WHEY PROTEIN ISOLATE: A POTENTIAL FILLERFOR THE LEATHER INDUSTRY

I))

EIMJARI) HNRNANDEZ BALADA, MARYANN M. TAYLOR, ELEANOR M. BROWN, CIIEN;-KCN; LiuU. S. Department ofAgriculture, Agricultural Research Service

Eastern Rcc'ionai Research ('enter600 E,sr MERMAID LANE, WYNo1ooR, PA 19038 USA

EDI;ARD HERNANDEZ B..\I\D,\

Department of(hemicaI Engineering, University of BarcelonaMAR11 I FRANQIJS 1, 08028 BARCELONA, SPAIN

JAUME COT6onsejo Superior de Inuestigaciones Gent/fleas (('S'IG

Research and Development ('enter, Ecotechnologies DepartmentJORDI GIRONA 18-26, 08034 BARCELONA, SPAIN

122

ABS TRACt

The upgrading of leather that presents loose areasand poor grain break is one of the most valueadding Opportunities for a tanner. Typically,petroleum-based products are used to improve thefinal appearance and feel of crust leather. In thisstudy, we demonstrate that blends composed ofwhey protein isolate (WPI), a byproduct of theCheese industry, and small amounts of gelatin, abyproduct of the leather industry, could beeffectively used as fill jug agents for both shoeLipper and upholstery leather. Wet blue leatherfrom three different areas in the hide (butt, bellyand neck) was treated with the WPI-gelatin blend,retanned, colored and fatliquored, and theirsubjective and mechanical properties evaluated.The effect of pretreatment of the wet blue sanipleswith various concentrations of the enzyme microbialtransglutaminase (mTGase) was also examined. Itwas found that the rate of uptake of the WPI-gelatinblend by upholstery wet blue increased four-foldwhen it was pretreated with a 2.5% mTGasesolution. Conversely, this rate was decreased whenshoe upper was pretreated with increasing amountsof inTGase. The subjective properties (e.g. handle,fullness, color and grain break) of both shoe upperand upholstery leather that were treated with theWPI-gelatin blend were significantly improvedover the controls. Importantly, the grain break ofthe belly area of samples that were pretreated withenzyme (both upholstery and shoe upper) was

remarkably improved. Hence, fillers mainlycomposed by the less expensive WPI weredemonstrated to be effective filling agents for bothupholstery and shoe upper leather.

RESUM EN

La mejorIa en l)ieles que presentan iireas vacIas ypoca lirmeza de for es uno de los mayores desafIosclue tiene el curtidor para afiadirles valor.TIpicamente, materias primas derivadas del petróleoson uti I izadas para mejorar el aspecto final y tactode la piel. Ell presente estudio, demostramosque mezclas compuestas por suero concentrado deproteina (WPI, de sus siglas en inglés), LinsubprodLlcto de la industria del qLleso, Junto conpequeñas cantidades de gelatina, tin subproducto deIa indListria del cuero, se puedcn utilizar de formaefectiva como agentes de relleno para cueros detapicerIa y de empeine (calzado). Diferentes zonasde pieles curtidas al cromo (croupOn, falda y cuello)fueron tratadas con la mezcla de WPI-gelatina,recurtidas, teñidas y engrasadas y las muestrasevaluadas con respecto a las propiedades subjetivasy mecanicas. El efecto del pretratamiento de la pielcon varias concentraciones de la enzimatransglutaniinasa microbial (mTGase) fiie tambiénexaminado. Se halló clue la velocidad de absorciónde la mezcla de WPI-gelatina por pilrte del cuero detapiceria incrementaba cuatro veces cuando erapretratado con una solucidn de 2.5% mTGase. Encambio, esta velocidad disminula cuando el cuero

Corresponding Author e-mail address: maryann.taylor@ars.usda.gov"Mention of trade names or commercial products in this article is solely for the purpose of providing specific

information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.Manuscript received September 8, 2008, accepted for publication September II, 2008

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WPI Fiii

de empeine era pi-etratado coil crecientesde mTGase. Las propiedades subjetivas (toque,Ilenura, tirmeza de Ilor y color) de las pieles detapicerIa y de empeine que fueron tratadas coilmezcla de \VPI -gelati na mejolaron sensi blementeen comparacion a los controles. En particular. Iatirmeza de for dc las muestras de la falda quefueron pretratadas con la enzima (ambos tapiceria yempei ne) fue niejoracla notablemente. Porconsiguiente, agentes de relleno compuestos por larnãs económica WPI fueron eficazmentecornprobados de increnientar ci rendimiento, tantopara cueros de tapiccrIa como de empeine.

INTRODUCTION

The presence of loose areas with poor grain break in finishedleather is one of many concerns that tanners are facing intoday's leather processing. This problem becomes particularlysignificant III neck and belly areas of the hide with thebelly area exhibiting a looser break.' Fillers are materialsused to till the interstices of the leather and make thelooseness less pronounced which in turn should improvecutting yields.

Over the last several years, this laboratory has exploredalternatives to petroleum-based fillers. Chen et al.2demonstrated that collagen hydrolysate crosslinked withglutaraldeliyde could he suitable for filling low qualityleather. More recent research has focused oil use ofgelatin polymerized by the action of microbialtransglutaniinase (mTGase) (EC 2.3.2.13), an enzymecapable of forming crosslinks in a wide variety of proteins.Both commercial and experimental alkali-extracted gelatinswere effectively crossl inked with mTGase, yielding productswith improved functional properties. This enzyme alsoproved to be effective in the crosslinking of gelatin withsodium caseinate, a dairy industry byproduct.' Enzymaticallymodified gelatin and casein were successfully applied asfillers in wet blue leather. It was later found that thesemodified proteins were not removed during the washingprocess. Nevertheless, the relatively elevated cost of gelatinarid casein encouraged the search for a cheaper source ofrenewable proteins. Whey and whey protein isolate (WPI),byproducts of the cheese manLifacturing industry, fulfill thatcondition and were also effectively reacted with mTGaseyielding viable products for use as filling agents.9'°

We recently demonstrated that the addition of small amountsof gelatin to whey protein isolate (WPI) in the presence ofmTGase arid the reducing agent dithiothreitol (DTT) yieldednovel products with improved physical properties (e.g.,viscosity, gel strength, degree of polymerization) over eitherprotein component.'' The main goal of that study was toobtain biopolymers with unique properties at low cost,hence using WPI as the majority component of theWPI-gelatin blend.

In the present study. we examine the suitability ofbiopolymersproduced by combining WPI with small amounts ofcommercial low Bloom gelatin as a tilling agent for shoeLipper or upholstery leather. The effectiveness of the varioustreatments was assessed by measuring the physical,mechanical and subjective properties of crust leather fromthree different areas of the hide (butt, belly and neck).

EXPERIMENTAL

MaterialsMicrobial transgl utaminase. Activa TG-TI (approximately100 units/(, ), a commercial mTGase formulation containing99% maltodextrose as a carrier, with an active range of p1-14.0 to 9.0 at 0 to 70°C, was obtained from Ajinomoto USAInc. (Paramus, NJ) and used without further purification.Type B gelatin, alkaline extracted from bovine skin, andcharacterized in this laboratory as I I 5 g Bloom, was obtainedfrom Sigma (St. Louis, MO). WPI, Alacen 895, containing93.2% protein (manufacturer's data), was generously suppliedby NZMP (formerly New Zealand Milk Products; Lemoyne,PA). Dithiothreitol (DT'T) 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 fromthe former Pilar River Plate Corp. (Newark, NJ); HavanaDye (Derma Havana R Powder) was obtained from ClariantCorporation (Charlotte. NC); Altasol-CAM, Altasol 310-Land Eureka 400R were obtained from Atlas Refinery. Inc.(Newark, NJ). Basyntan NNOL and Basyntan 1)LE wereobtained from BAS F Corporation (Clirlotte,NC). Upholsteryand shoe upper wet blue leather was obtained from commercialtanneries. All other chemicals were reagent grade and usedas received.

Preparation of W1't-Gelatin BlendsOne day prior to the treatment, the required amounts of WPIand gelatin powders were suspended in water (200% float),mixed well and allowed to sit at room temperature for at leasttwo h. The amount of protein powder was calculated on thebasis of the weight of wet blue. Next, a 10% D'VF (w/v)solution was prepared and the volume necessary to give aconcentration of 10 mg DTT per g of WPI was added. ThePH was then adjusted to 7.5 with I N NaOH or I N HCI andheated at 38 °C for one h, cooled to room temperature andstored overnight at 4 'C. It is worth noting that all theproteinaceous blends discussed in the present paper wereprepared without adding mTGase to the mixture. By doingthis, the WPI-gelatin blend can be stored for an extendedperiod of time without danger of it becoming apermanent gel.

Application of WPI-Gelatin Blends to Wet Blue LeatherWet blue samples from the butt, belly, and neck weretumbled with water in a Dose drum (Model PFI 300-34.Dose Maschineiibau GmnbH, Lichtenau, Germany) for 30min at 50°C, drained and refloated (200% float, 50°C) in 417c

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sodium bicarbonate solution, based oil wet blue weight.Upon pH stabilization, the float was drained, mTGasesolution (2.5 1/c or 5 mTGase ) with a 200/c float was addedand samples were drummed for one h at room tenperature(22 ± 40 C). Note that the mTGase concentrations statedthroLighout the manLtscript are mTGase+carrierconcentration.The float was drained and a WPI-gelatin solution was added(200 1Yc float). Control pieces to which no enzyme or proteinmixture was added were also run with a 200 float of water.The samples were tumbled for one h at RT and then lot- liveh at 45°C. The floats were then drained and the sampleswashed twice for 10 rnin at 50°C (200% float), drained.patted dried and stored at 4°C. Previous work carried out inour laboratory typically employed a 400% float in all theabove mentioned processing steps ,bb0,12 By cutting back theIloat to a half we reduce the consunipt ion of water and alsoincrease the concentration of protein in the solution, whichultimately leads to a higher concentration gradient betweenthe solution and the leather.

Aliquots of 3 nil were extracted from the drum after varyingtime intervals throughout the treatment of the samples withthe ml'Gase or the WPI-gelatin solutions. AliqLtots of waterafter each wash were also collected to estimate the amount ofprotein that was removed from the wet blue. One drop of '5%sodium azide solution was added to each aliquot and theywere stored at4°C until needed to run the protein determinationassay. The following day, both treated and untreated sampleswere weighed and the amount of reagents needed for theRetail -Color-Fatliquor (RCF) calculated.

Retan/Color/Fatliquor (RCF)Control and test samples were retanned, colored andfatliquored in separate drums. Shoe upper and upholsterysamples followed slightly different procedures (Figure Iaand lb. respectively).

Figure la

Retan/Color• 150% float @ 30°C

'Add 2% Trutan PA-65 (20 mitt 30°C)

'Add 4% PRP. 77 (30 mm @ 30°C)

'Add 4% 400R (5 mm § 60°C)

'Add 6% Havana Dye (60 mm @ 60°C)

'Add 1% formic acid and tumble until dye is exhausted, @ 60°C

• Batch wash x 3(200% float @ 60°C, 10 mm)

Fatliquor• Refloat in 150% float @ 60°C

'Add 10% Altasol CAM and 2% Eureka 400R (60 ruin @ 60°C)

• Add 1.5% formic acid (target pH 3.0-3.5)• Drain and wash for 5 mm

Toggle dry, mill for 24 h, store for 48 h constant temp. & RHMechanical Properties & Subjective Evaluation

Figure lb

Retan ICoIor

• 75% float @ 43 'C

• Add 10% IDLE Syntan 2% Basyntan NNDL (5 mm @ 43 'C)

• Add 1% Havana dye in 25% float @60 "C (45 mum @ 60 C)

• Add 05% formic acid and tumble until dries exhausted, @60 C

• Batch wash x 3)100% flout (at '5 C I'J mum)

Fattiquor• Refloat ri foat © 55 C

• Add 5% Altasol CAM, 1% Eureka 400R

• Run 30 min © 55 C

'Add Altasol 310L in 10% water@ 55C

• Run 10 min @ 55 C

Haul, horse overnight, met Out, toggle dry, store for 48 h con slant temp. S RHMechanical Properties S Subjective Evaluation

Figure I: Flow diagram for retan. color and fatliquor fornittlationof (a) upholstery and (b) shoe upper wet blue.

DryingAfter RCF, samples were removed from the drum andallowed to dry. Shoe upper crust leather was allowed to hangfreely,whereas stretching (toggl i ng) was applied to upholsterycrust leather. Once dry, the leathers were conditioned, putinto plastic bags for one day and then staked twice. Shoeupper crust samples were not milled upholstery crustsamples were milled for about 24 11. All samples were thenkept on a shelf in the conditioning rooni at 20°C and 65%relative humidity for at least three days.

Mechanical PropertiesTensile strength, Young modulus and tear strength weredetermined as described in a previous publication .12

Subjective EvaluationExperimental and control crust leathers were assessed forhandle, fullness, grain tightness (break), color and generalappearance by hand and visual examination. Handle isdellned as the sensation or feeling of certain physicalproperties of leather, such as flexibility and smoothness,which can be perceived by touch with lingers and hands.Fullness refers to the way a 1001) of leather feels in the palmof the hand when compressed. A full leather fills the palmwhile a flat leather has more of a cardboard effect. Grainbreak is the pattern of tiny wrinkles formed when the leatheris bent grain inward. Leather was rated oil scale of I to 5for each functional property by two experienced tanners,where higher numbers indicate a better property.

Protein Concentration DeterminationProtein concentrations in the float, at different stages of thetreatment, were determined using the bicinchoninic acid(BCA) assay" according to the directions supplied with thekit. Samples were centrifuged at 13,400 rpm for 30 mill in amicrocentrifuge (Eppendorf MiniSpin plLtS, Westbury, NY).

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Time (mm)

0 1 2 3 4 5 6

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WPI Fil - LER

One ml of protein supernatant was removed and typically a1:25 (v/v) dilution was prepared in order to fall within thelinear concentration range for the assay (200 to 1000 (,,/mlprotein). A 50 11 aliquot of the diluted solution was mixedwith 1.0 ml of RCA reagent and incubated at 37°C for 30minutes. The absorbance of a sample solution at 562 nniminus a reagent blank was compared with a standard curveusing known concentrations of bovine serum albumin.

RESULTS AND DiscussioN

Shoe Upper Wet BlueWe first investigated the uptake of mTGasc and WPI-gelatinblends by shoe upper wet blue at both 2.5% and 5% mTGascconcentration levels. Similar trends for the uptake of mTGasewere obtained for samples that were pretreated with 2.5 or5% mTGase. In both cases, the curve leveled off after only20 minutes and the bath was not exhausted after one hour ofdrtimming (Figure 2a). After draining the mTGase solution.a blend of 5% WPI and 0.5% gelatin, with respect to weightof wet blue, was added and drummed one hour at RTfollowed by 5 h at 45°C. A protein Uptake of 98% wasachieved with wet blue that was not treated with mTGase,whereas wet blue pretreated with 2.5% and 5% mTGasereduced the percentage to 86% and 83%, respectively(Figure 2b).

The absorption of the protein by the wet blue follows firstorder reaction kinetics. Hence,

[A]In = —ic • t

^

[A]()

where [A] and [A] 0 are the protein concentration remainingin the drum at time r and t = 0, respectively and k is theuptake rate coefficient. Table I shows k and the correlationcoefficient values for the uptake of WPI-gelatin by shoeupper wet blue and shoe upper wet blue pretreated with 2.517,or 5% mlGase. The most rapid absorption, reflected by thehighest value of k, was obtained for the wet blue that was notpretreated with enzyme (k = 0.608 h'), followed by the onepretreated with 2.5% (k = 0.413 h') and 5% mTGase(k = 0.323 h'), respectively.

The wet blue was washed twice immediately after drainingthe proteinaceous solution. No detectable level of proteinwas found ill of wet blue pretreated with 5% mTGase.At 2.5% and 0% mTGase there was a protein removal ofapproximately 6% and 8%, respectively, and approximately75% of that protein was washed out in the first wash. Part ofthat washed out protein could he due to the unbound proteinadhered to the hide or to the insufficient draining of the drumbefore the addition of water.

All crust samples were evaluated with respect to handle,fullness, grain tightness (break) and color. The samples wererated oil scale of I to 5, with 1 being the worst and 5 beingthe best. Front ratings, all rating in which the

Figure 2a

60

506-

404J

30'B

E 10

0

Figure 21)

100

80

'B

'B4-J

20

Time (h)

Figure 2: (a) niTGzrse arid (h) pIotei Fr uptake Profi le ,, by shoe upper wetblue pretreated with a solution containing 0. 2.5 or 5 17o nilGase andtreated with a solution of 5% WPI + 0.5 17o gelatin. All percentageswere calculated with respect to the weight of wet blue and added ina 200% lIoat.

grain break was weighted more than the other ratings wasalso presented. Table 11 reports the results on the abovementioned subjective properties of shoe upper wet bluesubjected to treatments A, B or C. Values that were equal toor better than controls are underlined. In all treatments, thetest pieces were found to be equal to or superior to thecontrol pieces. Only the handle of the leather pretreated with2.5% mTGase was rated slightly lower than the control.Focusing oil grain break, it is important to note that thewet blue samples used for the 0% and 2.5% mTGase batcheshad a good break before the treatment while the 5% mTGasewet blue samples exhibited a poor break. The samples thatalready exhibited a good break showed neither a significantimprovement in break nor any detrimental effect. The 5%

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TABLE I

Uptake Rate Coefficient k for Various Treatments

Treatment Wet blue % mTGase' % WPFIGelatin k (h1 ) R2

A Shoe upper 0 5 0.5 0.608 0.994

B Shoe upper 15 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 15 2.5 0.25 1.377 0.956

"Percentages calculated with respect to weight of wet blue.

TABLE H

Property

HandleFullnessBreak

Color

Overall

Handle

Fullness

BreakColor

Overall

Handle

Fullness

Break

Color

Overall

Subjective EvaIuationTreatment A Treatment B

Control Test Control Test

4

5

4

55

5

S

3

5

3

5

4

5

4

5

3

4

3

7

3

5

4

5

4

5

55

3

Sn 5

4

5

3

4

3

7

4

5

4

5

5

5

57

5

7 5

3

4

1.5

4.5

Hide area

Litt

Belly

Neck

Treatment C

Control Test

4

4.5

1.5

3.54

4

3

4

2

4.5

2

4.5

4

2.5

3.5

1.5

4.5

3.5

4.5

4

4.5

2.5

4

3

3.5

2.5

4.5

e 1-s, I=worst, =best

bn=2CA , B and C stand for treatments of shoe upper with a solution containing 5% WPI + 0.5% gelatin andpretreated with a solution of 0, 2.5 or 5% mlGase, respectively. All percentages were calculated withrespect to the weight of wet blue and added in a 200% float.

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0 1 2 3 4 5 6

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WPI Fiui

mTGase wet blue sample clearly showed significantimprovement in leather from all areas of the hide whencomparing 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 thatexhibited a poor break. Approximately 80% of the proteinwas taken up by the vet blue, with a rate coefficient rate ofk = 0.262 It 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 thecontrol, the improvement was not as dramatic as when a 517cniTGase treatment followed by 5%WPI + 0.5% gelatin wasused (data not shown). These results suggest that 5% WPI +0.5% gelatin filled the leather better than 2.5% WPI + 0.5%gelatin, particularly ]it belly area.

Upholstery Wet BlueThe ability of WPI and gelatin to 1111 and improve upholsterywet blue was examined. Given the smaller thickness ofupholstery (1.0-1.2 mm) compared to shoe upper (2.0-2.4mm), a lower concentration of WPI and gelatin was selectedto make up the proteinaceoLls blend (2.5% WPI + 0.25%gelatin). The effect of allenzymatic pretreatment of thesamples with 2.5% mTGase prior to the addition of theproteinaceous blend was also evaluated. About half theamount of the enzyme was picked up by the wet blue withinthe first 30 minutes, and the curve leveled off thereafter(Figure 3a). A complete uptake of protein was reachedwithin the first 3 h of tumbling for wet blue that waspretreated with 2.5% mTGase. Conversely, the proteinuptake trend for samples that were not pretreated with theenzyme leveled off at approximately 90% after 4 h ofdrumming (Figure 3b). A remarkably faster uptake ofprotein was observed for samples that were pretreated withmTGase, as can be seen from the uptake coefficient values(Table I). Approximately 5 17o of the protein was removed inthe first wash regardless of the enzymatic pretreatment. Anon detectable amount of protein was removed duringsubsequent washes.

The treatment of upholstery wet blue with 2.5% WPI +0.25 11'o gelatin considerably improved the handle, fullness,and color of the resulting crust leather. Most importantly,the break of the belly and butt areas was significantlyimproved when the wet blue was pretreated with 2.5%mTGase prior to the addition of the proteinaceous blend(Figure 4).

Mechanical PropertiesLeathers from three different areas of the hide, neck, bellyand butt, were tested for mechanical properties. This reportwill present the test results from belly area only, the primaryarea of concern. The three areas demonstrated the sametendency towards the change of two major variables: percentWPI and percent mTGase. A 3-D regression plot of theresultant tensile strength as a function of percent WPI and

Figure 3a

60

40

3

rr 20

10

0

Figure 3h

120

100

80III

11

60

40

i20

0

Time (h)

Figure 3: (a) in I'Gasc and (h) protein uptake profiles by upholsterywet blue pretreated with a solution containing 0 or 2.57v rnTGaseand treated with a solution of 25 17c WPI + 0.25 17c. All percentageswere calculated with respect to the weight of wet blue and added ina 200% float.

percent mTGase simultaneously for both upholstery andshoe upper leather samples was developed (Figure 5). Thetensile strength decreases slightly with increasing percentWPI for both upholstery and shoe upper leather. However,for upholstery leather, the tensile strength increasessignificantly with percent mnTGase, whereas for shoe upperleather, the tensile strength shows little change with percentmTGase. It is worthy to note that as demonstrated in Figure5, shoe upper leather hasgreater tensile strength thanupholstery leather and this is ascribable to the fact that shoeupper leather is thicker and has more fiber network to resistthe fracture.

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WPI FILLER

Figure 6

Handle- Fullness

BreakColorOverall

128

Figure 4

5

4

3

2

0

5

4

3

2

0Control

Trotrr,o',t E

.1-il-Treatment D Treatment E

Handle- Fullness

Breakil Color

Overall

Handle- Fullness

BreakEtJ Color

Overall

Fi g ure 4: Subjective properties of upholstery crust leather. D and Fstand for treatments of upholstery with a solution coutai ti iii g 2.5 7 WPI.- 0.25; gelatin and pretreated with a solution of 0 or 2.5 ittTGase,respectively. All percentages were calculated with respect to theweight of wet blue and added in a 200 11e float.

Figure 5

Figure 5: Effect of the various treatments of leather with WPI andmTGase oil tensile strength of (a) upholstery and (b) shoe uppercrust leather. The regression graphic corresponds to samples from thebelly area.

Fi gure 6: Effect of the various treatments of leather with WPI andm F( iase oil Young's modulus of (a) upholstery and (b) shoeupper crust leather. The regression graphic corresponds to samplesfrom the belly area.

Figure 7

Figure 7: Effect of the various treatments of leather with WPI andmTGase oil tear strength of (a) upholstery and (h) shoe upper crustleather. The regression graphic corresponds to samples from thebelly area.

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Young's Modulus is a value indicating the stiffness ofleather. Youn g 's modLilus of upholstery leather increasessignificantly with both percent WPI and percent niTGase(Figure 6a). On the other hand, for shoe upper leatherYoung's modulus also increases significantly with percentmTGase, but changes very little with percent WPI (Figure6b). Looking closely at Figure 6, one can notice that theranges of Young's modulus values are higher for shoe upperleather than upholstery leather. Besides the fact that shoeleather is thicker, this variation could he due to the use ofdifferent types of fatliquors.

Tear strength decreases with both percent WPI and percentmlGase for upholstery leather (Figure 7a). However, forshoe upper leather the tear strength responds quite differentlyto the changes of percent mTGase and percent WPI. Asdemonstrated in Figure 7b, tear strength of shoe upperleather decreases pronouncedly with percent rnTGase but isrelatively unchanged with percent WPI.

CONCLUSIONS

Current prices of sodium casemate ($5.8/lb)'4 and gelatin($2.6/lb)'5 emphasized the need for research into cheapersources of protein to generate fillers for leather. The lessexpensive whey protein isolate ($1 .05/lb)' 4 along with smallamounts of gelatin were successfully applied as a tillingagent for upholstery and shoe upper leather. Subjectiveproperties such as fullness, handle and color of the resultingcrust leather were significantly improved. Furthermore,grain break for upholstery and shoe tipper leather faredmarkedly better when samples were pretreated with niTGase.The enzymatic pretreatment of wet blue leather with inTGasealso affected the protein uptake ratio. The uptake coefficientfor upholstery leather pretreated with 2.5% mTGase increasedfour-fold over samples that were not enzyme pretreated. Thetrend was reversed for shoe upper leather with a drop in theuptake coefficient value from 0.608 h-' to 0.323 h' forsamples that were not pretreated with inTGase or pretreatedwith 5% mTGase, respectively. We further demonstratedthat a 200% float satisfactorily enabled the proteins to betaken up by the wet blue. If this technology is to betransferred to the industry, use of a shorter float could befeasible due to a stronger mechanical action. Importantly,the proteins are not considerably removed by washing,regardless of the enzymatic pretreatment. Another advantagepresented herein is that the proteinaceous blend was added tothe drum without any enzyme pretreatment, thus itspreparation becomes more convenient and feasible. Eventhough the various treatments did not negatively affect themechanical properties of the crust leather, tilled sampleswere a little stiffer and presented slight lower tear strengththan the controls. Further research that explores thepossibility of using even cheaper sources of protein as a rawmaterial for bio-based leather products is an interestingoption currently being examined in our laboratory.

ACKNOWLEDGEMENTS

The authors would like to thank Lorelie Bumanlag, Gary DiMaio, Rafael Garcia, Nicholas Latona, Renée Latona and JoeLee for their technical support and assistance.

REFERENCES

I. Tancous, J. J., Roddy, W. T., and OFlaherty, F.; Defectsdue to natural characteristics of the skin or hide. In: Skin,Hide and Leather Defects (The Western Hills PublishingCompany), pp. 2-18. 1959.

2. Chen, W., Cooke, P. H., DiMaio, G. L., Taylor, M. M.,and Brown. E. M.; Modified colla gen fiydrolysate. potentialfor use as a filler for leather. JALCA 96, 262-267, 2001.

3. Taylor, M. M., Caheza, L. F., Marmer, W. N.. and Brown,E. M.: Enzymatic modification of hydrolysis productsfrom collagen using a microbial transglutaminase. I.Physical properties. JALCA 96, 319-332, 2001.

4. Taylor, M. M., Liii, C. K., Latona, N. P., Mariner, W. N.,and Brown, E. M.; Enzymatic modification of hydrolysisproducts from collagen using a microbial transghutaminase.II. Preparation of films. JALCA 97, 225-234, 2002.

5. Taylor, M. M., Liu, C. K., Marmer, W. N., and Brown, E.M., Enzymatic modification of hydrolysis products fromcollagen using a microbial trans g lutaminase . 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 ofenzymatically modified commercial and experimentalgelatins. JALCA 99, 129-141, 2004.

7. Taylor, M. M., Marmer, W. N., and Brown. E. M.;Characterization of biopolyniers prepared from gelatinand sodium caseinate for potential use in leather processing.JALCA 100, 149-159,2005.

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