

appearance of high molecular weight bands due to inter-protein crosslinking in

SDS-PAGE gel patterns suggested that gelatin chains crosslinked the whey

proteins to form a network.

� The synergistic effect found between WPI and gelatin suggested that new

biopolymers, with improved functionality, could be developed by mTGase

treatment of protein blends containing small amounts of gelatin with the less

expensive WPI.

� Bioproducts composed of WPI along with small amounts of gelatin were

successfully applied as filling agents for upholstery and shoe upper leather.

� Wet blue leather treated with the WPI-gelatin blend, retanned, colored and

fatliquored, exhibited a significant improvement in fullness, handle and color,

with respect to the samples that were not treated.

� Grain break for upholstery and shoe upper leather fared markedly better when

wet blue samples were pretreated with mTGase, prior to the treatment with the

WPI-gelatin blend.

� The protein uptake rate coefficient was affected by whether the wet blue was

pretreated with mTGase or not. The coefficient for upholstery leather pretreated

with 2.5% mTGase increased four-fold over samples that were not enzyme

pretreated (from 0.365 h-1 to 1.377 h-1). A reverse trend was observed for shoe

upper leather, with a drop in the uptake coefficient value from 0.608 h-1 to 0.323


133

h-1 for samples that were not pretreated with mTGase or pretreated with 5%

mTGase, respectively.

� The proteins were not largely removed by washing, regardless of the enzymatic

pretreatment.

� The mechanical properties of the filled crust leather were not adversely modified

with respect to the sample that did not undergo the treatment. However, treated

samples were a little stiffer and presented slight lower tear strength than

untreated samples.


Recommendations

� The enzyme microbial transglutaminase has the potential of polymerizing a wide

variety of proteins and the ability of crosslinking dissimilar proteins for

generating novel products. Therefore, the research for other sources of protein

and the study of their reactivity towards the mTGase is strongly encouraged. In

the case of the production of potential fillers for leather, it would be interesting

to look into the possibility of using keratin, the main protein in the cattle hair

and wool, as a cheap and readily available source of protein.

� The reaction between the proteinaceous substrate and a carbodiimide (e.g. 1-

ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC) prior to the

enzymatic treatment with mTGase would be an interesting approach to increase

the reactivity of the proteins towards the enzyme.

� Scaling up of the filling process. Once the new biopolymer has been succesfully

tested as a filling agent for leather in the lab scale, it should be scaled up to a

pilot plant size, prior to eventually transferring the technology to the industry.

By scaling up the process, lesser concentrations of reactants and volume of float

would be needed due to a stronger mechanical action. A continuous monitoring

of the protein concentration in the drum should be carried out in order to assess

the efficiency of the process and whether the proteinaceous solution should be

dumped or reutilized for another batch.

� From an industrial point of view, it would be interesting to make the filler from

the tannery'ssolid waste (e.g. chrome shavings). By doing this, they would not

only adding value to a byproduct that it is usually landfilled, but they would also

Chapter 8

134

be increasing the value of low quality hides that will be treated with the product.

Also, they would be closing the loop within the industry itself.

CHAPTER 9

References

Chapter 9

136

9. REFERENCES

Alexander, K.T.W. (1988). Enzymes in the tannery – catalysts for progress? Journal of

the American Leather Chemists’ Association�83(9): 287-316�

Anandan, A., Marmer, W.N., Dudley, R.L. (2007). Isolation, characterization and

optimization of culture parameters for production of an alkaline protease isolated

from Aspergillus tamari. Journal of Industrial Microbiology and Biotechnology�

34(5): 339-347��

Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H.,

Motoki, M. (1989). Purification and characteristics of a novel transglutaminase

derived from microorganisms. Agricultural and Biological Chemistry 53(10): 2613-

2617.

Babiker, E. F. E., Khan, M.A.S., Matsudomi, N., Kato, A. (1996a). Polymerization of

soy protein digests by microbial transglutaminase for improvement of functional

properties. Food Research International 29(7): 627-634.

Babiker, E. F. B., Fujisawa, N., Matsudomi, N., Kato A. (1996b). Improvement in the

functional properties of gluten by protease digestion or acid hydrolysis followed by

microbial transglutaminase treatment. Journal of Agricultural and Food Chemistry

44(12): 3746-3750.

Bailey, D.G., Birbir, M. (1993). A study of the extremely halophilic microorganisms

found on commercially-brined cured cattle hides. Journal of the American Leather

Chemists’ Association 88(8): 291-299.

Bailey, D.G., Gosselin, J.A. (1996). The preservation of animal hides and skins with

potassium chloride. Journal of the American Leather Chemists’ Association 91(12):

317-333.

Bailey, D.G. (1999). Gamma radiation preservation of cattle hides. A new twist on an

old story. Journal of the American Leather Chemists’ Association 94(7): 259-267.

Bailey, D.G., DiMaio, G.L., Gehring, A.G., Ross, G.D. (2001). Electron beam

irradiation preservation of cattle hides in a commercial-scale demonstration. Journal

of the American Leather Chemists’ Association 96(10): 382-392.

Bailey, D.G. (2003). The preservation of hides and skins. Journal of the American

Leather Chemists’ Association 98(8): 308-319.

Brown, E.M., Dudley, R.L. (2005). Approach to a tanning mechanism: Study of the

interaction of aluminum sulfate with collagen. Journal of the American Leather


References

137

Cabeza, L.F., Taylor, M.M., Brown, E.M., Marmer, W.N. (1997). Influence of pepsin

and trypsin on chemical and physical properties of isolated gelatin from chrome

shavings. Journal of the American Leather Chemists’ Association 92(8): 200-207.

Cabeza, L.F., Taylor, M.M., Brown, E.M., Marmer, W.N. (1999). Potential applications

for gelatin isolated from chromium-containing solid tannery waste:

Microencapsulation. Journal of the American Leather Chemists’ Association 94(5):

182-189.

Catalina, M., Antunes, A.P.M., Attenburrow, G., Cot, J., Covington, A.D., Phillips, P.S.

(2007). Sustainable management of waste-reduction of the chromium content of

tannery solid waste as a step in the cleaner production of gelatin. Journal of Solid

Waste Technology and Management 33(1): 43-50.

Cayot, P., Lorient, D. (1997). Structure-Function relationships of whey proteins. In

(Damodaran, S., Paraf, A. eds.) Food proteins and their applications, pp. 225-256.

Chambers, J.V., Ferretti, A. (1979). Industrial applications of whey/lactose. Journal of

Dairy Science 62(1): 112-116.

Chattopadhyay, B. (1998). Molecular biology of collagen. Part I. Journal of the Indian

Leather Technologists' Association 48: 473- 492.

Chen, W., Cooke, P.H., DiMaio, G.L., Taylor, M.M., Brown, E.M. (2001). Modified

collagen hydrolysate, potential for use as a filler for leather. Journal of the American


Covington, A.D. (1997). Modern tanning chemistry. Chemical Society Reviews 26: 111-

126.

Daniels, R. Overview: Avoiding salts. World Leather, May 1998, pp. 66-70.

de Carvalho, R.A., Grosso, C.R.F. (2004). Characterization of gelatin based films

modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids

18(5): 717-726.

de Wit, J.N., Swinkels, G.A.M. (1980). A differential scanning calorimetric study of the

thermal denaturation of bovine �-lactoglobulin. Thermal behaviour at temperatures

up to 100 ºC. Biochimica et Biophysica Acta 624(1): 40-50.

de Wit, J.N. (1998). Nutritional and functional characteristics of whey proteins in food

proteins. Journal of Dairy Science 81:597-608.

Chapter 9

138

Didato, D.T., Bowen, J., Hurlow, E. (1999). Microorganisms control during leather

manufacture. In: Leafe MK (Ed.) Leather Technologists’ Pocket Book. Society of

Leather Technologists and Chemists, UK, pp. 339-352.

Ding, K., Taylor, M.M., Brown, E.M. (2007). Effect of genipin on the thermal stability

of hide powder. Journal of the American Leather Chemists’ Association 101(10):

362-367.

Eigel, W.N., Butler, J.E., Ernstrom, C.A., Farrell Jr., H.M., Harwalkar, V.R., Jenness,

R., Whitney, R. McL. (1984). Nomenclature of proteins of cow's milk: fifth

revision. Journal of Dairy Science 67: 1599-1631.

Færgemand, M., Otte, J., Qvist, K.B. (1997). Enzymatic cross-linking of whey proteins

by Ca2+-independent microbial transglutaminase from Streptomyces lydicus. Food

Hydrocolloids 11(1): 19-25.

Folk, J.E. (1980). Transglutaminases. Annual Review of Biochemistry 49: 517-531.

Glicksman, M. (1969). Gum Technology in the Food Industry. Academic Press Inc.,

New York.

Godsalve, E. Personal communication, WE CO., 1991, Inc. Research Liaison

Committee, May 1st 2007, USDA Eastern Regional Research Center, Wyndmoor,

PA.

Grant, W.D., Kamekura, M., McGenity, T.J., Ventosa, A. (2001). Class III

Halobacteria class nov. In: Bergey’s manual of systematic bacteriology (Boone, R.,

Castenholz, R., Garrity, G., Eds.), pp. 294-334.

Grazú, V., Ovsejevi, K., Cuadra, K., Betancor, L., Manta, C., Batista-Viera, F. (2003).

Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins.

Applied Biochemistry and Biotechnology – Part A Enzyme Engineering and

Biotechnology 110(1): 23-32.

Gustavson, K.H. (1956). The chemistry of tanning, definition of tanning, the

hydrothermal stability of vegetable-tanned collagen. Academic Press Inc., New

York, pp. 177-181.

Harris, D. (1974). Dictionary of terms used in the hides, skins and leather trade, USDA,

Foreign Agricultural Service, Agricultural Handbook #465.

Jelen, P. (2002). Whey Processing. In: Encyclopedia of Dairy Sciences (Roginski, H.,

Fuquay, J.W., Fox, P.F.,Eds.), pp. 2739-2751.

References

139

Jongjareonrak, A., Soottawat B., Visessanguan W., Tanaka, M. (2006). Skin gelatin

from bigeye snapper and brownstripe red snapper: Chemical compositions and

effect of microbial transglutaminase on gel properties. Food Hydrocolloids 20(8):

1216-1222.

Kanagaraj, J., Chandra Babu, N.K., Sadulla, S., Suseela Rajkumar, G., Visalakshi, V.,

Chandra Kumar, N. (2001). Cleaner techniques for the preservation of raw goat

skins. Journal of Cleaner Production 9(3): 261-268.

Kanagaraj, J., Chandra Babu, N.K. (2002). Alternatives to salt curing techniques – A

review. Journal of Scientific and Industrial Research 61(5): 339-348.

Kanagaraj, J., John Sundar, V., Muralidharan, C., Sadulla, S. (2005). Alternatives to

sodium chloride in prevention of skin protein degradation – a case study. Journal of

Cleaner Production 13(8): 825-831.

King, G., Brown, E.M., Chen, J.M. (1996). Computer model of a bovine type I collagen

microfibril. Protein Engineering 9(1): 43-49.

Kinsella, J.E. (1984). Milk proteins: physicochemical and functional properties. Critical

Reviews in Food Science and Nutrition 21(3): 197-262.

Klein, J.D., Guzman, E., Kuehn, G.D. (1992). Purification and partial characterization

of transglutaminase from Physarum polycephalum. Journal of Bacteriology 174(8):

2599-2605.

Kolomazník, K. Biodiesel from tannery waste. Paper presented at the 104th ALCA

Annual Meeting, June 19th - 22nd 2008, Greensboro, NC.

Leather Facts. (1965). A picturesque account of one of nature’s miracle. Published by

New England Tanners Club.

Leather International. Traders enjoy a hide bonanza. April 2006.

Mandl, I., Zipper, H., Ferguson, L.T. (1958). Clostridium histolyticum collagenase: its

purification and properties. Archives of Biochemistry and Biophysics 74(2): 465-

475.

McLaughlin, G.D., Rockwell, G.E. (1922). The bacteriology of fresh steer hide. Journal


McDermott, M.K., Chen, T., Williams, C.M., Markley, K. M., Payne, G.F. (2004).

Mechanical properties of biomimetic tissue adhesive based on the microbial

transglutaminase-catalyzed crosslinking of gelatin. Biomacromolecules 5(4): 1270-

1279.

Meat Industry. Profitable hide curing. April 1979, pp. 51-53.

Chapter 9

140

Morr, C.V., Foegeding, E.A. (1990). Composition and functionality of commercial

whey and milk protein concentrates and isolates: a status report. Food Technology

44(4): 100-112.

Morr, C.V., Ha, E.Y. (1993) Whey protein concentrates and isolates: processing and

functional properties. Critical Reviews in Food Science and Nutrition 33(6): 431-

476.

Munz, K.H. (2007). Silicates for raw hide curing. Journal of the American Leather

Chemists’ Association 102(1):16-21.

Nonaka, M., Sakamoto, H., Toiguchi, S., Kawajiri, H., Soeda, T., Motoki, M. (1992).

Sodium caseinate and skim milk gels formed by incubation with microbial

transglutaminase. Journal of Food Science 57(5): 1214-1218.

Onwulata, C.I., Smith, P.W., Konstance, R.P., Holsinger, V.H. (2001). Incorporation of

whey products in extruded corn, potato or rice snacks. Food Research International

34(8): 679-687.

Orthmann, A.C., Higby, W.M. (1929). The cause of vein like protuberances on finished

leather. Journal of the American Leather Chemists’ Association 24(12): 654-656.

Pallavicini, C., Alloggio, V., de Leonardis, A. (1992). Action of a transglutaminase

from plant tissues on some milk proteins. Industrie Alimaentari 31(310): 1130-

1134.

Peng, B., Shi, B., Ding, K., Fan, H., Shelly, D.C. (2007). Novel titanium (IV) tanning

for leathers with superior hydrothermal stability. Journal of the American Leather


Preethi, V., Rathinasamy, V., Kannan, N., Babu, C., Sehgal, P.K. (2006). Azardirachta

Indica: a green material for curing of hides and skins in leather processing. Journal


Ramachandran, G.N., Kartha, G. (1955). Structure of collagen. Nature 176(4482): 593-

595.

Ramasami, T., Prasad, B.G.S. (1991). Environmental aspects of leather processing.

Calcutta Proceedings of the LEXPO XV, pp. 43-71.

Ramasami, T., Sreeram, K. J, Gayatri, R. (1998). Emerging leather processing strategies

for waste minimisation. Unit 1. Background information and cleaner technologies in

raw material preservation and in the beamhouse processes, UNIDO pp. 183-197.

References

141

Rao, J. R., Chandrababu, N. K., Muralidharan, C., Nair, B. U., Rao, P. G., Ramasami,

T. (2003). Recouping the wastewater: A way forward for cleaner leather processing.

Journal of Cleaner Production 11(5): 591-599.

Rao, B.R., Henrickson, L. (1983). Short-term preservation of cattlehide. Journal of the

American Leather Chemists’ Association 78(2): 48-53.

Rich, A., Crick, F.H.C. (1955). The structure of collagen. Nature 176(4489): 915-916.

Reddington, J.J. Personal communication. Research Liaison Committee, April 29th

2008, USDA Eastern Regional Research Center, Wyndmoor, PA.

Rüegg, M.P., Morr, U., Blanc B. (1977). A calorimetry study of the thermal

denaturation of whey proteins in simulated milk ultrafiltrate. Journal of Dairy

Research 44: 509-520.

Sakamoto, H., Kumazawa, Y., Motoki, M. (1994). Strength of protein gels prepared

with microbial transglutaminase as related to reaction conditions. Journal of Food

Science 59(4): 866-871.

Saravanabhavan, S., Aravindhan, R., Thanikaivelan, P., Rao, J.R., Chandrasekaran, B.,

Nair, B.U. (2003). An integrated eco-friendly tanning method for the manufacture

of upper leather from goatskins. Journal of the Society of Leather Technologists and

Chemists 87(4): 149-158.

Sharphouse, J.C. (1971). Types of hides and skins and principal uses. In Leather

Technician’s Handbook (Leather Producer’s Association eds.) Northampton, UK,

pp. 20-36.

Sharphouse, J.H., Kinweri, G. (1978). Preservation-formaldehyde of raw hides and

skins. Journal of the Society of Leather Trade Chemists. 62: 119-123.

Stuart, L.S., Frey, R.W. (1938). On the spoilage of stored salted calf skins. Journal of

the American Leather Chemists’ Association 33(4): 198-203.

Tancous. J.J. (1959). Skin, hide, and leather defects, 1st ed. Published by Leather

Industries of America, Cincinnati, OH.

Tanimoto, S.-Y., Kinsella, J. E. (1988). Enzymatic modification of proteins. Effect of

transglutaminase on some physical properties of �-lactoglobulin. Journal of

Agricultural and Food Chemistry 36(2): 281-285.

Taylor, M.M., Cabeza, L.F., Marmer, W.N., Brown, E.M. (2000). Use of Tryptec

enzyme preparations in treatment of chrome shavings. Journal of the American


Chapter 9

142

Taylor, M.M., Cabeza, L.F., Marmer, W.N., Brown, E.M. (2001). Enzymatic


transglutaminase. I. Physical properties. Journal of the American Leather Chemists’

Association 96(9): 319-332.

Taylor, M.M., Liu, C.K., Latona, N., Marmer, W.N., Brown, E.M. (2002). Enzymatic


transglutaminase. II. Preparation of films. Journal of the American Leather


Taylor, M.M., Liu, C.K., Marmer, W.N., Brown, E.M. (2003). Enzymatic modification

of hydrolysis products from collagen using a microbial transglutaminase. III.

Preparation of films with improved mechanical properties Journal of the American


Taylor, M.M., Marmer, W.N., Brown, E.M. (2004). Molecular weight distribution and


gelatins. Journal of the American Leather Chemists’ Association 99(3): 129-141.

Taylor, M.M., Marmer, W.N., Brown, E.M. (2005). Characterization of biopolymers

prepared from gelatin and sodium caseinate for potential use in leather processing.

Journal of the American Leather Chemists’ Association 100(4): 149-159.

Taylor, M.M., Bumanlag, L., Marmer, W.N., Brown, E.M. (2006a). Use of

enzymatically modified gelatin and casein as fillers in leather processing. Journal of


Taylor, M.M., Marmer, W.N., Brown, E.M. (2006b). Preparation and characterization

of biopolymers derived from enzymatically modified gelatin and whey. Journal of


Taylor, M.M., Marmer, W.N., Brown, E.M. (2007). Evaluation of polymers prepared

from gelatin and casein or whey as potential fillers. Journal of the American Leather


Taylor, M.M., Marmer, W.N., Brown, E.M. (2008). Effect of fillers prepared from

enzymatically modified proteins on mechanical properties of leather. Journal of the


Tegtmeyer, D., Vorlaender, O., Zeyen, W., Tysoe, C., Silberkuhl, F. (2007). New

micro-sphere application in the leather industry. Journal of the American Leather


References

143

Thomson, R.S. (1981). Tanning. Man´s first manufacturing process? Transactions of the

Newcomen Society, 53: 139-156.

Thorstensen, T.C. (1993). Practical leather technology, 4th ed. Krieger Publishing

Company. Malabar, Florida.

USDA Agricultural Marketing Service. http://www.ams.usda.gov/AMSv1.0/.

van der Rest, M., Garrone, R. (1991). Collagen family of proteins. The Journal of the

Federation of American Societies for Experimental Biology, 5: 2814-2823.

Von Hippel, P.H. (1967). Structure and stabilization of the collagen molecule in

solution. In: Treatise on collagen (Ramachandran, G.N. ed.). Academic Press, Inc.,

New York, pp. 253-338.

Wagner, M., Bailey, D.G. (1999). Structure of bovine skin and hair root – A scanning

electron microscope investigation. Journal of the American Leather Chemists’

Association 94 (10): 378-383.

Webb, E.C. (1992). Enzyme Nomenclature. Recommendations (1992) of the

Nomenclature Committee of the International Union of Biochemistry and Molecular

Biology. Academic Press, Inc. San Diego, CA.

Yasueda, H., Kumazawa, Y., Motoki, M. (1994). Purification and characterization of a

tissue-type transglutaminase from Red Sea Bream (Pagrus Major). Bioscience

Biotechnology and Biochemistry 58(11): 2041-2045.

Yi, J. B., Kim, Y. T., Bae, H. J., Whiteside, W. S., Park, H. J. (2006). Influence of

transglutaminase-induced cross-linking on properties of fish gelatin films. Journal

of Food Science 71(9): 376-383.

Yildirim, M., Hettiarachchy, N.S., Kalapathy, U. (1996). Properties of biopolymers

from cross-linking whey protein isolate and soybean 11s globulin. Journal of Food

Science 61(6): 1129-1131.

Yildirim, M., Hettiarachchy, N.S. (1997). Biopolymers produced by cross-linking

soybean 11s globulin with whey proteins using transglutaminase. Journal of Food

Science 62(2): 270-275.

Yildirim, M., Hettiarachchy, N.S. (1998). Properties of films produced by cross-linking

whey proteins and 11s globulin using transglutaminase. Journal of Food Science

63(2), 248-252.

CHAPTER 10

Notation

Chapter 10

145

10. NOTATION

SYMBOL /

ACRONYM DESCRIPTION UNITS

a Pore half length of the skin (m)

[A] Protein concentration remaining in the drum at time t (g dm-3)

[A]0 Protein concentration remaining in the drum at time t = 0 (g dm-3)

Ancova Analysis of covariance (-)

b Thickness of cured hide (m)

BCA Bicinchoninic acid (-)

BSA Bovine serum albumin (-)

c Concentration of sodium chloride in the hide moisture, at a

distance x from the boundary ( > 0) (mol m-3)

c0 Concentration of sodium chloride in the bath ( > 0) (mol m-3)

c0n Concentration of saturated sodium chloride solution at 25 °C (mol m-3)

c0p Initial concentration of sodium chloride in the bath ( = 0) (mol m-3)

c0� Equilibrium concentration of sodium chloride in the bath (mol m-3)

C Dimensionless concentration integral average (-)

C, C0 Dimensionless concentrations (-)

CV Coefficient of variation (-)

D Diffusion coefficient of sodium chloride in the hide (m2 s-1)

D’ Effective diffusion coefficient of sodium chloride in the hide (m2 s-1)

DSC Differential scanning calorimetry (-)

DTT Dithiothreitol (-)

F0 Fourier number/dimensionless time (-)

G’ Elastic or storage modulus (Pa)

GLR Gray level range (-)

HLB Hydrophilic-lipophilic balance (-)

Ig Immunoglobulins (-)

k Protein uptake rate coefficient (h-1)

LF Lactoferrin (-)

MAFB Moisture and ash free basis (-)

mTGase Microbial transglutaminase (-)

MW Molecular weight (Da, kDa)

Notation

146

SYMBOL /


Na Soaking number (-)

R2 Correlation coefficient (-)

RCF Retan-Color-Fatliquor (-)

RH Relative humidity (%)

RT Room temperature (ºC)

S Outer surface of the solid phase (skin) (m2)

SAL Brine saturation degree (ºSAL)

SD Standard deviation (-)

SDS-PAGE Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (-)

SEM Scanning electron microscopy (-)

SEM-BSE Back-scattered low vacuum scanning electron microscopy (-)

SL Sophorolipid (-)

t Time (min, h)

TD Hide’s denaturation temperature (ºC)

TDS Total dissolved solids (kg/ton hide)

TGase Transglutaminase (-)

USDEC United States Dairy Export Council (-)

USHSLA United States Hide Skin & Leather Association (-)

UV Ultraviolet (-)

V Volume of skin (m3)

V0 Volume of brine solution (m3)

WPC Whey Protein Concentrate (-)

WPI Whey Protein Isolate (-)

x Distance (m)

X Dimensionless distance (-)

Chapter 10

147

Greek symbols

SYMBOL /


�-La Alpha-lactalbumin (-)

�-Lg Beta-lactoglobulin (-)

� Porosity of solid state (skin) (-)

� Transport coefficient (s-1)

Time (s)

ξ Tortuosity (-)

CHAPTER 11

Glossary of terms

Chapter 11

149

11. GLOSSARY OF TERMS

– A –

Adipose tissue. Form or connective tissue in whose cell fat is deposited and stored.

More frequently found in the flesh layer of hide or a skin.

– B –

Back (of animal). Main portion of hide, obtained by cutting off the two bellies. Note

that in North America a back is a half cattle hide (or side) after the removal of the head

and belly.

Bacteria. Bacteria are the smallest organisms that can complete their life cycle

independently. They lack a nucleus and membrane-bound organelles. They may be

autotrophic or heterotrophic and occur in a wide range of environments. They are

abundant on the remains of dead plants and animals and some cause disease in other

living organisms. Bacteria are also responsible for such process as fermentation and

decomposition.

Bacterial damage. Hides and skins damaged and rendered evil-smelling by bacterial

damage.

Bactericide. Agent or treatment that specifically kills bacteria.

Belly. The underside of a hide between the fore and the hind legs.

Break. Tiny wrinkles formed on the grain side of the leather when it is bent inward.

Brine. A strong solution of salt and water used for preserving raw stock in the

preparation of leather.

Butt. The part of the hide after the bellies and shoulders have been removed.

Glossary of terms

150

Byproduct. A secondary or incidental product deriving from a manufacturing process,

a chemical reaction or a biochemical pathway, and is not the primary product or service

being produced (e.g, gelatin, whey).

– C –

Casein. A nitrogenous substance prepared by precipitation of skim milk. Large

quantities of casein are used by the tanning industry in making leather finishes.

Cattle hide. The skin of a fully grown bovine animal.

Chrome leather. A bluish-green leather which has been chrome tanned. Also known

as wet blue.

Coarse grain. Grain surface that is somewhat rough, due to the nature of the pelt and

the method of treatment during tannage etc., and in which the hair or wool follicles are

large, forming a prominent pattern (see also loose and pipey grain).

Collagen. Protein contained in connective tissue, cartilage and bones, the chief protein

of raw hides and skins.

Crosslink. Chemical links between the molecular chains of polymers.

Crosslinking agent. Highly reactive products like polyisocyanates or polyfunctional

aziridine compounds to achieve film-forming properties of different finish formulations.

Crosslinking reaction. Process of joining free polymer chains with each other by side

linkages to form a two or three dimensional network.

Crust leather. Leather which, after tanning, has not been further processed.

Curing. The treatment of raw hides and skins after flaying to retard bacterial action and

putrefaction.

Chapter 11

151

– D –

Degrease. To remove grease by any method.

Delayed salting. Salting which has been delayed for so long a period after flaying that

damage may have been caused through putrefaction, etc.

– F –

Filler. Any substance which is capable of entering into the voids that exist between the

fibers of leather and remain there.

Filling. Introduction of conditioning substances into the leather to give weight and

body.

Fine grain. Leather whose grain is smooth and the hair follicles are minute.

Fleshing. Removal of any adipose tissue on the flesh side of the skins.

Float. Aqueous liquor in which a process such as curing, pickling or tanning is

performed.

Fresh hide. Undressed, uncured hides taken directly from an animal’s carcass.

Fullness. Property of a leather to have pleasing handle and not being flat and empty.

To achieve fullness or an improved, pleasing handle, filling agents are used in finish

formulations. They deposit additional material between the leather fibres and are used

especially for leathers which feel thin or empty.

– G –

Gelatin. An organic colloidal substance made from animal bones, skins or hide

fragments.

Glossary of terms

152

Grain. Indicates the outer or hair side of hide or skin in cases where it is split into two

or more thicknesses, or to unsplit skins which are finished on the grain side.

Grain layer. The top layer of the corium including the hair follicles.

Green fleshing. Fleshing in the raw state.

Green hides. Hides which have not been salted, dried, or otherwise cured for

preservation.

– H –

Hair side. The side of the skin or hide on which the hair grows; the grain side.

Hair slip. Slipping or loosening of the hair in hides or skins due to putrefaction.

Halotolerant bacteria. Class of bacteria that do not require salt (NaCl) for growth but

can tolerate salt and are capable of growth in 20% salt environment. Halotolerant

bacteria are frequently equated with halophiles, a group of archaeobacteria that require

salt for growth.

Handle. Sensation or feeling of certain physical properties of leather, such as flexibility

and smoothness, which can be perceived by touch with fingers and hands.

Hide. The outer covering of a mature, fully grown animal of the larger kind (e.g. cattle,

horses, buffaloes, camels, elephants).

– L –

Leather. The hide or skin of an animal which has been treated chemically so as to

make it a non-putrescible substance impervious to and insoluble in water.

Chapter 11

153

Loose and pipey grain. Grain layer which is loosely attached to the underlying main

corium layer and forms folds or wrinkles when the leather is bent grain inwards. Often

caused by staleness and can also be caused by mechanical processes.

– P –

Paddle. Semi-cylindrical vessel (of metallic, wood, concrete or plastic materials) fitted

with a revolving paddle wheel for keeping skins and liquors in motion. This type of

vessel could be used in different beamhouse processes such as soaking, liming, and

rinsing (see also vat).

Pelt. A hide or skin, usually when raw, with the hair or wool left on. Most frequently

used to designate the skins of fur bearing animals.

Pipey. Leather whose grain layer separates away from the corium creating a void

between them.

Preservative. An agent used to prevent decay, decomposition, putrefaction, etc.

Preserve. To treat something in such a way as to protect it against harmful influences.

Putrefaction. Hides and skins damaged and rendered evil-smelling by bacterial

damage.

– R–

Raceway brining. Raceway, such as a tank shaped like a racecourse, in which brine

solution and hides are moved around by a paddle, for brine curing of hides.

Raw stock. The hides and skins used for making leather, as referred to in a completely

untanned state.

Rawhide. The usual American name for cattle hide that has been dehaired and limed.

Glossary of terms

154

Red heat. Red colouration found on the flesh side of salted hides after storage. Caused

by salt tolerant (halophilic) bacteria that are aerobic so they stay on the surface of the

hide. Long term storage of hides with red heat can lead to pitting of the surface.

– S–

Salometer. An instrument for measuring the weight of a salt solution per unit volume

and thus giving a measure of its salt content. Saturation equals to 100°SAL.

Salting. Any treatment of hides and skins with a salt for preservation.

Salt diffusion. Penetration of salt into the fibrous tissue of the hide or skin.

Salt uptake. Amount of salt taken up, or absorbed, when hides or skins are treated with

salt.

Saturated brine. Saturated solution of sodium chloride; used for brining hides.

Shoe upper leather. A shoe leather used for the upper portions. Predominantly from

cattle hides and calfskins, although a high variety of skins are used.

Short term curing. Treatment of hides and skins by a method which will preserve

them for a few days only.

Shoulder. The part of the hide that is bounded by the face and cheeks on the top, the

butt on the bottom, and the two bellies on the side.

Skin. A general term of the outer covering of an animal. The raw skins of a mature

fully-grown animal of the smaller kinds (e.g. sheep, goat, pigs).

Sodium chloride. Colourless crystalline compound, NaCl, occurring naturally as halite

and in sea water; common salt. Sodium chloride is used in great quantities for the

conservation of raw hides and skins and in leather making (pickling).

Chapter 11

155

Surfactant. Substance introduced into a liquid to alter (usually to increase) its

spreading, wetting and similar properties (particularly properties depending on surface

tension); can cause foaming and hinder biological activity.

– T –

Tanner. People whose job is to convert animal hides or skins into leather by any

process.

Tannery. An establishment where hides or skins are converted into crust, wet tanned

or finished leather.

Tanning. The processing of perishable rawhides and skins by the use of tanning

materials, into the durable and permanent form of leather.

– U –

Unsaturated brine. Solution of water and salt (brine) that has, at 25 oC, a specific

gravity under 1,198 or contains less than 36,0 g of salt per 100,0 g of water.

Upholstery leather. A general term for leathers used for furniture, airplanes, buses and

automobiles. The staple raw material consists of spready cattle hides, split at least once

and in many cases two or three times. The top or grain cuts go into the higher grades

and the splits into the cheaper.

– V –

Vat. Water-tight vessel, of wood, brick, concrete, etc., usually above ground level,

for storing liquids, preparing solutions, giving liquid treatments, etc.

Vat curing. Method of curing in which hides are laid one by one in a pit and covered

by salt, the pit being finally filled with brine.

Veininess. A prominent vein pattern in hides which becomes visible in the finished

leather, often due to poor bleeding which encourages bacterial growth.

Glossary of terms

156

Veins. Tube through which blood circulates in an animal body. When visible in

leather the cause is usually poor bleeding or staleness.

Veiny. Leather in which the pattern of the blood vessels is visible, or unusually

prominent, on the grain or flesh side, usually through use of stale hides or skins.

CHAPTER 12

Resum en català

Chapter 12

158

12. RESUM EN CATALÀ La recerca presentada a la present dissertació doctoral va ser desenvolupada en la seva

totalitat al Departament d’Agricultura dels Estats Units (USDA), Eastern Regional

Research Service (ERRC) a Wyndmoor, Pennsilvània.

Durant la meva estada al ERRC des del Juliol de 2005 fins a l’Octubre de 2008, se’m

van assignar dos projectes CRIS (Current Research Information System). Els principals

objectius de cada projecte es detallen a sota. Més informació pot ser trobada a la pàgina

web de l’ERRC (http://cris.csrees.usda.gov/).

1. Nous i eficaços processos en la producció de cuir de qualitat

Projecte Nº: 1935-41440-013-00D

Científics en cap: Dr. William N. Marmer i Dr. Cheng-Kung Liu

Objectius: Desenvolupar noves tecnologies per la preparació de pells per l’adobatge.

Establir els processos de secat i acabament així com mètodes no destructius amb la

finalitat de millorar la qualitat i durabilitat de la pell. Preparació de la pell: fons

addicionals destinats a la recerca i optimització de la preservació de pells amb salmorra.

2. Tecnologies sostenibles pel processat de pells, cuirs, llana i subproductes

associats

Projecte Nº: 1935-41440-014-00D

Científics en cap: Dr. Eleanor M. Brown

Objectius: 1. Modificació funcional de pells i subproductes de la pell: desenvolupar una

fundació en l’ús de noves tecnologies químiques i bioquímiques (a) en la producció de

cuirs d’alta qualitat lliures de crom; (b) expandir les aplicacions de subproductes

proteics de la indústria pelletera en el camp dels biomaterials. 2. Modificació funcional

Resum en català

159

de la llana: modificar la llana per tal de millorar-ne la funcionalitat i expandir-ne el seu

camp d’aplicacions.

1. Nous i eficaços processos en la producció de cuir de qualitat

En un món globalitzat, és cada cop més comú trobar articles fabricats a l’estranger amb

matèries primeres provinents de tercers països. En el cas particular del mercat de les

pells i cuirs als Estats Units d’Amèrica, més de la meitat de les pells provinents dels

escorxadors americans són exportades a països com Xina, Corea, Taiwan o Mèxic.

Prèviament a la seva exportació, les pells crues han de ser sotmeses a un procés de

preservació o conservació.

L’objectiu de la preservació és prevenir de forma temporal el deteriorament de les pells

des del moment que són extretes de l’animal fins que són convertides en un producte

que ja no és putrescible (Bailey, 2003). La putrefacció de la pell és deguda als enzims

proteolítics produïts per bactèries, microorganismes unicel·lulars que es multipliquen

molt ràpidament quan les condicions són favorables. La presència de menjar (les

proteïnes de la pell) i aigua, junt amb les condicions adequades de pH (neutral) i

temperatura (entre 20 i 37 ºC), fa que la pell crua sigui un substrat idoni per al ràpid

creixement bacterià, i per tant per a la putrefacció de la pell. Una pell que no ha estat

preservada de forma apropiada tindrà com a conseqüència l’obtenció d’un producte

acabat (cuir) de baixa qualitat.

La preservació de pells crues amb clorur de sodi (NaCl) rep el nom de curat i és el

mètode més tradicional i econòmic. A Europa i als Estats Units, el curat es duu a terme

en uns tancs de gran capacitat omplerts amb una solució quasi saturada de clorur de sodi

(salmorra). Les pells es fan circular en els tancs durant un mínim de 18 hores, després

de les quals són retirades, escorregudes i apilonades. En el curat es dóna un doble

procés de difusió; per una banda, el clorur de sodi migra cap a l’interior de la pell,

mentre que per l’altra, part de l’aigua continguda inicialment a la pell es desorbeix i

passa a formar part de la solució de salmorra continguda en el tanc. Típicament, l’aigua

és el component majoritari de la pell crua (60-70%), i el contingut de sals minerals és

només d’un 0.5-1% (Sharphouse, 1971). Després del procés de curat, el percentatge

d’aigua disminueix fins a un 45-50%, i el de sals minerals incrementat fins a un 10-

Chapter 12

160

15%. Mitjançant aquest parell de valors, hom pot trobar el nivell de saturació de sal de

la pell.

100359.01

%%

% ×��

��

�⋅��

��

�=

AiguaSal

Saturació

O’Flaherty va assegurar el 1953 que una pell estava adequadament preservada quan el

contingut d’aigua remanent a la pell després del curat era com a màxim del 50%, i amb

un grau mínim de saturació de sal del 70%. Més endavant, la organització americana

U.S. Hide, Skin & Leather Association va assegurar que el nivell de saturació mínim de

l’aigua remanent a la pell després del curat havia de ser igual o superior al 85%. Tot i

així, a data d’avui no s’ha publicat cap article que estudiï exhaustivament el grau de

saturació mínim que es necessita assolir per tal de garantir la correcta preservació de les

pells, ni l’efecte que les condicions d’emmagatzematge de les pells (temperatura, temps,

humitat...) hi exerceixen.

El procés de curat té dues variants. Una, anomenada green fleshing, en la qual les pells

son descarnades primer i curades posteriorment. El descarnat consisteix en eliminar

gran part del teixit adipós de la pell, la qual cosa provoca una absorció més ràpida de la

sal durant el curat. L’altra opció és curar les pells sense haver estat descarnades

prèviament, la qual cosa facilita l’eliminació del fang i fems adherits al pèl de l’animal,

i també un menor ús d’aigua en comparació al green fleshing.

La preservació de pells crues amb salmorra presenta una sèrie d’avantatges, la més

important de les quals és el relatiu baix cost de la matèria primera emprada (1 kg de

clorur de sodi val aproximadament 11 cèntims de dòlar). Tot i així, el preu del clorur de

sodi s’ha vist incrementat en un 10-15% durant els darrers anys (Ed Godsalve, 2007).

L’àcid bòric (Kanagaraj et al., 2005), el clorur de potassi (Bailey i Gosselin, 1996), el

formaldehid (Sharphouse i Kinweri, 1978) o el gel de sílica (Kanagaraj et al., 2001;

Munz, 2007) són exemples de substàncies que han estat assajades com a agents

conservants de pells crues, però el seu cost, sensiblement superior al del clorur de sodi,

ha provocat que no s’apliquessin a nivell industrial. A part de la vessant econòmica, el

curat de pells amb NaCl és una tecnologia segura, que no requereix amplis

coneixements ni una preparació complicada, i que permet processar varis milers de pells

Resum en català

161

diàries. Com qualsevol altre procés, el curat de pells amb clorur de sodi presenta alguns

inconvenients. Un d’ells és la pol·lució de l’aigua, ja que la sal absorbida durant el

procés de curat és en gran part abocada als rius en el procés de remullat de les pells.

L’elevada salinitat dels efluents pot acabar afectant la salinitat del sòl provocant una

reducció del rendiment de les collites (Daniels, 1998). Des del punt de vista

operacional, el fet que es tractin tantes pells al dia i que el procés sigui discontinu, pot

provocar que hi hagi pells que siguin retirades del tanc abans de les 18 hores establertes,

i per tant amb un grau de curat insuficient.

Tot i que el procés de curat ha estat modelitzat en substrats tals com el formatge

(Turhan i Kaletunç, 1992) o la carn (Bertram et al., 2005), mai no ho ha estat en el cas

de les pells crues. Per tal de buscar les condicions òptimes del procés de curat, tals com

la concentració de salmorra o el volum de bany, es va desenvolupar un model

matemàtic que descriu la difusió del clorur de sodi a la pell durant el procés de curat. El

model proposat s’anomena de reacció contínua i assumeix que el clorur de sodi forma

un gradient de concentració no estacionari dins la pell a mesura que s’hi va difonent

(Figura 12.1).

Figura 12.1 – Model matemàtic del procés de curat d’una pell crua (veure notació en el capítol 10).

La difusió de sal a la pell es va caracteritzar pel coeficient de transport �, el valor del

qual es va trobar de l’ordre de 10-5 s-1. Els resultats obtinguts eren del mateix ordre de

magnitud que els trobats en el model matemàtic del remullat (Blaha i Kolomazník,

1988), la qual cosa va suggerir que la difusió del clorur de sodi no diferia gaire entre les

operacions inverses de curat i remullat.

Chapter 12

162

Mitjançant el model es va esbrinar que els valors més alts de � s’obtenien al utilitzar una

salmorra inicialment saturada (35.9 g/100 ml aigua) així com un mínim de 500% de

volum de bany. Els resultats obtinguts corroboraren una norma generalment acceptada

que estipula que es necessiten un mínim de 5 kg de salmorra per cada kg de pell crua

per tal d’assolir un bon nivell de preservació (Bailey, 2003). El model també revelà que

quan s’empraven salmorres diluïdes de 20 ó 25 g NaCl/100 ml aigua, les pells no rebien

un bon nivell de curat, independentment del volum de bany aplicat o el temps. El

model també va evidenciar que els resultats obtinguts depenien altament del nivell de

saturació de sal que havia d’assolir la pell, que actualment està establert al 85%. Es pot

concloure que el model matemàtic presentat pot ser utilitzat per tal d’estimar la quantitat

de sal, volum de bany i temps que es necessiten per preservar eficaçment pells crues

sota unes condicions d’operació donades.

Un altre objectiu important del projecte d’investigació era trobar maneres d’accelerar

l’absorció de clorur de sodi a la pell crua durant el procés de curat. Assolint aquest

objectiu, el temps de processat en els banys es veuria reduït i per tant es crearia una

capacitat addicional de processat.

En primer lloc, es va dur a terme un estudi estratigràfic que permetés fer un seguiment

de les quantitats d’aigua i clorur de sodi a la pell durant el procés de curat. Es va

observar que la sal entrava a la pell majoritàriament per la cara de la carn, mentre que

l’aigua es desorbia per totes dues cares, amb l’epidermis actuant com una membrana

semipermeable. L’epidermis, en altres paraules, era permeable a l’aigua però no al

clorur de sodi. Aquestes observacions van ser corroborades pels resultats obtinguts amb

microscopia epifluorescent i microscopia d’escaneig electrònic.

Les variables susceptibles de ser modificades en el procés de curat són les següents:

concentració de salmorra, volum de bany, temps, acció mecànica, temperatura i

utilització d’additius. Donat que les tres primeres variables ja havien estat estudiades i

optimitzades en el model anteriorment presentat i que l’efecte de l’acció mecànica i

temperatura no eren de l’interès de la indústria, es va decidir optar per l’estudi

d’utilització d’additius en el procés de curat.

Resum en català

163

Tal i com s’ha apuntat prèviament, la presència de teixit adipós adherit al costat de la

carn de la pell es va demostrar que disminuïa la velocitat de penetració de la sal (Stuart i

Frey, 1940). Tenint en compte la relació directa entre la quantitat de greix a la pell i la

quantitat de sal absorbida, es va decidir emprar tres desengreixants comercials així com

un tensioactiu glucolipídic experimental (soforolípid, SL), desenvolupat als laboratoris

de l’ERRC, com a additius del procés de curat. Els desengreixadors comercials estaven

compostos per una barreja de tensioactius no iònics de la família dels etoxilats

d’alcohol. Els desengreixadors seleccionats eren comercialitzats per a la seva utilització

en la indústria d’adobatge de pells, tot i que eren concebuts per a altres operacions com

ara el remullat o encalcinat de les pells. Les propietats més destacables dels SL són la

seva biodegradabilitat, nul·la ecotoxicitat i baixa escumositat. Els SL estan actualment

emprant-se en la indústria cosmètica i també com a ingredient actiu dels sabons per a

rentavaixelles (Solaiman, 2005). A més, el cost d’obtenció d’aquests productes és

relativament baix, d’entre $1 i $3/kg (Sun et al., 2004). Un dels majors inconvenients

que presenten els SL és la seva limitada solubilitat, tot i que s’està duent a terme una

recerca extensiva per tal d’augmentar i millorar la funcionalitat i aplicabilitat d’aquests

productes.

Un dels tres desengreixadors comercials potenciava significativament l’absorció de sal,

al mateix temps que disminuïa la quantitat de greix a la pell. Els assajos amb el SL van

demostrar que si era emprat per sobre del límit de solubilitat, la capacitat

desengreixadora era comparable a la dels desengreixadors comercials. La implantació

dels SL en el els processos de producció de cuir no s’ha dut a la pràctica encara, però els

esperançadors resultats aquí presentats així com les seves propietats antimicrobials el

converteixen en un candidat molt prometedor. Malauradament, la seva limitada

solubilitat fa que més recerca sigui necessària per tal d’obtenir un producte que pugui

competir amb els que estan actualment al mercat.

Chapter 12

164

2. Tecnologies sostenibles pel processat de pells, cuirs, llana i subproductes

associats

La presència de venes i d’àrees buides i sense cos en el cuir acabat és un dels problemes

més importants que els adobadors han de fer front avui en dia. En cas que el cuir

presenti venes prominents o àrees buides, l’àrea aprofitable es pot veure notablement

reduïda, així com el benefici econòmic de l’adobador. Per tant, la millora del cuir que

presenta aquests defectes és una bona oportunitat per augmentar el valor del producte

final.

Una vena es pot comparar a un tub que tan pot estar ple com buit de sang i que està

localitzat a una zona profunda o superficial de la dermis. Tot i que al principi es creia

que estava causada per un dessagnament deficient de l’animal (Orthmann i Higby,

1929), posteriors teories aposten també per altres motius com l’edat, dieta o raça de

l’animal. Normalment, els flancs i coll de l’animal són les àrees més susceptibles de

patir aquest tipus de problema.

Un altre dels defectes més recurrents que apareix algunes vegades en el cuir acabat és la

mala qualitat de flor, caracteritzada per la formació d’arrugues gruixudes a la superfície

del cuir quan aquest es torça. Un bon toc de flor està caracteritzat per la formació d’un

gran nombre d’ arrugues fines, molt juntes les unes a les altres, i que resulten més

plaents a la vista (Figura 12.2). Un exemple gràfic d’aquesta característica seria el plec

que es forma a la part de davant de les sabates de pell al caminar. Típicament, l’àrea

dels flancs i el coll acostumen a tenir una qualitat de flor pitjor que d’altres àrees de

l’animal (esquena, part posterior de l’animal).

Figura 12.2 – Mostres de cuir amb un toc de flor (a) gruixut i (b) fi.

a b

Resum en català

165

Un mètode extensament aplicat per solventar o minimitzar els esmentats problemes és

mitjançant l’aplicació d’una substància anomenada filler o reblidor. L’objectiu dels

reblidors és donar més cos i substància al cuir tot reduint-ne la buidor i disminuint-ne la

prominència de venes. Per tal d’aconseguir aquest propòsit, els reblidors s’introdueixen

en els buits que existeixen entre les fibres del cuir.

La naturalesa dels reblidors ha canviat durant els darrers anys. Inicialment, s’empraven

extractes de tanins vegetals, compostos de bari o glucosa (Harris, 1974). Més

recentment, altres substàncies com polímers i resines han estat utilitzades com a

reblidors. L’augment de preu dels productes derivats del petroli juntament amb una

major conscienciació mediambiental dels adobadors va provocar que les teneries

busquessin altres fonts per a la fabricació de reblidors. Si fos possible emprar

subproductes d’altres processos o indústries, no només es solucionaria el problema dels

cuirs de baixa qualitat, sinó que també s’estaria donant valor afegit a aquests

subproductes.

Un dels primers intents en aquesta línia de recerca va ser emprant gelatina, un

subproducte de la indústria d’adobatge de pells (Chen et al., 2001). Es va demostrar

que la gelatina, prèviament reticulada amb glutaraldehid, podia ésser utilitzada de forma

efectiva com a reblidor de cuirs. La citotoxicitat del glutaraldehid i l’elevat cost de la

gelatina ($5.7/kg) va reconduir la recerca cap a la búsqueda d’altres fonts renovables de

proteïna i agents reticulants no tòxics. L’elevat preu de la caseïna ($12.8/kg), que havia

estat una de les proteïnes més àmpliament emprades en les teneries americanes,

desaconsellava seguir aquesta línia. Finalment, es va optar per una de les proteïnes de

rebuig més econòmiques en el mercat americà actual, com és el sèrum de proteïna i els

seus derivats, que al seu torn són subproductes de la indústria del formatge. El preu de

mercat d’aquests productes depèn en gran part de la concentració de proteïna, i van des

de $0.66/kg pel sèrum de proteïna (aproximadament un 15% en pes de proteïna) fins als

$2.30/kg pel concentrat de sèrum de proteïna, WPI per les seves sigles en anglès (al

voltant d’un 90% en pes de proteïna).

Als laboratoris de l’ERRC es va treballar durant els darrers anys en la modificació de

subproductes proteics amb transglutaminassa microbiana (mTGase), un enzim que

catalitza la reacció entre un grup �-amino d’un residu de lisina i un grup �-carboxiamida

Chapter 12

166

d’un residu de glutamina, obtenint un entrecreuament (crosslinking) covalent de les

proteïnes, que pot ser inter o intramolecular (Figura 12.3).

Figura 12.3 – Reacció de crosslinking entre molècules proteiques amb l’enzim mTGase

S’ha demostrat que l’actuació d’aquest enzim sobre proteïnes de naturalesa molt diversa

provoca l’obtenció d’estructures polimèriques amb propietats reològiques i funcionals

modificades, la qual cosa permet ampliar els seus camps d’aplicació. La gelatina, tant

la comercial com la obtinguda a partir de residus sòlids de les teneries, el sèrum de

proteïna i els seus concentrats, i la caseïna, així com barreges binàries d’aquestes

substàncies, van ser reaccionades de forma efectiva amb l’enzim mTGase, i els seus

productes de reacció van ser aplicats amb èxit com a reblidors (Taylor et al., 2004;

2005; 2006a; 2006b; 2007). A més, els reblidors, un cop adherits al cuir, no n’eren

despresos en etapes posteriors de processat. Finalment, les propietats mecàniques del

cuir acabat no van ser modificades sensiblement pels diferents reblidors estudiats

(Taylor et al., 2008).

En els casos estudiats, la solució proteica estava formada per un component majoritari

dels considerats cars (gelatina, caseïna) i un de minoritari més econòmic (sèrum de

proteïna, WPI). Per tal de continuar aquesta línia de recerca i aconseguir produir un

bioproducte més econòmic, es va estudiar l’efecte d’afegir una quantitat petita de

gelatina al més econòmic WPI, i reaccionar-los amb mTGase. Es va trobar que

mitjançant l’addició d’una petita quantitat de gelatina al WPI i posterior reacció amb

l’enzim, s’obtenien uns productes les propietats físiques dels quals (força de gel,

viscositat, mòdul elàstic) eren millorades respecte a si la gelatina o WPI eren

Resum en català

167

reaccionats amb l’enzim de forma individual (Figura 12.4). També es va concloure que

feia falta disposar d’un ambient reductor per tal que els grups reactius de les dues

majors proteïnes constituents del WPI, �-lactoglobulina i �-lactalbúmina, amagats a

causa de la geometria esfèrica de les molècules, fossin susceptibles de reaccionar amb

l’enzim mTGase.

Un cop caracteritzats els nous biopolímers es va procedir a estudiar-ne la seva

aplicabilitat com a reblidors de pells de baixa qualitat. En la present tesi, es va estudiar

l’eficàcia d’aquests productes tant en cuirs per tapisseria (d’un gruix que oscil·la entre

1.0 i 1.2 mm) com per calçat (gruix entre 2.0 i 2.4 mm), ambdues adobades amb sals de

crom (wet blue). Es van introduir dues importants diferències respecte als estudis

realitzats prèviament a l’ERRC, amb l’objectiu d’optimitzar el procés des del punt de

vista econòmic. Per una banda, no es va afegir mTGase en la preparació de les

solucions proteiques; així, es retallava el cost de matèries a emprar i també s’evitava

que el producte esdevingués massa viscós abans de ser utilitzat. Per l’altra banda, es va

decidir dur a terme el procés de rebliment amb un volum de bany del 200%, la meitat

0,0

20,0

40,0

60,0

80,0

0 0,5 1 1,5 2 2,5 3% gelatina (w /w )

For

ça d

e ge

l (g)

Control

Test

0,0

20,0

40,0

60,0

0 0,5 1 1,5 2 2,5 3% gelatina (w /w )

Vis

cosi

tat (

cP)

Control

Test

0,1

1

10

100

1000

10000

0 2 4 6 8 10 12 14 16 18 20Temps (h)

Mòd

ul e

làstic G

' (Pa)

Test

Control

0,0

20,0

40,0

60,0

80,0

0 0,5 1 1,5 2 2,5 3% gelatina (w /w )

For

ça d

e ge

l (g)

Control

Test

0,0

20,0

40,0

60,0

0 0,5 1 1,5 2 2,5 3% gelatina (w /w )

Vis

cosi

tat (

cP)

Control

Test

0,1

1

10

100

1000

10000

0 2 4 6 8 10 12 14 16 18 20Temps (h)

Mòd

ul e

làstic G

' (Pa)

Test

Control

a a

b

c

Figura 12.4 – (a) Força de gel, (b) viscositat i (c) mòdul elàstic (G’) d’una barreja proteica composta per 10% (w/w) de WPI i diferents quantitats de gelatina, en un ambient reductor (1% DTT, respecte el pes de WPI). Control: 0 U mTGase/g proteïna; Test: 2 U mTGase/g proteïna. Nota: els valors de viscositat dels tests que contenien un 1.5% o més de gelatina excedien el límit del rang de mesura del viscosímetre (1,800 cP). El test del gràfic c correspon a una barreja de 10% (w/w) WPI + 3% (w/w) gelatina, amb 2 U mTGase/g proteïna, en un ambient reductor (1% DTT, respecte el pes de WPI).

Chapter 12

168

del que havia estat aplicat típicament. Finalment, també es va analitzar l’efecte que el

pretractament del wet blue amb diferents quantitats de mTGase exercia en la qualitat

final del cuir.

L’estudi va concloure que l’aparença general del cuir, tant el destinat a tapisseria com el

destinat a calçat, fou significativament millorada després del tractament amb l’agent de

rebliment, obtenint un cuir més ple, de color més intens i un toc de flor més plaent a la

vista i al tacte. Tot i que aquestes propietats són subjectives i van ser avaluades per dos

adobadors experts, la millor qualitat del cuir que havia estat tractat respecte el que no ho

havia estat era evident fins i tot per a persones aleatòries sense coneixements específics

en aquesta matèria.

El seguiment de l’absorció de proteïna durant l’operació de rebliment va concloure

tendències inverses pel casos del wet blue per tapisseria o per calçat. El coeficient

d’absorció del wet blue per tapisseria pretractat amb una solució de mTGase era quatre

vegades superior al del que no ho havia estat; en canvi, el coeficient d’absorció del wet

blue per calçat que no havia estat pretractat amb mTGase era quasi el doble del d’aquell

que sí que havia estat pretractat amb la solució de l’enzim. Independentment de

l’enzim, un cop les proteïnes eren absorbides pel wet blue, el percentatge desprès en les

posteriors etapes de rentat era poc significatiu (entre un 0 i un 6%).

L’absorció de proteïnes pel wet blue també es podia visualitzar mitjançant microscopia

epifluorescent (Figura 12.5). En aquest cas, les diferents proteïnes van ser complexades

amb uns colorants que fluorescien a una determinada longitud d’ona. Aquesta tècnica,

tot i ser només qualitativa, permetia localitzar les proteïnes de rebliment en el sí del wet

blue i també avaluar la seva presència després del procés de rentat.

Resum en català

169

Figura 12.5 – Imatges de microsopia epifluorescent del wet blue després d’haver estat tractat amb una solució etiquetada de WPI i gelatina. La fluorescència del complex WPI-colorant fluorescent s’aprecia amb un intens color vermellós (imatge superior) i la de la gelatina-colorant fluorescent adopta un color verd intens (imatge inferior). La flor del wet blue es troba a la part superior de les imatges.

Addicionalment, es va demostrar que un volum de bany del 200% era suficient per

aconseguir que les proteïnes fossin absorbides pel wet blue. És molt probable que si

aquesta tecnologia acaba essent implantada a la indústria, sigui factible dur a terme

aquesta operació en un volum de bany fins i tot inferior al 200%, a causa d’una major

acció mecànica. També es important destacar que les propietats mecàniques dels cuirs

sotmesos al procés de rebliment, tot i ser una mica més rígids, no es van veure

significativament afectats.

Chapter 12

170

Referències

Bailey, D.G., Gosselin, J.A. (1996). The preservation of animal hides and skins with

potassium chloride. Journal of the American Leather Chemists’ Association

91(12): 317-333.

Bailey, D.G. (2003). The preservation of hides and skins. Journal of the American


Bertram, H.C., Holdsworth, S.J., Whittaker, A.K., Andersen, H.J. (2005). Salt diffusion

and distribution in meat studied by 23Na nuclear magnetic resonance imaging

and relaxometry. Journal of Agricultural and Food Chemistry 53: 7814-7818.

Blaha, A., Kolomazník, K. (1988). Mathematical model of soaking. Part I. Journal of

the Society of Leather Technologists & Chemists 73: 136-140.

Chen, W., Cooke, P.H., DiMaio, G.L., Taylor, M.M., Brown, E.M. (2001). Modified

collagen hydrolysate, potential for use as a filler for leather. Journal of the


Daniels, R. Overview: Avoiding salts. World Leather, May 1998, pp. 66-70.

Godsalve, E. Personal communication, WE CO., 1991, Inc. Research Liaison

Committee, May 1st 2007, USDA Eastern Regional Research Center,

Wyndmoor, PA.

Harris, D. (1974). Dictionary of terms used in the hides, skins and leather trade, USDA,

Foreign Agricultural Service, Agricultural Handbook #465.

Kanagaraj, J., Chandra Babu, N.K., Sadulla, S., Suseela Rajkumar, G., Visalakshi V.,

Chandra Kumar, N. (2001). Cleaner techniques for the preservation of raw goat

skins. Journal of Cleaner Production 9(3): 261-268.

Kanagaraj, J., John Sundar, V., Muralidharan, C., Sadulla, S. (2005). Alternatives to

sodium chloride in prevention of skin protein degradation – a case study.

Journal of Cleaner Production 13(8): 825-831.

Munz, K.H. (2007). Silicates for raw hide curing. Journal of the American Leather

Chemists’ Association 102(1):16-21.

O’Flaherty, F. (1959). Curing study part 1: Moisture to ash relationship. Leather and

Shoes 138: 31-34.

Orthmann, A.C., Higby, W.M. (1929). The cause of vein like protuberances on finished

leather. Journal of the American Leather Chemists’ Association 24 (12): 654-

656.

Resum en català

171

Sharphouse, J.C. (1971). Types of hides and skins and principal uses. In Leather

Technician’s Handbook (Leather Producer’s Association eds.) Northampton,

UK, pp. 20-36.

Sharphouse, J.H., Kinweri, G. (1978). Preservation-formaldehyde of raw hides and

skins. Journal of the Society of Leather Trade Chemists. 62: 119-123.

Solaiman, D.K.Y. (2005). Applications of microbial biosurfactants. Inform 16: 408-410.

Stuart, L.S., Frey, R.W. (1940). Effect of adipose tissue fat on the green-salting of

heavy hides. Journal of the American Leather Chemists’ Association 35: 414-

418.

Sun, X.X., Choi, J.K., Kim, E.K. (2004). A preliminary study on the mechanism of

harmful algal bloom mitigation by use of sophorolipid treatment. Journal of

Experimental Marine Biology and Ecology 304: 35-49.

Taylor, M.M., Marmer, W.N., Brown, E.M. (2004). Molecular weight distribution and


gelatins. Journal of the American Leather Chemists’ Association 99(3): 129-141.

Taylor, M.M., Marmer, W.N., Brown, E.M. (2005). Characterization of biopolymers

prepared from gelatin and sodium caseinate for potential use in leather

processing. Journal of the American Leather Chemists’ Association 100(4): 149-

159.

Taylor, M.M., Bumanlag, L., Marmer, W.N., Brown, E.M. (2006a). Use of

enzymatically modified gelatin and casein as fillers in leather processing.

Journal of the American Leather Chemists’ Association 101(5): 169-178.

Taylor, M.M., Marmer, W.N., Brown, E.M. (2006b). Preparation and characterization

of biopolymers derived from enzymatically modified gelatin and whey. Journal


Taylor, M.M., Marmer, W.N., Brown, E.M. (2007). Evaluation of polymers prepared

from gelatin and casein or whey as potential fillers. Journal of the American


Taylor, M.M., Marmer, W.N., Brown, E.M. (2008). Effect of fillers prepared from

enzymatically modified proteins on mechanical properties of leather. Journal of


Trade Practices for Proper Packer Cattlehide Delivery, 3rd ed., Leather Industries of


Chapter 12

172

Turhan, M., Kaletunç, G. (1992). Modeling of salt diffusion in white cheese during long

term brining. Journal of Food Science 57: 1082-1085.

Ph.D Thesis - Improvement of Conventional Leather Making Processes

Technology

Transcript of Ph.D Thesis - Improvement of Conventional Leather Making Processes