UTILIZATION OF FISH MARKET WASTE FOR PREPARATION OF SILAGE … · With regard to the thesis...

UTILIZATION OF FISH MARKET WASTE FOR PREPARATION

OF SILAGE

THESIS

Submitted to the

Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli

In partial fulfillment of the requirements for the degree of

MASTER OF FISHERIES SCIENCE

IN

FISH PROCESSING TECHNOLOGY

BY

NIKHIL DILIP PALKAR

B. F. Sc.

Under the guidance of

Dr. J. M. Koli

Associate professor,

Department of Fish Processing

Technology & Microbiology

College of Fisheries, Shirgaon, Ratnagiri - 415 629

(Maharashtra state, India)

May, 2017

UTILIZATION OF FISH MARKET WASTE FOR PREPARATION

OF SILAGE

THESIS

Submitted to the

Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli

In partial fulfillment of the requirements for the degree of

MASTER OF FISHERIES SCIENCE

IN

FISH PROCESSING TECHNOLOGY

BY

NIKHIL DILIP PALKAR

B. F. Sc.

Approved by the Advisory committee

Chairman and Research Guide: Dr. J. M. Koli




Members

: Dr. S. B. Patange Professor, Department of Fish Processing

Technology & Microbiology.

: Shri. S. T. Sharangdhar




: Dr. R. K. Sadawarte


Department of Fisheries

Engineering and Technology

Date:

Place: Ratnagiri

Dr. BALASAHEB SAWANT KONKAN KRISHI VIDYAPEETH, DAPOLI

COLLEGE OF FISHERIES, RATNAGIRI

(Certificate to be submitted by the supervisor of the candidate supplicating for

M. F. Sc. (F.PTM) degree along with thesis)

With regard to the thesis entitled “Utilization of fish market waste for

preparation of silage” submitted by Mr. Nikhil Dilip Palkar for the degree of this

university.

I certify that:

1. He has carried out research work under my direct supervision and guidance

during the academic year 2015-2017 and that the manuscript of the

dissertation has been scrutinized by me.

2. The entire thesis comprises the candidate’s own work and it is his own

achievement. It has not previously formed the basis for the award of any

degree, diploma, associate-ship, fellowship or other similar title of

recognition.

3. The thesis does not contain any conjoint research work with me or anyone

else.

4. He has completed his research work to my entire satisfaction.

5. The final typed copy of the thesis, which is being submitted to the University

office, has been carefully read by me for its material and language and it is to

my entire satisfaction.

Dr. J. M. Koli

Chairman (SAC)

Associate professor, Department of Fish Processing


Date:

Place: Ratnagiri

CANDIDATE’S DECLARATION

I hereby declare that the thesis entitled “Utilization of fish market waste for

preparation of silage” is an authentic record of the research work done by me and

that no part thereof has been submitted by me or any other person to any other

University or institute for a degree or diploma, associate-ship, fellowship or other

similar title.

Date:

Place: Ratnagiri (Mr. Nikhil Dilip Palkar)

DEDICATED

TO Aai- Baba

AND

Dr. J. M. Koli sir

ACKNOWLEDGEMENTS

I would like to express a deep sense of gratitude and thanks to authorities of Dr.

Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli for granting me the permission to pursue

my post graduate studies and providing me all the necessary facilities at College of fisheries,

Ratnagiri. I, wish to record my sincere thanks to Dr. Tapas Bhattacharya Hon’ble, Vice

Chancellor, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli for the same.

I feel gratifying to express my respectful gratitude to Dr. R. K. Pai, Associate Dean,

College of Fisheries, Ratnagiri for providing me all the necessary facilities and for giving needful

support during my post graduate studies.

I wish to express my appreciations and deep sense of gratitude to beloved teacher and

research guide Dr. J. M. Koli, Associate Professor (Department of Fish Processing Technology and

Microbiology), College of Fisheries, Ratnagiri for giving me invaluable and scholarly guidance

with constant encouragement throughout the period of the research work, for critically going

through the manuscripts of this thesis and making many constructive improvements. His altruistic

view and astute perception have been highly instrumental in making this stupendous work a

success. I hold the deepest respect and thank him for creating interest in field of research and

hunger for knowledge in me.

It is my pleasure to express my deep sense of gratitude to Dr. V. R. Joshi Professor and

I/C. Head, Department of Fish Processing Technology and Microbiology. I must gratefully

acknowledge the ever willing and sincere help offered by advisory committee members, Dr. S. B.

Patange, Professor (F.PTM), shri. S. T. Sharangdhar, Assistant professor (F.PTM) and Dr. R. K.

Sadawarte, Associate Professor (F.ENGG) for his, constant inspirations of this talented advice,

gracious encouragement, appreciable and constructive criticism right from the suggestion of this

investigation till the completion of this manuscript.

I take this opportunity to express my sincere regards to shri. A. S. Desai, Assistant

professor (F.PTM), Shri. V. V. Vishwasrao, Assistant professor (F.PTM) and Shri. S. S. Sawant,

Assistant professor (F.PTM) for their invaluable advice, encouragement and ever willing help to

carry out the research work.

I wish to thank Dr. A. S. Mohite, Professor and Head (F. ENGG.) and official in charge

for ARIS cell, for providing me the internet facilities. I wish to express my deep sense of gratitude

to Dr. S. D. Naik Professor and Head of the (F. Biology), Dr R. K. Pai, Professor and Head

(Department of Aquaculture), Dr. M. M. Shirdhankar, Professor and Head (Department of

F.RESE), and Dr. S. T. Indulkar, Professor and Head (Department of Hydrography) for providing

me the necessary facilities during the research work.

My gratefulness is due to Mrs. Manisha Sawant, Assistant Librarian and Mr. Mangesh

Chopade for going out of their way in providing me all the books and references needed in my

work. I am also thankful to Mrs. Meenal Kale and Mrs. Prajwala Sawant for their support and

guidance in lab operations. I am obliged to various academic and non-academic staff of the College

of Fisheries Shirgaon, Ratnagiri for their kind corporation in all field and special thanks to

Talekar kaka, Mahesh kaka, Manoj, Tejas, Ram dada, Ghadshi dada, Pinky tai, Madhavi, Salvi

madam for help during my work.

I wish to express thanks to my seniors Ajay Sonavne, Ranjeet dada, Rohini Mugale,

Akshay Akhade, Bahar Mahakal, Sushil Kamble, Amit Kokate, Raghini Bharankar, Rasika

Sawant, Sanna Shah, Bhavesh Gaikwad for their suggestions and kind help during this research

work.

My special appereciation goes to my batch mates and friends Dipali, Mrunali, Pradnya,

Priyanka, Jagruti, Madhu, Mayuri, Shital, Akash, Vishal, Gaurav, Utkarsh, Digambar,

Sudharshan, Akshay, Ketan, Kishan, Malhari, Sagar, Rutwij and Jayesh who were always there

when I really needed. Thank you doesn’t seem sufficient but it is said with appreciation and

respect to both of them for their support, encouragement, care, understanding and precious

friendship.

It is my duty to honestly thank to my juniors Sonal, Tejaswita, Neha, Supria, Praras,

Gurudev, Neeraj, Rameshwar, Nikhil, Amol, Shekhar , Pratik, Vikrant, for their co-operation and

creation of friendly environment.

I express my heartfelt regards for the invaluable help of my seniors for their inestimable

help during the entire period of thesis. It is my duty to honestly thank to all my B. F. Sc., M. F.

Sc. and D. F. Sc. juniors who intermingled with me easily and became my good friends. I will

never able to forget their ever-smiling faces and caring nature.

And finally, I am indebted to the constant encouragement, love and affection given all the

time by my father Mr. Dilip Govind Palkar, My mother Mrs. Dipali Dilip Palkar, dear uncle Mr.

Pradip Palkar, Mr. Rajendra Palkar and aunty Mrs. Pallavi Palkar, Mrs. Sharvari Palkar and

dear brother Pratik and sister Mugdha and all my family members who gave me constant

sustenance and motivation during my research career. I am really lucky to have their whole hearted

help and support during the entire course of my study period. Without their co-operation it would

have been practically impossible for me to complete my master degree. I am very much thankful to

god Ganesha for blessing me with such loving and caring family members, relatives, Friends,

teachers and research guide.

Date: (Mr. Nikhil Dilip Palkar)

Place Ratnagiri

CONTENTS

LIST OF TABLES

II-IV

LIST OF FIGURES

V-VI

LIST OF FLOW CHARTS

VII

LIST OF PLATES

VIII

ABBRIVIATIONS

IX

ABSTRACT

X-XI

1. INTRODUCTION

1-5

2. REVIEW OF LITERATURE

6-25

3. MATERIAL AND METHODS

26-43

4. RESULTS

44-89

5. DISCUSSION

90-104

6. SUMMARY

105-107

7.REFERENCES

108-116

LIST OF TABLES

Table No. Particulars Page No.

4.1 Proximate composition of Fish waste 45

4.2 Biochemical and microbiological analysis of fish waste. 45

4.3 Proximate composition of Rice bran 45

4.4 pH Changes in different treatment of sulphuric acid silage

during storage

49

4.5 Two-way ANOVA for pH changes in different treatment of

Sulphuric acid silage

49

4.6 pH Changes in different treatment of Formic acid silage

during storage

50

4.7 Two-way ANOVA for pH Changes in different treatment of

Formic acid silage

50

4.8 SNK Test for pH Changes in different treatment of Formic

acid silage

50

4.9 pH changes in different treatment of Biological silage during

storage.

51

4.10 Two-way ANOVA for pH changes in different treatment of

Biological silage

51

4.11 SNK Test for pH changes in different treatment of Biological

silage.

51

4.12 AAN (mg-N100g-1)

changes in different treatments of

sulphuric acid silage

55

4.13 Two-way ANOVA for AAN changes in different treatments of


55

4.14 SNK Test for AAN changes in different treatment of sulphuric

acid silage

55

4.15 AAN (mg-N100g-1

) changes in different treatments of formic

acid silage

56

4.16 Two-way ANOVA for AAN changes in different treatments

of formic acid silage

56

4.17 SNK Test for AAN changes in different treatments of formic

acid silage

56

4.18 AAN (mg-N100g-1)changes in different treatment of

biological silage

57

4.19 Two-way ANOVA for AAN changes in different treatment of

biological silage

57

4.20 SNK Test for AAN changes in different treatment of

biological silage

57

4.21 TVB-N (mg-N100g-1

) changes in different treatment of


62

4.22 Two-way ANOVA for TVB-N changes in different treatment

of Sulphuric acid silage

62

4.23 SNK Test for TVB-N changes in different treatment of

Sulphuric acid silage

62

4.24 TVB-N (mg-N100g-1

) Changes in different treatment of

Formic acid silage.

63

4.25 Two-way ANOVA for TVB-N Changes in different

treatment of Formic acid silage

63

4.26 SNK Test for TVB-N Changes in different treatment of

Formic acid silage

63

4.27 TVB-N (mg-N100g-1

) Changes in different treatment of

Biological silage

64

4.28 Two-way ANOVA for TVB-N changes in different treatment

of Biological silage

64

4.29 SNK Test for TVB-N Changes in different treatment of

Biological silage

64

4.30 TPC (cfu/g) changes in different treatment of Biological

silage

68

4.31 Two-way ANOVA for TPC changes in different treatment of

Biological silage

68

4.32 SNK Test for TPC changes in different treatment of

Biological silage.

68

4.33 LAB Changes in different treatment of Biological silage 69

4.34 Two-way ANOVA for LAB Changes in different treatment

of Biological silage

69

4.35 SNK Test for LAB Changes in different treatment of

Biological silage

69

4.36 Proximate composition of the different types of Fish silages

after 60 days.

72

4.36a One-way ANOVA for protein content of different methods of

silages.

72

4.36b SNK test for protein content of different methods of silages. 72

4.37 Drying rate of fish silage powder containing different

concentration of rice bran.

76

4.38 Two-way ANOVA for Drying rate of fish silage powder

containing different concentration of rice bran.

76

4.39 SNK Test for Drying rate of fish silage powder containing

different concentration of rice bran.

77

4.40 Proximate composition of powder fish silage made with

different quantity of rice bran.

79

4.40.1a One-way ANOVA for protein content of powder fish silage

made with different quantity of rice bran

80

4.40.1b SNK for protein content of powder fish silage made with

different quantity of rice bran

80

4.40.2a One way ANOVA for fat content of powder fish silage made

with different quantity of rice bran

81

4.40.2.b SNK for fat content of powder fish silage made with different

quantity of rice bran

81

4.40.3a One way ANOVA for moisture content powder fish silage


82

4.40.3b SNK for moisture content of powder fish silage made with


82

4.40.4a One-way ANOVA for Ash content of powder fish silage


83

4.40.4b SNK for ash content of powder fish silage made with


83

4.41 Biochemical and microbial changes in 30 % Rice bran

packed fish silage powder during storage.

88

LIST OF FIGURES

Figure No. Particulars Page No.

4.1 Proximate composition of Fish waste 46

4.2 Proximate composition of Rice bran 46

4.3 pH Changes in different treatment of Sulphuric acid silage

during storage

52

4.4 pH Changes in different treatment of Formic acid silage

during storage.

52

4.5 pH Changes in different treatment of Biological silage

during storage

53

4.6 AAN changes in different treatment of Sulphuric acid

silage during storage

58

4.7 AAN changes in different treatment of Formic acid silage

during storage.

58

4.8 AAN changes in different treatments of Biological silage

during storage

59

4.9 TVB-N changes in different treatment of Sulphuric acid


65

4.10 TVB-N changes in different treatment of Formic acid


65

4.11 TVB-N changes in different treatment of Biological silage

during storage

66

4.12 TPC changes in different treatment of Biological silage

during storage

70

4.13 LAB changes in different treatment of Biological silage

during storage

70

4.14 Proximate composition of the different types of Fish

silages after 60 days.

74

4.15 Drying rate of fish silage powder containing different


78

4.16 Proximate composition of powder fish silage made with


79

4.17 Protein content of powder fish silage made with different 84


4.18 Lipid content of powder fish silage made with different


84

4.19 Moisture content of powder fish silage made with different


85

4.20 Ash content of powder fish silage made with different


85

4.21 Biochemical and microbial changes in 30 % Rice bran

packed powder silage during storage.

89

LIST OF FLOW CHARTS

Flow Chart

No

Particulars Page No.

3.1 Method for preparation of acid silage

(Mousavi et al., 2013)

39

3.2 Method for preparation of Biological fish silage

(Palekar , 2009)

40-41

3.3 Method for preparation of fish silage powder

(Hossain and Alam, 2015)

42-43

LIST OF PLATES

Plate No. Particulars

Plate 1 Fish market waste and viscera

Plate 2 Sulphuric acid silage with weight percentages of 2.5, 3.5 and 4.5 %

Plate 3 Formic acid silage with weight percentages of 2.5, 3.5 and 4.5 %

Plate 4 Biological silage with weight percentages of 5, 10 and 15 % molasses

Plate 5 Solar tunnel Dryer

Plate 6 Drying of semidry fish silage

Plate 7 Powder fish silage made with different quantity of rice bran

Plate 8 Powder fish silage with 30% rice bran

Plate 9 Packed and Stored 30 % rice bran powder silage

ABBREVIATIONS

AAN : Alpha Amino Nitrogen

ANOVA : Analysis of Variance

AOAC : Association of Official Analytical Chemist

cfu : Colony forming unit

CMFRI : Central Marine fisheries research institutes

CS : Curd Silage

DOF : Department of Fisheries

FAS : Formic Acid Silage

FPC : Fish Protein Concentrate

LAB : Lactic Acid Bacteria

NSD : Not Significantly Difference

SD : Significantly Difference

SNK : Student’s Newman Keul test

TN : Total Nitrogen

TPC : Total Plate Count

ABSTRACT

In the present study an attempt was made to transformation of fish market

waste into silage by using three different methods viz. Inorganic (98% sulphuric acid

with weight percentage of 2.5, 3.5 and 4.5%), organic (98% formic acid with weight

percentage of 2.5, 3.5 and 4.5%) and biological method (molasses with weight

percentage of 5, 10, 15% and curd used as lactic acid bacteria source for

fermentation).The chemical, microbiological and nutritional properties of the

differently preserved fish silages were estimated during a storage period of 60 days at

ambient temperature. The important findings are summarised as: the chemical

analysis of fish market waste was observed on wet basis to be moisture 77.09 ± 0.14

%, crude protein 15.20 ± 0.15 %, lipid 4.03 ± 0.07 % and ash 3.30± 0.11 %. Rice bran

was used as co-drying material for drying liquid silage. The proximate composition of

rice bran contained moisture, crude protein, fat and ash as 9.45 ± 0.19 %, 16.05 ± 0.08

%, 13.42 ± 0.15 % and 10.44 ± 0.14 % respectively on dry weight basis. The rate of

pH, AAN and TVB-N were gradually increasing trends and then stable. In case of

biological silage 10% and 15 % molasses, pH was decreased from 6.56 to 4.45 and

6.75 to 4.14 at the end of 6th

day. pH was decreased below 4.5 after 72 hours in both

silages. The rate of autolysis in sulphuric acid silage was slow compared to formic

acid and biological silage. In case of bio-fermented silage (15% molasses) a steady

supply of nutrients from molasses showed a steady increase in LAB from 2.24×106 to

3.67×109 cfu/g and decrease in TPC from 4.50×10

6 to 8.20×10

3 cfu/g. In present

study, Sulphuric acid 2.5%, Formic acid 2.5%, biological silage 5 and 10% silages

were corrupted at the end of 24, 24, 12 and 30 days respectively. In all three methods

of fish silage production (mineral acid, organic acid, and biological method), the

optimum amount of sulphuric acid, formic acid and molasses were determined 3.5%,

3.5% and 15%,respectively.At the end of experiment, biological silage shown higher

protein content compared to inorganic and organic acid silage.

Powder fish silage was prepared by mixed liquid silage with different quantity

of rice bran. For neutralizing the liquid fish silage 1.5% sodium carbonate was added

and measured pH value was 6.45. Mixtures were dried within 2-3 days in solar tunnel

drier. The proximate composition of powder fish silage made with different quantity

of rice bran 10, 20, 30, 40 and 50% contained moisture content as 13.74 ± 0.12, 12.54

± 0.30, 10.91 ± 0.13, 10.18 ± 0.05, 9.18 ± 0.02 % respectively; crude protein content

as 29.50 ± 0.22, 28.56 ± 0.12, 27.66 ± 0.10, 26.53 ± 0.18, 25.73 ± 0.08 %

respectively; fat content as 16.28 ± 0.11, 15.71 ± 0.14, 14.45 ± 0.11, 13.73 ± 0.17,

12.60 ± 0.10% respectively; ash content 14.21 ± 0.12, 14.55 ± 0.11, 15.27 ± 0.13,

15.65 ± 0.15, 15.99 ± 0.12 % respectively. Considering all the limitation, powder fish

silage with 30 % rice bran was found to be better and carried for further storage study.

During storage, moisture content of powder silage was increased in the range from

10.91 ± 0.14 to 11.15 ± 0.10 ,whereas the protein content was decreased in the range

from 27.66 ± 0.10 to 27.04 ± 0.06 %, fat content decreased in the range from 14.45 ±

0.11 to 13.42 ± 0.10 %, ash content decreased in the range from 15.27 ± 0.11 to 14.60

± 0.09 %, Fiber content decreased in the range from 9.89 ± 0.06 to 9.64 ± 0.07 %,

Carbohydrate content increased in the range from 21.82 ± 0.07 to 23.14 ± 0.11 %, pH

increased in the range from 6.45 ± 0.05 to 6.59 ± 0.14 and TPC increased in the range

from 2.10×104

to 2.36 ×104

cfu/g during the storage period from 0 to 90 days.

From above study it can be concluded that fish market waste is suitable for

preparation of fish silage powder. At room temperature powder fish silage could be

stored up to more than 3 months without loss of major nutrient components.

1.0 INTRODUCTION

In India, the fisheries sector plays an important role in the national economy.

Fish is today considered as the most promising inexpensive alternative source of

animal protein consumed by the man and it offers relatively high amount of essential

amino acid, minerals and fatty acids as compared to live stock (Huisman et al., 1989).

In 2015, total marine fish landing of Maharashtra was 2.65 lakh tonnes (CMFRI,

2015). An average of 30% of the catch was sold locally as a fresh fish, 20-30 % was

used in canning industry and the rest was used mainly in fish meal production (Anon,

1999).

In the tropical countries, every efforts must be made to preserve fish for human

consumption, but seasonal variation in catch, transport difficulties, inadequate

processing facilities etc., resulted in the important quantities of fish has been wasted

(Disney et al.,1977).

During processing of sea foods large amount of fish waste and deteriorated

whole fishes are daily discarded daily in the fish markets (Faid et al., 1996). Fish

waste, includes frames and rests from trimming, guts, skins, fats, viscera, roes/eggs,

heads, breasts, scales and deteriorated filets. A fish contain 45 % flesh, 24-27 % head,

12 % skeleton, 3 % skin, 4 % cut off and 12 % viscera including eggs, milts and liver

of its total body weight (DOF, 2013). These wastes are a potential source of pollution

and contamination of the environment, as they degrade rapidly in warm temperatures.

If it is not appropriately stored or managed, it creates aesthetic problems and strong

odours due to bacterial decomposition. On the other hand, they contain high amount

of nutrients such as protein, fat and minerals (Djazuli et al., 2007) which is available

in low cost So that, there is need for developing new methods for biotransformation

of this fishery waste into animal feed to reduce aqua production cost.

Fish meal production is most commonly used method to recover the

nutrient loss in discarding the fish processing by products and it was used as animal

feed. If there is absence of any fish meal plant in the area due to restriction on fish

meal production to avoid fish odours, no transport facility is available towards the

nearest fish meal plant and increasing price of fish meal due diminishing fish stock,

then one has to look for alternate process (Zynudheen, 2003). These fish waste can

cause disposal problem for the processors and retail markets, especially in the

monsoon season (Raghunath and Gopakumar, 2002).

The best alternative solution is to utilize the wastes for the production of by-

products (Ramasubburayan et al., 2013). Many essential and costly value added

products like fish meal, FPC, fish oil, isinglass, fish glue, fish maw, chitin, chitosan,

fish finger, fish stick, fish ball, soup powder, fish sauce, fish cake, fish roll, omega -3

enriched tooth paste, liver oil, vitamin premix, ladies purse, binder, gelatin, cosmetics,

flavor, glucosamine, fertilizer etc. can be produced from fish wastes like head,

collarbone, cut-off, skin, backbone, roe, milt, liver, bile, viscera, cheek and tongue,

shell etc.

The best alternative way of utilizing fish waste material is the production of

fish silage which does not release any off odour during preparation (Pagarkar et al.,

2005).The product has a good nutritive quality and can be sufficient for animal

feeding. This procedure is safe, cost effective, eco-friendly and has a good nutritive

quality which can be adequate for animal feeding (Hanafy and Ibrahim, 2004). Fish

silage preparation usually depends on the locally available raw materials and

conditions (Hasan, 2003).

Silage production acquires potential importance compared to fish meal with

the following advantages: the process is virtually independent from the scale, the

technology is simple, the investment is little and the place of preparation is anywhere

even in fish farm (Arruda et al., 2007)

The name "silage" is derived from the process of storing chopped up green

plants such as grass, oats or corn, without drying, in a silo. A better word to describe

fish silage would perhaps be "liquid fish" or "liquefied fish protein" (Jangaard, 1987).

Fish silage is defined as a liquid product produced from the whole fish or

parts of it, to which acids, enzymes or lactic acid-producing bacteria are added, with

the liquefaction of the mass provoked by the action of enzymes from the fish (FAO,

2007).

The principle of preservation by acids was first explored in the 1920s by A.

I. Virtanen in Finland who treated green fodder with a mixture of sulphuric and

hydrochloric acids. Experiment on fish started in Sweden in 1936 carried out using,

sulphuric acid and molasses, and formic acid (Tatterson and Windsor, 1974). Acid fish

silage has been produced commercially for 30 years in Denmark. Fish silage

production is currently being introduced in Southeast Asian countries for utilizing the

waste, by catch and surplus fish (Raa and Gildberg, 1982).

Fish silage is the liquefied product rich in protein and free amino acids

(Martin, 1996). The liquefaction of fish mass carried out by enzymes already present

in fish (Tatterson and Windsor, 1974). This is obtain by action of the naturally

occurring enzymes presence in the whole fish, fish minced or fish offal. The enzymes,

mainly from the digestive organ, break down protein into smaller soluble unit and the

acid helps to speed up their enzyme activity while preventing bacterial spoilage (Al-

Abri et al., 2014).

Depending on the process employed in the preservation of fish waste, fish

silage can be categorised in two methods, viz. acid silage and bio-fermented silage

(Raa and Gildberg, 1982). Acid silage is produced by mixing fish waste with inorganic

(sulphuric acid, hydrochloric acid) and organic acid (formic acid, propionic acid) or

mixture of both organic and inorganic acid, while bio-fermented silage is obtain by

adding fermentable sugar to fish biomass. Fermentation is carried out by lactic acid

bacteria (LAB) which are already present in a fish mass or added externally for

successful fermentation (Raa and Gildberg, 1982).Using lactic acid bacteria(LAB) for

fermentation of biological silage can provide the recovery of various biomolecules and

also prevent fat oxidation (Rai et al., 2010). Bio-fermented fish silage has been shown

to have a good nutritional value with significantly better than acid fish silage and fish

meal (Pagarkar et al., 2005).

The disadvantage of fish silage is that it has high water content make it

bulky and difficult to transport and store. The most effective method is to co-dried fish

silage with other dry ingredients or other filler materials for convenient use. Co-drying

is a process where dry products are added to the wet silage to absorb the solublized

protein and some of the moisture (Fagberno et al., 1994). Co-dried fish silage used as

an aqua feed ingredient that is easy to package, stored, and transport.

Due to the similarity of the protein source with the raw material and low

cost, especially when compared to fish meal, silage has a potential use in aquaculture

(Vidotti et al., 2003).

Silage can be effectively used for animal feeding, like powder fish silage is

used to feed beef cattle, milk cow, swine, sheep, mink and many other terrestrial

animals (Rahmi et al., 2008, Al-Abri et al., 2014, Anuraj et al., 2014). Fish silage can

be used as a protein source for broiler chicks. Replacing fish meal protein by fish

silage protein resulted in similar or increased weight gain and feed conversion ratio in

broiler chicks, slaughtered at 4 or 5 weeks of age (Kjos et al., 2000). In many

countries, it is used as bird feed (Arruda et al., 2007).

It can be very vitally used as a feed supplement in aquaculture to convert

nutrients into flesh. Powder fish silage was found to give better growth than fish meal

when fed to carp (Djajasewaka, 1986).) In case of pink abalone, Haliotis fulgens,

powder fish silage has been used as an alternative to natural food (microalgae) (Viana

et al., 1999). Fish silage could also find use as a fertilizer, and trials with various

vegetables on different types of soil have been underway for several years at research

station of many countries (Blatt, 1983-86).

In view of above facts, the present research work was carried out with

following two broad objectives:

1. To study different methods of producing high quality of fish silage.

2. To evaluate nutritional quality and shelf life of powder fish silage during storage.

2.0 REVIEW OF LITERATURE

Acid silage was developed in 1920 by A. I. Virtanen, using hydrochloric and

sulphuric acid for the conservation of forages. At that time, fermentation of crops was

very difficult as crops do not have enough starch or sugar for natural fermentation to

yield acids. Virtanen was first used acid for fermentation. He found that acid addition

caused an instantaneous stop in the respiratory processes in the plants, preventing loss

of organic carbon from the material, notably sugars and acid-tolerant bacteria

survived in the acidified grass silage slowly convert sugars into lactic acid and thus

kept the pH low (Tatterson and Windsor, 1974).

Experiments with fish to prepare silage began in Sweden in 1936, using

hydrochloric, sulphuric, and formic acids and sugars (Tatterson and Windsor, 1974).

The fish silage process is cheap and simple. The raw material, which can be either

whole fish or fish waste, is minced, and then organic acid, mineral acid or a

carbohydrate source is added. Fish silage made by adding mineral or organic acid is

called acid fish silage whereas silage produced with a carbohydrate source and

anaerobic conditions is called fermented fish silage (Perez, 1995).

2.1. Acid silage:

Tatterson and Windsor (1974) prepared six types of fish silage were made

using the different raw materials: sprats, herring, herring offal, sand-eels, white fish

offal and mackerel. The silages were stored for a period of one year at temperatures of

2 ºC and 23 ºC. Results indicate that there are rapid deteriorative changes in the fish

oils and increases in soluble nitrogen.

Hall et al. (1985) prepared silage from tropical fish: Proteolysis. Acid silages

were prepared from silver belly (Leiognathus sp.) with 3 % (w/w) of 98 % formic acid

and stored at 30°C. The limited autolysis in silver belly silage is compared with that

found in cold water fish silages and attributed principally to the resistance of warm

water fish collagen to the effects of acid pH and temperature during ensilation.

Alwan et al. (1993) studied chemical aspects of fish silage. Fresh fish waste

consisted of flat and round fish frames, heads, whole fishes, fillets and offal in

different proportions, sizes and from various species was manufactured into fish

silage using formic acid to reduce pH to 3.5. The pH remained below 4.0 for up to 30

days. Fish silage contains 72.6 to 80.3 (mean 76.4) % moisture, 3 to 7.1 (mean 3.8) %

oil, 3.5 to 7.2 (mean 5.1) g ash, 14.4 to 16.1 (mean 14.7) g protein.

Goddard and Al-yahyai (2001) analysed the chemical and nutritional

characteristics of dried sardine silage. Acid silage was prepared by mixing fresh

Indian oil sardines, Sardinella longiceps, with a mixture of 1.5 % (v/w) mixture (1:1)

of formic and propionic acid and (200 ppm) Ethoxyquin were added to reduce lipid

oxidation. The silage was stored outside at ambient temperature (range 28-39ºC).

After 48 hour fish silage were co-dried with wheat bran and dried in a solar cabinet.

The results indicate that sardine silage, dried in a solar cabinet, could be prepared

within 4-5 days. During the liquefaction the pH of the silage remains constant and

non-protein nitrogen increased. The process of liquefaction was carried out within the

range of pH from 4.0-4.5.

Vidotti et al. (2003) studied the Amino acid composition of processed fish

silage using different raw materials. Commercial marine fish waste, commercial

freshwater fish waste, and tilapia filleting residue were used to produce fish silage by

acid digestion (2 % formic acid and 2 % sulphuric acid) and anaerobic fermentation

(5 % Lactobacillus plantarum, 15 % sugar cane molasses). Results indicate that

marine fish waste had higher crude protein content compared to freshwater fish waste

and tilapia filleting residue.

Arruda et al. (2007) investigate the use of fish silage as a substitute for protein

ingredients in rations for aquatic organisms is an alternative to solve sanitary and

environmental problems caused by the lack of adequate disposition for the waste from

the fish industry. Besides, it is also a way of decreasing feeding costs, and,

consequently, fish production costs, since feeding corresponds to about 60% of the

overall expenses with production. The objective of this review was to discuss the use

of fish waste, the elaboration of chemical silage and the use of this ingredient in feed

for aquaculture.

Preparation of silage from whole Spanish mackerel (Scomberomorus

maculatus) found on both the Pacific and Gulf of Mexico coasts were studied by

Delgado et al. (2008). The liquid silage obtained was mixed with sorghum (1:2 w/w),

and dried in the sun. During the silage process the NPN (Non Protein Nitrogen)

increased significantly by 85% from the original total nitrogen at 96 h. The protein

content in the dried silage and in the silage-sorghum mix was 653 and 169 g kg−1 on

a dry matter basis, respectively. Silage sorghum mix is a good alternative to use a fish

waste or undesirable marine species in poultry feeding.

Ramasubburayan et al. (2013) investigate the characterization and nutritional

quality of formic acid silage developed from marine fishery wastes and their potential

utilization as feed stuff for common carp (cyprinus carpio) fingerlings. Acid silages

were prepared using fishery wastes supplemented with three different concentrations

(2, 2.5 and 3%) of formic acid. Results indicated that pH, dry matter, ash, protein

contents and aerobic plate count showed a declining trend during ensilaging day’s

interval. Lipid and mineral contents were gradually increased up to 30th

day.

Majumdar et al. (2014) studied the effect of co-dried silage from fish market

waste as substitute for fish meal on the growth of the Indian major carp Labeo rohita

fingerlings. Acid silage was prepared by mixing the chopped fish entrails with 90 %

formic acid (2 % w/w) and concentrated sulphuric acid (2 % w/w).Potassium sorbate

solution (1 % v/w) was sprayed on the surface of silage to prevent formation of mould

growth. Liquid silage was neutralised with sodium hydroxide and co-dried with rice

bran (1:1 dry weight basis).The results indicate that moisture, crude protein, lipid, ash

content of analysed from fish market waste were ranged from 68.42 %, 15.59 %,

12.81 % and 2.97 %, respectively and co-dried silage had moisture, crude protein,

lipid, ash content range from 4.06 %, 16.1 %, 5.58 % and 3.33 %, respectively.

Tanuja et al. (2014) studied the shelf life of acid added silage produced from

fresh water fish dressing waste with and without the addition of antioxidants. Acid

silage was prepared by acidifying the paste with 1.5% formic acid and 1.5%

hydrochloric acid. The silage was divided into 2 lots. In one lot of FHB, 200 ppm

Butyl Hydroxy Toluene (BHT), 0.1 % Potassium sorbate and in other lot of FH, only

0.1 % Potassium sorbate was added. Results indicate that addition of antioxidant did

not significantly (p>0.05) alter the proximate composition of the silage (FHB). The

addition of BHT significantly reduced the rate of oxidation in FHB.

Hossain and Alam (2015) investigated the suitability of using fish wastes

(viscera) as raw material for powder fish silage production. Fish viscera contained

14.01% protein, 20.00 % lipid, 4.75 % ash, 60.62 % moisture and 0.62 % Nitrogen

Free Extract (NFE) on wet weight basis. For liquid fish silage production, 2%, 3%,

4% and 5% formic acid were added in blended viscera, of which 4% formic acid was

found better. The pH value of liquid fish silage was 3.77 when mixing with 4%

formic acid. For neutralizing liquid fish silage 1, 2, 3, 4, 5 and 6 % Na2CO3 were

added. The pH value was better (6.33) when mixing with 4 % Na2CO3. Liquid silage

mixed with 30% rice bran was found better and had pH value 6.55. Packaged powder

fish silage contained 20.84 % crude protein, 33.73 % lipid, 14.05 % ash, 10.83 %

moisture, 6.61% crude fibre and 13.94 % carbohydrate. A slow decrease in protein,

lipid, ash and crude fibre and a little increase in carbohydrate, moisture and pH were

observed during the storage period.

Gullu et al. (2015) producing silage from the industrial waste of fisheries. Fish

silage was produced by acid hydrolysis. The fish silage was ripened and became half

liquid, a room temperature in 12 days. Its odour became less pungent and was deemed

to have an acceptable malt smell. The results indicate that, the use of silage instead of

fish meal reduces the cost of feed by 21 %. The chemical composition of the silage

mixture was moisture 64.04 %, crude protein 16.84 %, crude lipid 10.81 % and crude

ash 3.95 %.

2.2. Biological silage:

The production of naturally fermented fish silage using various lactobacilli

and different carbohydrate sources were studied by the Van Wyk and Heydenrych

(1985). The suitability of eight different Lactobacillus cultures for fish silage-making

was investigated. The industrial by-products whey powder and molasses, as well as

refined sugar, were tested as fermentation substrates. Using the information obtained

two batches of fish silage were made at local white fish processing plants on a semi-

commercial basis and subsequently successfully used on local cattle and pig farms.

Hassan and Heath (1987) investigate the chemical and nutritive characteristics

of fish silage produced by biological fermentation. Fish silage was compared before

and after biological fermentation with L. plantarum. Trout had a larger per cent

protein and fat and less ash than white perch both before and after fermentation. Total

amino acids increased (P < 0.05) as a result of fermentation and the increase was

reflected in changes in the amino acid profile. A decrease (P < 0.05) in moisture and

an increase (P < 0.05) in fat were found after the silage was stored at both ambient

temperature and 37° C for 35 days. There was an increase (P < 0.05) in water-soluble

nitrogen at both temperatures as storage time increased.

The changes in microbial population during fermentation of tropical

freshwater fish viscera were studied by the Ahmed and Mahendrakar (1996b).

Freshwater fish viscera (FV) was homogenized, mixed with 10 % (w/w of FV)

molasses and 0, 2 or 4 % salt and allowed to ferment at ambient temperature under

micro-aerophilic conditions. Inclusion of either 0.5 % propionic acid, 0.3 % calcium

propionate or 0.1 % sorbic acid suppressed growth of yeasts and moulds with

propionic acid being the most effective. The study indicated that a microbiologically

stable product could be prepared by ensiling fish viscera with 10 % molasses and 0.5

% propionic acid.

Biotransformation of fish waste into a stable feed ingredient evaluate by the

Faid et al. (1996). Fish waste of (Sardina pilchardus) including viscera, heads and

tails, were mixed with 25 % molasses and inoculated with a starter culture composed

of Saccharomyces sp. and Lactobacillus plantarum. The silage was incubated at

22°C. Results indicated that the pH decreased considerably and remained constant at

4.2 and 4.5 in the two trials. The total nitrogen decreased while the non-protein

nitrogen and total volatile nitrogen increased significantly. The microbiological

characteristics showed a rapid decrease of coliform and Clostridium counts to reach a

low level after 5-7 days.

Espe and Lied (1999) studied the chemical changes during storage in fish

silage prepared from different cooked and uncooked raw materials at different

temperatures. Four silage types were prepared from raw and cooked whole herring

(Clupea harengus), whole mackerel (Scomber scombrus), by products from the

filleting-line of cod (Gadus morhua) and saithe (Pollachius virens), and from the

viscera of cod by adding 2.2 %(v/w) formic acid (85 %).Silage prepared from each

raw material, except from the viscera, were heated for about 20 minutes, giving a core

temperature of about 90° C in a conventional microwave oven prior addition of

formic acid. Each silage type was stored at 4, 20 and 50°C for 48 days. Dry matter,

crude protein and total fat were not affected by the different storage temperatures or

the length of the storage period. Cooked raw materials did not show any change in

hydrolysis during storage.

Conditions for the natural fermentation during ensilage sardines or their waste

in sugarcane molasses (60:40 w/w) at different temperatures (15 ºC, 25 ºC, 35 ºC) in

open and closed jars were evaluated by the Zahar et al. (2002). Successful natural

fermentation took place in sardine silages incubated at 25 or 35ºC in open jars to

reach a pH of 4.4 in about 2 and 1 week, respectively. For samples kept at 15ºC, the

pH decline was very slow and pH did not decrease below 5.5 after one month of

incubation. At 25ºC, the most favourable conditions for silage of sardine waste in

cane molasses, as evidenced by the fastest decline in pH to a stable value of about 4.4,

were achieved in closed jars and with daily stirring of the mix. The pH 4.4 was

reached in one week with an advance of at least 3 day compared to the other

conditions (open jars and closed jars without daily stirring). Addition of salt at 5%

w/w in the mix before incubation inhibited the fermentation process.

Neethiselvan (2002) studied the lactic acid fermentation of minced meat of

silverbelly (Leiognathus splendens) using three different lactic acid bacterial sources

viz. pure culture of Lactobacillus plantarum, fermented cabbage and curd revealed

fermented cabbage. 48 h fermented cabbage could bring down the pH of the minced

meat mixture to 4.4 within 48 h of fermentation whereas the other two minced meat

mixtures took nearly 84 h to reach the same pH. Free sugar, lactic acid content, NPN,

TVBN and lactic acid bacterial count were found to be good indices of quality of the

fermented product under storage.

Hasan (2003) investigated the suitability of Lactobacillus pentosus for fish

silage fermentation. Three kinds of fish silage were prepared respectively using pure

culture of L. pentosus (LBPN), liquid fermented bamboo shot containing L. pentosus

(LFBS) and aged silage prepared by L. Pentosus (ALPN) as fermentation starter. The

starter and molasses were added to fish mince (short-bodied mackerel, Rostreliger

brachysoma) and stored at 30 ºC for 60 days. Addition of starter initiated a rapid

decrease in pH of the silage. A desired pH level (<4.5) was reached within 24 hours,

and low pH was maintained throughout the fermentation. Overall, NPN production of

fish silage increased and then remained stable throughout storage. The lowest NPN

concentration was found in silage prepared by addition of L. pentosus pure culture.

Percentage of crude protein and fat was lower in fermented silage than that acid silage

due to addition of molasses or lower proportion of fish mince in fermented silage than

acid silage.

2.3. Comparative study

Dapkevicius et al. (1998) studied the changes in lipids and protein during

storage (15 days) of acid silages (with 0, 0.25 and 0.43 % w/w of formaldehyde) and

biological silages (with 10 and 20 % molasses or dehydrated whey) prepared from

blue whiting (Micromesistius poutassou Risso). A remarkable reduction in protein

solubilisation values was achieved by adding formaldehyde. Addition of

formaldehyde led to an increase in the peroxide value of the oil extracted from the

silages. In addition, the oil from biological silages had lower peroxide values than the

oil from acid silages with added formaldehyde.

Babu et al. (2005) compared the biochemical and microbial quality of formic

acid silage and lactobacillus fermented silage. Acid silage (AS) were prepared by

mixing formic acid with silver belly (Leiognathus sp.) minced at 2 %, 2.5 %, 3 %

(v/w) and fermented silage (FS) were prepared by mixing Lactobacillus plantarum

culture with fish mince at (5 % v/w) and molasses at 10 %, 12 % (v/w).Sodium

benzoate was added at 0.5 % w/w level to FS to inhibit mould growth. pH of 2.5 %

AS and 3 % AS fell below 4.5 within two days and stabilized at 4.24 and 4.01,

respectively. pH of 2 % AS reached a minimum value of 4.68. In 10 % FS and 12 %

FS, the pH dropped to less than 4.5 by the end of 1st day indicating good lactic acid

fermentation by Lactobacillus plantarum. Crude protein of the silage ranged between

18.22 % and 19.17 %. Fat was lower in FS (1.0-1.14 %) than in AS (3.67-5.13 %) on

wet basis. NPN of FS silage was found to be lower than the AS silage which indicate

the lower protein break down alpha amino nitrogen in FS changed to 21% of TN in

10% FS and 12% FS from an initial concentration of 11% of TN, which was lower

than that in AS than in FS. Microbiological quality of AS and FS was found to be

good as indicated by the absence of total Coliforms, faecal coliforms, E.coli,

salmonella, Vibrio cholerae, coagulase positive staphylococci and H2S producing

bacteria. Total yeast mould count was highest in 2 % AS.

Pagarkar et al. (2006) prepared bio fermented and acid silage from fish waste

and studied its biochemical characteristic. The fish waste silage prepared from

sulphuric acid (3 %) and a mixture of 1.5 % (w/w) formic acid and 1.5 % (w/w)

propionic acid showed an increase in pH from second day onwards and a sign of

spoilage. The pH of the silages prepared from sulphuric acid (4%) and a mixture of

2% (w/w) formic acid and 2 % (w/w) propionic acid and bio-fermented silage using

pure culture of lactic acid bacteria (L. Plantarum), recorded on alternate days showed

a decreasing trend and at the end of experiment of 15 th

days, the final pH values were

recorded as 3.20, 3.60, 4.38 respectively. In case of bio fermented silage a steady

supply of nutrients from molasses showed a steady increase in lactic acid bacteria

from 1.8×108 to 4.3×10

10 cfu /g, which produced lactic acid. Constant production of

lactic acid showed a gradual decrease in pH at the end of 15 days.

Palekar (2009) studied the transformation of fish waste into fish waste silage

in an eco-friendly way by incorporating curd as a source of lactic acid bacteria.

During standardization the ratio of fish waste to molasses 100:15 (w/w) and fish

waste to curd 100:10 (w/w) were found suitable for preparation of fish waste silage.

By using standardised ratios of molasses and curd with other ingredients such as

water, salt, butylated hydroxyl toluene (BHT) and sodium benzoate, fish waste silage

(CS) was prepared and kept in a airtight containers for storage periods of 90 days with

L. Plantarum from same fish waste. Fish waste silage prepared molasses and curd was

having good keeping quality for a period of 90 days and had almost same

characteristics like LPS but better quality as compared to FAS.

Mousavi et al. (2013) produced silage from fish waste in cannery factories of

Bushehr city using mineral acid, organic acid, and biological method. Waste of

cannery factories from three main species of economic tuna including Thunnus

tonggol, Euthunnus afinis, and Kasuwonus pelamis, as raw material for fish silage

production, were combined with 95% sulphuric acid and 90% formic acid with weight

percentages of 2.5, 3.5, and 4.5. In order to produce silage by biological method,

fermentable sugar was prepared from sugar beep pulp with weight percentage 5, 10,

and 15. In all three methods of fish silage production (mineral acid, organic acid, and

biological method), the optimum amount of sulphuric acid and formic acid and sugar

beet molasses were determined 3.5% and 15%,respectively. If mineral acid is used,

pH neutralization must be done. Comparing mineral acid and organic acid methods, it

can be concluded that the use of mineral acid with weight percentage of 3.5 is

economically more preferred than organic acid with the same amount, although

neutralization is needed.

Ozyurt et al. (2015) studied biotransformation of seafood processing wastes

fermented with natural lactic acid bacteria; the quality of fermented products and their

use in animal feeding. They used Enterococcus gallinarum for producing fermented

products. In this study, chemical and microbiological qualities of fish silage by acid or

fermented methods were assessed after ripening of silages. It was observed that

ripening was completed in maximum two weeks for all silage groups. Then, acid and

fermented fish silages were spray dried and analysed for chemical and nutritional

properties. As results of the study, these bacteria can be used as starter cultures in

fermented products, especially for fish silage. In respect to essential/nonessential

amino acid ratio (E/NE), the best groups among the spray-dried fish silages were

prepared with formic acid, Lb. plantarum and Pd. acidilactici, respectively.

According to the in-vitro gas production assessment, spray-dried fish silages generally

had considerably high rate of digestibility. It was determined that the acid and

fermented fish silage powders had high digestibility and valuable feed sources.

2.4 Proximate composition:

2.4.1. Fish silage:

The composition of fish silage is very similar to that of the material from

which it is made. A typical analysis of white fish offal contain 80 % water, 15 %

protein, 4.5 % ash and 0.5 % fat and the composition of silage from offal is virtually

the same. Whole fatty fish like sprats and sand eels have a higher protein and fat

content, and correspondingly lower water and ash content (Tatterson and Windsor,

1974).

Arason et al. (1990) in their experiment produced silage from fish by-catch

office fish trawler and factory trawler and compared the nutritional value. They found

that silage made from fish of ice fish trawler contained 12.5 % protein and 18.9 % fat

and factory trawler contained 14.5 % protein and 10.7 % fat.

Gullu et al. (2015) reported that chemical composition of the silage mixture

contained moisture 64.04 %, crude protein 16.84 %, crude lipid 10.81 % and crude

ash 3.95 %.

Fish silage made from fish wastes contains 13.62 % protein, 8.8 % lipid, 17.35

% ash and 31.67 % dry matter (Rahmi et al., 2008). Palekar (2009) also reported that

fish waste contain 15.78 % protein, 2.13 % fat, 79.47 % moisture and 2.32 % ash.

Abowei and Tawari (2011) prepared silage from viscera of different fish.

Silage made from herring offal contained 13.5 % crude protein, 8.7 % lipid, 75.4 %

moisture and 2.6 % ash. Silage made from white fish offal contained 15.0 % crude

protein, 0.5 % lipid, 78.9 % moisture and 4.2 % ash.

2.4.2. Rice bran:

The proximate composition of rice bran varies with grain size. Tao et al.

(1993) analysed the proximate composition of rice bran from the long grain and

medium grain. The protein, lipid, ash, moisture, crude fibre and NFE were 16.07 %,

19.20 %, 9.23 %, 11.20 %, 8.49 %, and 47.01 % in rice bran from long grain and

16.20 %, 21.97 %, 9.46 %, 10.83 %, 8.41 % and 44.07 % in rice bran from medium

grain on dry weight basis.

Rosniyana et al. (2007) analyse proximate composition of rice bran produced

at 2, 4, 6 and 8 % (MD). Bran produced at 2 % MD had the highest value for protein

content (13.12 %). Carbohydrate, fat and moisture content of analysed rice bran were

ranged from 28.71 % to 33.54 %, 21.53 % to 26.05 % and 9.53 % to 10.24 %,

respectively. 8% MD, the bran had significantly higher carbohydrate content than the

other bran due to endosperm breakage during milling. There is significant difference

in crude fibre and ash content between bran produced at 2, 4, 6 and 8 % MD. Bran

produced at 8% MD (11.29 %) had the lowest crude fibre while bran at 2% MD

(13.20 %) had the highest. Bran produced at 8 % MD (11.08 %) had the highest ash

while bran at 2 % MD (10.53 %) had the lowest.

Rice bran can be of three types: 1. De-oiled rice bran (DORB), 2. Grade A rice

bran (comprising 85–90 % bran and 15–10 % husk) and 3. Grade B rice bran

(comprising 60–50 % bran and 40–50 % husk). The protein content of rice bran varies

with type. The protein content of DORB and grade A rice bran is approximately 12–

17 % and 10–13 %, respectively (Rashid et al., 2013).

Satter et al. (2014) analysed the nutritional composition and stabilization of

local variety rice bran BRRI-28 that contained 6.54 to 9.48 % moisture, 7.24 to10.63

% ash, 12.26 to 14.01 % proteins, 23.53 to 27.86 % fats, 2.5 to 10.10 % fibres, 42.19

to 45.74 % carbohydrates.

Bhosale and Vijayalakshmi (2015) evaluate the processing and nutritional

composition of rice bran. Macronutrient composition of stabilized and probiotic

treated rice bran contained moisture, protein, fat, ash and carbohydrate were 4.30 and

5.40, 17.50 and 19.25, 13.10 and 17.20, 4.92 and 4.64, 52.33 and 48.55 g/100g

respectively and it contained 7.85 and 4.96g crude fibre, 21.17 and 13.10g insoluble

dietary fibre, 2.17 and 1.80g soluble dietary fibre and 23.34 and 14.90g total dietary

fibre.

2.5. Biochemical parameters

2.5.1. pH :

James (1966) recorded a pH 4.4 and 5.0 in cooked ensiled silver belly and

uncooked ensiled silver belly within 72 hours of fermentation. According to Haland

and Naja (1989), deamination reactions probably caused slight changes in pH during

storage.

Hassan and Heath (1987) reported that pH of the silage did not changes during

storage at either ambient temperature or 37°C but the silage stored at ambient

temperature was more acidic than stored at 37°C.

Faid et al. (1996) showed that a regular decrease from 5.8 to 4 after 5 days of

fermentation for trial 1 and from 6.2 to 4.5 for trial 2. These values remain constant

for 7 days and started to increase slightly to 4.2 for trial 1. Espe and Lied (1999) no

changes in pH were detected when stored at different temperatures. pH remain at 3.7±

0.1 during 48 days of storage.

Palekar (2009) reported that the pH of the different types of silages viz,

Formic acid silage (FAS), Lactobacillus plantarum silage (LPS) and Curd silage (CS)

was 3.81, 6.74, 7.28 initially which reached up to 4.11, 4.38 and 4.42 at the end of

90th

day respectively.

Tanuja et al. (2014) reported there was significantly dropped in pH of FH and

FHB to 3.3 and 3.12, respectively on the second day. Both the lots shows fluctuations

in till the 180th

day of storage, it never increased above 3.5. The pH at the end of 180th

day of storage was 3.44 and 3.41 of FH and FHB respectively.

2.5.2. Alpha Amino Nitrogen (AAN):

Fagberno and jauncey (1998) observed that NPN of tilapia fish silage

increased gradually from 16 % and attained a maximum of 46.5 % after 30 days

which was lower than obtained in acid preserved silage. Faid et al. (1996) showed that

AAN increased in trial 1 during 11 days of fermentation to reach 1.3 % and remain

constant for the remaining period. A similar increase in AAN was observed in trial 2.

Hassan (2003) reported that NPN production of fish silage increased from

18.37- 61.40 % of total nitrogen up to 30 days of storage (P<0.05) and then remained

stable throughout storage.

Babu et al. (2005) had reported differences in AAN levels of AS and FS. It

was observed that the maximum AAN value as (% of TN) obtained was lower in 2.5

% AS (21.21% of TN) than in 3% AS (24.33% of TN) and 2% AS (27.30% of TN).

The AAN values in FS were constant at 21% of TN by 15th day in both the

concentrations (10 % FS and 12% FS) from an initial concentration of 11% of TN.

The maximum AAN levels as % of TN were almost identical in 2.5 % AS, 10% and

12% FS. Delgado et al. (2008) reported that, during silage process the NPN (Non

Protein Nitrogen) increased significantly by 85%from the original total nitrogen at 96

h.

2.5.3. Total Volatile Base Nitrogen (TVBN):

Hassan and Heath (1987) reported that silage stored at 37°C had a higher Total

Volatile Base value than silage stored at ambient temperature. Total Volatile Bases

increased (P< 0.05) as storage times increased up to 18 days and then started to

decline.

Haaland and Njaa (1989) in their analytical study investigated the significance

of total volatile nitrogen (TVN) consisting mainly of tri-methylamine (TMA) and

ammonia (NH3) portion as quality criteria for fish silage. They prepared formic acid

silages from fresh raw material of mackerel and of capelin for their study and reported

that, in properly preserved silage (stable pH) TVN and NH3-N increased moderately

during storage but they found large increases in TVN and NH3-N when the acid

addition was too low and pH was higher.

Ahmed and Mahendrarkar (1996b) had reported low TVB-N value of 9 mg %

for fermented carp visceral silage. Faid et al. (1996) showed that TVN increased in

trial 1 from 71.26 mg per 100 g to reach 95.03 mg per 100 g after1 day and remain

constant around 132 mg per 100 g after 15 days of fermentation at 22º C. In case of

trial 2 TVN increased from 71.26 mg per 100 gram to 141.4 mg per 100 g after 15

days.

Palekar (2009) reported that the TVB-N steadily increased during storage

irrespective of the types of silages. TVB-N content in FAS was 18.22 mg-N 100 g-1

initially which increased to 57.67 mg-N100g-1

at the end of 90th

day which was

lowest among the all of silage. CS showed initial value of TVB-N with 20.13 mg-n

100g-1

initially but increased in slower rate with 133.28 mg -N 100 g-1

at the end of

90th

day. In case of LPS, it had TVB-N content of 128.91 mg- N 100g-1

at the end of

90th

day from 18.84 mg-N 100 g -1

.

Tanuja et al. (2014) showed that TVB-N level in both the treatments were well

below the limit of acceptability except on the 90th day of storage. The maximum

TVB-N value reached was 46.73 mg/100gm for the lot of without synthetic

antioxidant.

2.5.4. Total Plate Count (TPC):

Ahmed and Mahendrarkar (1996b) reported that there was a reduction in total

viable count and the number of spores, coliforms, Escherichia coli, staphylococci and

enterococci and an increase in yeasts and moulds and lactic acid bacteria during

fermentation. Coliforms and E. coli were found to be absent after 6 day and

enterococci on 8th

day.

TPC was lower than lactic acid bacterial count indicating the inability of lactic

acid bacteria on nutrient agar medium (Neethiselven et al., 2002). Bhaskar and

Mahendrarkar (2007) have also reported a significant reduction in total bacterial count

of fish viscera till 4 week of storage. Standard plate count decreased, and Coliforms

and clostridia were eliminated.

Palekar (2009) had reported that curd shown TPC of 8.70 ×10 5 cfu/g initially

which decrease to 4.26×103 cfu/g to the end of 90

th day. On 10

th day onward FAS had

not shown any detectable colony up to end of 90th

day. TPC decrease from initial

1.65x106 cfu/g up to 1.86×10

3 cfu/g at the end of 90

th day.

2.5.5. Lactic acid bacteria (LAB):

Pagarkar et al. (2006) reported that bio fermented silage showed a steady

increase in lactic acid bacteria from 1.8×108 to 4.3×10

10 cfu /g, which produced lactic

acid. Rahmi et al. (2008) reported an increased lactic acid bacterial count from

3.2×106 to 4.8×10

9cfu/g. Palekar (2009) had reported that curd shown LAB of

1.07×106 cfu/g initially which decrease to 1.20×10

7 cfu/g to the end of 90

th day. In

case of LPS, LAB count shown value with 5.49×106

cfu/g initially and decreased up

to 2.13 x107 cfu/g at the end of 90

th day.

2.6. Use of fish silage

The use of fish silage in the feeding of fish has been widely studied. Due to

the similarity of this protein source with the raw material and low cost, especially

when compared to fish meal, silage has a high potential use in aquaculture (Fagbenro

et al., 1994; Vidotti et al., 2003).

Diets with silage, especially the ones including silage and soy meal, could be

used to feed tilapias Oreochromis niloticus and the North African catfish Clarias

gariepinus with no changes in its performance, use of protein and carcass composition

(Fagbenro et al., 1994; Fagbenro and Jauncey ,1998).

Viana et al. (1999) evaluated the use of both unheated (US) and heated (HS)

silage as an alternative feed for the pink abalone (Haliotis fulgens). Abalone that were

fed the US and HS containing diets were replace two third of the total amount of

silage with fish meal. Results indicate that abalone originally fed the US and HS diets

had composite growth rate of 18.6 µm/d., while abalone that continue to fed with

commercial diet had growth rate of 18.6 µm/d.

Fish silage is used as a protein rich ingredients source for sheep feeding. In a

study, sheep were fed with control diet and diet with silage up for 9 weeks. The

results indicated that trial feeding studies with young sheep using fish silage showed a

net increase in weight above controls as well as a good enhancement of meat

characteristics and carcass shape (Rahmi et al., 2008).

Ramasubburayan et al. (2013) were used individual silage products as one of

the major ingredients to prepare formulate diets (E1: 2 %; E2: 2.5 % and E3: 3 %) to

replace 8 % of a control diet. The prepared diets were fed to C. carpio fingerlings for

45 days. The growth performance of 2 %, 2.5 % and 3 % acid silages incorporated

diets fed C. carpio displayed 2.38, 2.01 and 1.87 g weight gain and 1.49, 1.31 and

1.26 % SGR, respectively. At the same time, the control diet fed common carp had

the weight gain of 1.49 g and the SGR of 1.06 %.

Hassan and Heath (1987) reported that broiler chicks fed with a ration

containing 5 % and 10 % fish silage had better feed efficiency than chicks fed a ration

with no silage. Their results indicated that up to 10 % fish silage could be included in

broiler rations without adversely affecting feed efficiency or body weight.

Widjastuti et al. (2000) studied the effect of adding tuna (Thunnus atlanticus)

fish silage in ration on the meat protein conversion of broiler. They prepared four

diets. The diets were a control diet Ro (without any silage used) and three others R1,

R2 and R3 contain 4%, 6% and 8% tuna fish silage respectively. They reported that

final body weight gain by using these diets form the broiler chickens were 1755.03 g,

1844.87 g, 1654.84 g and 1439.53 g respectively and the meat protein conversion of

broiler (R0= 0.85, R1=0.86 ,R2= 0.82 and R3=0.78 ). They concluded that until 6 %

tuna fish silage had no significant effects on final body weight and meat protein

conversion while 4 % tuna fish silage gave the best result.

Kjos et al. (2000) studied the Effects of dietary fish silage and fish fat on

performance and egg quality of laying hens. The results indicated that fish silage did

not affect feed intake, egg production, and fatty acid composition of yolk, yolk colour

or sensory quality of eggs, compared with the control. The diet with 16.8g kg-1 fish

fat resulted in more intense egg albumen whiteness as measured by the sensory study,

compared with other diets (P<0.05).

Soltan and Fath El-Bab (2010) investigated the effect of partial or complete

replacement of fish meal (FM) by dried fermented fish by-product silage (FFS) in

diets of Nile tilapia (Oreochromis niloticus) fry.They found that dried FFS can

successfully replace up to 50 % of FM in catfish diets without any significant loss in

growth performance (body weight, body length, weight gain and specific growth rate).

Anuraj et al. (2014) studied the dried tuna waste silage as an alternate protein

source for swine feeding. An investigation was carried out in Large White Yorkshire

pigs to find out the effect of dietary incorporation of dried tuna waste silage on

growth. Pig lets (n=36) were randomly allotted to the three dietary treatments, T1

(standard ration with 10 % dried fish), T2 (ration with 50 per cent of protein of dried

fish replaced by dried tuna waste silage) and T3 (ration with 100 per cent of protein of

dried fish replaced by dried tuna waste silage) up to 70 kg body weight. There was no

significant difference between animals in the three dietary treatments with regard to

their average daily gain, fortnightly body weight and FCR.

3.0 MATERIAL AND METHODS

3.1 Materials:

3.1.1 Fish waste:

Fish market wastes were procured from fish market of Ratnagiri. The fish

mainly consisted of heads, tails, gills, fins and visceras of Sardine (Sardinella

Fimbriata, Sardinella longiceps), Mackerel (Rastrelliger kanagurta), Tuna

(Euthynnus affinis, Auxis thazard), Pink perch (Nemipterus japonicus), Ribbon fish

(Trichiurus lepturus, Lepturocanthus savala), Bombayduck (Harpadon nehereus),

Seer fish (Scomberomorus guttatus, Scomberomorus commerson), Mullet (Mugil

cephalus). The collected fish wastes were washed with potable water and stored at -20

°C until further used.

3.1.2 Sample preparation:

The fish waste was thawed, washed, and grinded into paste using mixer for

preparation of different types of silage.

3.1.3 Sulphuric acid:

Analytical quality grade Sulphuric acid (98%) was used for preparation of

inorganic acid silage.

3.1.4 Formic acid:

Analytical quality grade Formic acid (98%) was used for preparation of

organic acid silage.

3.1.5 Lactic acid bacteria source:

Curd was used as lactic acid bacteria source for preparation of fermented

silage. Curd was prepared fresh from boiled and cooled milk and kept for overnight.

3.1.6 Molasses:

Molasses obtained from Vishwasrao Sahakari Sakhar Karkhana, chikhali, Tal

Shirala, Dist, Sangli and it was used as a fermentable carbohydrate source in

preparation of fermented silage.

3.1.7. Rice bran:

Rice bran was collected from Joshi rice meal factory, Basani, Tal. Dist.,

Ratnagiri. Rice bran was used as co-dried material for preparation of dried fish silage

powder.

3.1.8 Antioxidant:

Butylated Hydroxy Toluene (BHT) of analytical grade was used as antioxidant

in all the silages.

3.1.9 Microbiological culture media:

All the bacteriological media used in this study were purchased from Hi-

media Ltd, Mumbai. Plate count agar and Dehydrated De-man Rogosa Sharpe (MRS)

agar were used for the enumeration of Total plate count and Lactic acid bacterial

count.

3.1.10 Diluent:

Physiological saline (0.85%) was used as diluents for all the microbiological

work.

3.1.11 Chemicals:

Most of the chemicals used for the analysis work were analytical grade.

3.1.12 Mixer:

Prestige made mixer was used to grinding the fish waste for preparation of

silage.

3.1.13 Glass wares:

Glass wares of 'Borosil' make used for analysis purpose.

3.1.14 Plastic containers:

Wide mouth plastic jars of 2 litre capacity with air tight lid were used for

storage of silage.

3.1.14 Equipment and machineries

3.1.14.1 Electronic weighing balance:

Electronic Monopan balance of ‘Milton’ make (Citizen Scale Pvt. Ltd.

Mumbai, India) was used for weighing fish waste.

3.1.14.2 Autoclave:

Autoclave of 'EQUITRON' brand was used for sterilization of media.

3.1.14.3 Hot air oven:

Hot air oven (Nishitronics instrument, Pune) was used for sterilization of

glassware and moisture estimation.

3.1.14.4 Muffle furnace:

Muffle furnace (Classic scientific, Mumbai) was used for estimation of ash.

3.1.14.5 Incubator:

Bacteriological ' YORCO' brand incubator was used for incubation of samples

in petri dishes with sample.

3.1.14.6 pH meter:

Digital pH meter of (EQUIPTRONICS brand, MODEL EQ 610) was used for

pH estimation (range 0 to 11).

3.1.14.7 Solar tunnel dryer:

The solar tunnel dryer developed by (College of Agriculture Engineering and

Technology, Dapoli) was used for drying fish silage.

3.1.14.7 Sealing machine:

Electronic sealing machine, a quality product of 'MOOSH' was used for

sealing purpose.

3.1.14.8 Packaging material:

Low density polythene (LDPE) pouches of 100 g. Capacity and 4 × 6 cm size

were used.

3.2 Methods:

Fish waste which includes heads, tails, fins, and viscera etc. were randomly

collected from local fish market, Ratnagiri in iced condition. The collected fish wastes

were washed with potable water and were grinded into paste by using mixer. The

resulting compound fish waste was used as raw material for preparation of different

silages.

3.2.1 Silage production using mineral acid (Mousavi et al., 2013)

1.5 kg of minced fish waste was poured in three plastic containers (each

container containing 500 gram of fish waste) and 98% sulphuric acid with weight

percentages of 2.5, 3.5, and 4.5 % (v/w) and 65 mg of Butylated Hydroxy Toluene

(BHT) were added to each sample. The mixture was stirred regularly with sterile glass

rod to ensure through mixing and inhibit growth of mould on the surface. Samples

were kept at room temperature (28°C to 32°C) for 60 days and stirred every 8 hours.

pH changes were measured by pH meter and recorded until it reached a stable level.

3.2.2 Silage production using organic acid (Mousavi et al., 2013)

1.5 kg of minced fish waste was poured in three plastic containers (each

container containing 500 gram of fish waste) and 98% formic acid with weight

percentages of 2.5, 3.5, and 4.5 % (v/w) and 65 mg of Butylated Hydroxyl Toluene

(BHT) were added to each sample. The mixture was stirred regularly with sterile glass

Plate 1: Fish market waste and viscera

Plate 2: Sulphuric acid silage with weight percentages of 2.5, 3.5 and 4.5 %

Sulphuric acid 2.5% Sulphuric acid 3.5%

3.5%2.5%

Sulphuric acid 4.5%

4444.54.5%3.5%

rod to ensure through mixing and inhibit growth of mould on the surface. Samples

were kept in room temperature (28°C to 32°C) for 60 days and stirred every 8 hours.

pH changes were measured by using pH meter and recorded until it reached a stable

level.

3.2.3 Silage production by biological method using Curd (Palekar, 2009)

1.5 kg of minced fish waste was poured in three plastic containers (500 grams

in each). Then, sugar cane molasses and water was added to each container with

weight percentages of 5%, 10%, 15% (v/w) and 30% (v/w) respectively. 65 mg of

butylated hydroxytoluene (BHT) were added to each sample. The mixture was stirred

using a sterile glass rod to ensure through proper mixing. After these samples were

heated in water bath for 15 minutes at 90 °C and then cooled at a room temperature.

Then curd (starter culture) with a weight percentage of 10 % (w/w) was added to

them. Mix the samples thoroughly and stored in airtight plastic container. pH changes

were measured by pH meter and recorded until it reached a stable level.

3.2.3.1 Preparation of starter culture

Curd was prepared fresh from boiled and cooled milk and kept for overnight.

Lactic acid bacterial (LAB) count of curd was estimated by pour plating technique on

MRS agar and Lactic Acid Bacteria (LAB) count of 4.20 x 106

cfu /gram used for

preparation of biological silage.

3.2.4. Preparation of dried fish silage powder (Hossain and Alam , 2015):

3.2.4.1 Preparation of biological silage:

500 g of minced fish waste was poured in plastic container. Then, sugar cane

molasses, Butylated Hydroxyl Toluene (BHT) and water were added in container with

weight percentages of 15% (v/w), 65 mg and 30% (v/w) respectively. Buckets were

stirred properly. After this sample was heated in water bath for 15 minutes at 90 °C

Plate 3: Formic acid silage with weight percentages of 2.5, 3.5 and 4.5 %

Plate 4: Biological silage with weight percentages of 5, 10 and 15 % molasses

Formic acid 2.5% Formic acid 3.5% Formic acid 4.5%

Biological silage 5% Biological silage 10% Biological silage 15%

and then cooled at a room temperature. Then curd (starter culture) with a weight

percentage of 10 % (w/w) was added to them. Mix the samples thoroughly and stored

in airtight plastic container. Silage was kept for 10 days at ambient temperature.

3.2.4.2 Neutralised pH:

After 10 days, 1.5 % of sodium carbonate Na2CO3 was used for neutralised pH

of silage.

3.2.4.3 Mixing with rice bran:

After neutralising the pH of the 15% of biological silage, rice bran were added

10%, 20%, 30%, 40%, 50% respectively to prepare a semidry feed.

3.2.4.4. Dry in solar tunnel drier:

The semidry fish silage was spread on the plastic film and dried in a solar

tunnel drier for further used.

3.2.4.5. Grinding:

Traditional mortar and pestle or mixer was used for grinded the dried fish

silage powder.

3.2.4.6. Selection of dried silage sample for packaging:

Proximate composition of the dried silage samples were analysed (10%, 20%,

30%, 40% and 50% respectively.).

3.2.4.7. Packaging:

Cooled powder mixture was packed in air-tight polythene (LDPE) packets

and packets were sealed by using sealing machine.

3.2.4.8. Quality evaluation during storage study:

Packets were kept at room temperature to study the shelf life of the product up

to 3 months. During storage, changes in proximate composition, pH and TPC were

observed.

Plate 5: Solar tunnel dryer

Plate 6: Drying of semidry fish silage

Plate 7: Powder fish silage made with different quantity of rice bran

Rice bran 10% Rice bran 20% Rice bran 30% Rice bran 40% Rice bran 50%

Plate 8: Powder fish silage with 30% rice bran

Plate 9: Packed and Stored 30 % rice bran powder silage

3.3 Proximate compositions:

Proximate composition i.e. Moisture, crude protein, crude fat, ash, fiber

content of prepared silages and dry silage powder were determine as per standard

methods of (AOAC, 2005).

3.3.1 Moisture content:

About 10 g of sample was taken in a glass dish or petri dish which was

previously dried in an oven and weighed and kept in air oven maintained at 100 ± 2ºC

to dry for 5 hrs. Cooled in desiccators and weighed. The process of heating, cooling

and weighing was repeated until the difference in weight between two successive

weighing was less than one milligram. The lowest weight was recorded.

The moisture content of the samples was estimated by using the following formula

W1 - W2

W0

Where,

W0: weight (gm) of test sample

W1: Weight (gm) of petridish + test sample

W2: Weight (gm) of petridish + test sample after drying.

3.3.2 Crude protein:

Kjeldhal method was used to determine crude protein content of fish waste,

liquid silage and powder fish silage samples. Sample (0.2 g) was taken and transferred

into pelican digestion flask. 3 g of digestion mixture [K₂SO₄: CuSO₄ (5:1)] was

added. Then 10 ml concentrated H₂SO₄ was added and mixed. Then it was heated

gently on pelican digestion instrument (Kel plus – Kes 04L, Pelican Instruments) for 1

hours at 420ºC, until the solution become clear.

X 100 Moisture content (%) =

The digested clear sample was transferred to Pelican digestion tube and

distillation was done with 50 ml 40% NaOH and 25 ml 4% boric acid solution. The

sample was distilled for about 15 min. After collection of distillate, solution turns

from blue violet to green. Distillate was titrated with 0.02N H₂SO₄ solution until the

colour of solution turns green to red.

14 x V x Normality of acid x100

Nitrogen (%) =

Protein (%) = Nitrogen (%) × 6.25

Where,

V=Volume (ml) of titrate

W=Weight (gm) of sample

3.3.3 Crude fat:

For estimation of fat, SOCS plus SCS 2 system (Pelican Instruments, Chennai)

was used. The beaker and sample were dried and kept in desiccators. Empty beaker

was weighted (Initial weight) and thimbles containing 2 gram sample were inserted in

thimble holders. Fat in the sample was collected using petroleum ether by boiling it

and final weight of beaker was recorded by taking out thimble holders from beakers.

The fat content was expressed in percentage and was calculated by formula:

Lipid content (%) =

Where,

W= Weight of sample in gm

W1= Initial weight of beaker in gm

W x 1000

X 100

W2 - W1

W

X 100

W2= Final weight of beaker in gm

3.3.4 Crude ash:

2 g of dried sample was weighed accurately in a porcelein crucible and was

placed in muffle furnace at 550ºC for 6 hours. Then the crucibles were cooled in

desiccators. The average in percentage of each sample of the remaining materials was

taken as ash. The ash content of the samples was estimated by using the following

formula:

Ash content (%) =

Where,

W=Weight (gm) of empty crucible

W1=Weight (gm) of crucible + dried sample

W2=Lowest weight (gm) of crucible with sample

3.3.5. Crude fibre:

Accurately weighed 0.5 g sample was taken in filter crucible. Then it was kept

on hot extractor unit. Hot 150 ml 0.128M Sulphuric acid and 3 drops N-Octanol were

added to filter crucible. Then it was boiled for 30 min at 230°C temperature and

solution was allowed to pass through the pipe. Sample was washed for three times

with hot distilled water. Then hot 150 ml 0.223M potassium hydroxide and 3 drops N-

octanol were added to filter crucible. It was boiled for 30 min at 230°C and solution

was allowed to pass through the pipe. Sample was washed for three times with hot

distilled water. Then sample plus filter crucible was kept on cold extractor unit and

washed for three times with acetone. After that, sample plus filter crucible was kept in

hot air oven for overnight. Sample plus filter crucible was taken away from hot air

W1 – W2

W1 – W

X 100

oven and kept in desiccators for cooling. Then weight was taken (X). Sample plus

filter crucible was kept in muffle furnace at 500°C for three hours. Finally sample

plus filter crucible was taken away from muffle furnace and kept in desiccators.

Weight was taken (Y). Amount of fiber was calculated from the difference of two

weights. The crude fiber content of the samples was estimated by using the following

formula:

Crude Fiber (%):

Where,

X- Initial weight

Y- Final weight

W – Weight of sample

3.3.6. Carbohydrate:

The carbohydrate content was determined by subtracting the summed up

percentage compositions of moisture, protein, lipid, fiber, and ash contents from 100

(Otitoju, 2009).

Carbohydrate content was determined by using the following formula:

Carbohydrate content (%) = 100 - (moisture % + protein % + lipid %+ % fiber

+ % ash)

3.4. Biochemical parameters.

3.4.1. pH measurement:

About (5 g) sample was ground with 45 ml distilled water and filtered using a

filter paper. The pH of filtrate was recorded using a pH meter (AOAC, 2005).

X - Y

W

X 100

3.4.2. Determination of Total Volatile Base Nitrogen (TVB-N)

Preparation of TCA

About 5 g of fish sample was taken and ground in a mortar with acid washed

sand and 10 ml of 20% TCA solution is added. Mixture was ground well. It is filtered

and residue is washed with distilled water containing a few drops of TCA. This

protein free filtrate was made up to 100 ml.

The TVB-N content of fish silage samples during storage was determined

according to Beatty and Gibbons (1936). The filtrate was then used for further

analysis (this filtrate was used for AAN estimation also).

Grease was applied on the edges of conway unit and 1 ml of 0.01 N standard

sulfuric acid was taken in inner chamber, 1 ml TCA extract and 0.5 ml saturated

potassium carbonate were taken in outer chamber of the unit. The unit was sealed

with glass lid and the contents were gently swirled. The unit was kept overnight

undisturbed. The amount of untreated acid in the chamber was determined by titration

against standard 0.01 N NaOH using Tashiro’s Indicator. A blank was run

simultaneously prepared with 1 ml of TCA solution. TVB-N was calculated as

mg/100 g of muscle as follows

(A – B) × 0.14 × Volume of extract × 100

Volume of sample taken × Sample weight

TVB-N (mg/100g) =

3.4.3 Determination of Alpha Amino Nitrogen (AAN)

The AAN content fish silages during storage were determined according to

Benjakul and Morrissey (1997). The filtrate extract prepared for TVB-N content was

used for analysis of AAN content.

About 10 ml of TCA extract was taken into 50 ml volumetric flask. Few drops

of thymolpthalein were added and extract was made alkaline by adding 1 N NaOH,

till a distinct blue colour appears. 1 part by volume of CuCl2 solution was mixed with

2 parts by volume for tri-sodium phosphate and 2 parts by volume of borate buffer.

These solutions were mixed well and 30 ml of suspension was added to alkaline

solution in standard flask which contains 10 ml of TCA extract. The volume was

made up to 50 ml with distilled water. After shaking, it was allowed to stand for 10

min and then filtered 10 ml of the filtrate was pipetted out to a conical flask; 0.5 ml of

glacial acetic acid (CH3COOH) was added followed by addition of 0.5 g of potassium

iodide. The liberated iodine was titrated against N/500 sodium thiosulphate using

starch as indicator, when yellow solution of iodine becomes faint yellow, few drops of

starch solution was added and titration was continued till blue colour disappeared.

The value of AAN was using a following value

1.0 ml of N/500 Na2S2CO3 = 0.056 mg of α Amino Nitrogen

3.5 Microbiological quality

3.5.1 Enumerations of total plate count (TPC)

Samples were analysed for total plate count (TPC) by the method

recommended by APHA (1992). Samples of FS were tested for total plate count

physiological saline (0.85%) was used as diluents for preparation of homogenate; 25

gram sample was aseptically weight and transferred to 225 ml of physiological saline

in homogenizer. Samples were homogenized using mortar and pestle. Appropriate

dilution were prepared from homogenate using physiological saline and plated on

plate count method as per Collins et al, (1984). The petridishes containing sample

were incubated at 37 °C for 24-48 hours. The colonies developed on agar were

counted and calculated. Dilution giving colonies between 30-300 ranges were

selected. Plates with crowded or spread colonies, which could not be counted, were

discarded.

The TPC was founded as under

Total plate count cfu /g =Avg. of Count in duplicate plates x dilution factor.

3.5.2 Lactic acid bacteria count:

Appropriate dilutions were plated by pour-plate technique in duplicate using

MRS medium (AOAC 1990) and were incubated at room temperature for 48 hours.

3.6 Statistical analysis:

The data were analysed to test significance difference by applying analysis of

variances (ANOVA) tool available in MS-EXEL 2010. The significant differences

were tested by 5% level of significances and are mentioned as p < 0.05 for

significance difference (Snedecor and Cocharn, 1967).

Flow chart 3.1 Method for preparation of acid silage

(Mousavi et al., 2013)

Fish market waste

Washing and draining

Blending in a blender

Addition of acids

Sulphuric acid 100:2.5, 3.5, 4.5 % (w/v)

Formic acid 100:2.5,3.5, 4.5 %(w/v)

Stored in airtight plastic container

Addition of BHT 65 mg

Mixing thoroughly

Weighing

Mixing thoroughly

Flow chart 3.2 Method for preparation of biological silage (Palekar, 2009)

Fish market waste

Washing, draining

Weighing


Addition of 100:5, 10, 15 %

(w/w) molasses

Addition of 100:30 (w/v) of

water and 65mg of BHT

Mixing thoroughly

Heating in water bath

90ºC for 15 minutes

Cooling at room temperature


Addition of curd 100:10(w/w)

Mixing thoroughly

Flow chart 3.3 Method for preparation of powder fish silage

(Hossain and Alam, 2015)

Fish market waste

Washing and draining

Weighing


Addition of 100:15 % (w/w)

molasses

Addition of 100:30 (w/v) of

water and 65mg of BHT

Mixing thoroughly

Cooling at room temperature

Heating in water bath at

90 ºC for 15 minutes

Addition of curd 100:10 (w/w)

Mixing thoroughly


Keeping for 10 days to transfer into liquid

Adding1.5% Na2Co3 to neutralize pH 6-7

Adding 30% rice bran to liquid silage

Mixing

Drying in a solar tunnel dryer

Grinding in a mixer

Packing in a polythene bag

Storage

4.0 RESULTS

4.1 Chemical analysis of Fish waste

The biochemical and microbiological characteristics of fish waste were

analysed during the initial stage of present work. The fish waste used for the present

work was having acceptable fishy smell and appearance.

4.1.1 Proximate composition

Proximate composition of fish waste on wet basis is shown in Table 4.1.and

Fig.4.1. Fish waste contained moisture 77.09 ± 0.14 %, crude protein 15.20 ± 0.15 %,

fat 4.03 ± 0.07 % and ash 3.30 ± 0.11 % and NFE (Nitrogen Free Extract) 0.38 %

±.0.06

4.1.2 Biochemical and microbiological analysis of fish waste

The pH, α- amino nitrogen, TVB-N and TPC of fish waste were 6.8 ± 0.49,

10.64 ± 0.13 mg-N100 g -1

18.64 ± 0.09 mg-N100g -1

, and 5.1× 106 cfu/g respectively

as shown in Table 4.2.

4.2 Proximate composition of rice bran.

Rice bran was used as dry ingredient for convenient use of liquid fish silage.

The proximate composition of rice bran was shown in Table 4.3 and Fig.4.2. The

moisture, crude protein, fat, ash content and NFE of rice bran were 9.45 ± 0.19, 16.05

± 0.08, 13.42 ± 0.15, 10.44 ± 0.14, 50.64 ± 0.19 (%) respectively.

4.3 Comparative analysis of Sulphuric acid silage, Formic acid silage and

Biological silage.

Changes in proximate composition, biochemical and microbiological quality

of different silages during storage are presented in this subsection.

Table 4.1 Proximate composition of Fish waste

Fish waste Proximate composition (%)

Moisture 77.09 ± 0.14

Crude protein 15.20 ± 0.15

Fat 4.03 ± 0.07

Ash 3.30 ± 0.11

NFE 0.38 ± 0.06

Table 4.2 Biochemical and microbiological analysis of fish waste.

Characteristic Fish waste

pH 6.8 ± 0.49

α-Amino nitrogen(mg-N100g -1

) 10.64 ± 0.13

TVB-N (mg-N100g -1

) 18.64 ± 0.09

TPC (cfu/g) 5.1 × 106

Table 4.3 Proximate composition of Rice bran

Rice bran Proximate composition (%)

Moisture 9.45 ± 0.19

Crude protein 16.05 ± 0.08

Fat 13.42 ± 0.15

Ash 10.44 ± 0.14

NFE 50.64 ± 0.19

Fig. 4.1 Proximate composition of Fish waste

Fig. 4.2 Proximate composition of Rice bran

15% 4%

77%

3% 1%

Protein

Fat

Moisture

Ash

NFE

16%

13%

10%

10%

51%

Protein

Fat

Moisture

Ash

NFE

4.3.1. Biochemical changes:

4.3.1.1 pH:

The changing pattern of pH in different methods of silages during storage

period is presented in Table 4.4, 4.6 and 4.9 respectively.

4.3.1.1.1 Sulphuric acid:

In case of 98 % Sulphuric acid, the initial pH of treatment A1, A2, A3 was

2.97, 1.94, and 1.33 respectively. Slight increase in pH was noticed in entire storage

study. Treatment A1 type silage was corrupted at the end of 24th day and last

recorded pH was 4.62. After 30th

days pH of treatment A2 and A3 were stable i.e.

2.66, 1.92 respectively. Finally no changes in pH of treatment A2 and A3 were

observed from day 30 to 60th

day storage and last recorded pH was 2.66 and 1.92.

(Shown in Table 4.4 and Fig.4.3)

The results of Two-way ANOVA (Table 4.5) indicated that there was no

significant difference in pH among the treatments of A1, A2, and A3respectively (p >

0.05).

4.3.1.1.2 Formic acid:

In formic acid, treatment B1, B2, B3 had the initial pH of 3.43, 3.14, and 2.73

respectively. Treatment B1 type silage was corrupted at the end of 24 days and last

measured pH was 4.64. The pH of treatment B2 and B3 silage were increase up to 24

days. Finally no changes in pH of treatment B2 and B3 were observed from day 24 to

60 day storage and last recorded pH were 3.65 and 3.41 respectively(Shown in Table

4.6 and Fig.4.4).

The results of Two-way ANOVA (Table 4.7) indicated that there was

significant difference in pH among the treatments (p<0.05). It was confirmed by SNK

test (Table 4.8). Further SNK test indicated that pH of treatment B1 was significantly

different from B2 and B3. Treatment B2 and B3 were not significantly different.

4.2.1.3 Biological silage:

In case of biological silage, treatment C1, C2, C3 had initial pH of 6.85, 6.56

and 6.75 respectively. There was decrease and increase in pH of all treatment. The

treatment C1 and C2 silage were corrupted within 12th

and 30th

days and last recorded

pH were 5.24 and 4.65 respectively. In C3 treatment silage pH was decreased from

6.75 to 4.04 and stable at 4.30 at the end of 24th

day and no changes were observed

from 24th

to 60th

days of storage (Shown in Table 4.9 and Fig.4.5).


significant difference in pH among the treatments (p<0.05). It was confirmed by SNK

test (Table 4.11). Further SNK test indicated that pH of treatment C3 was

significantly different from C1 and C2. Treatment C1 and C2 were not significantly

different.

4.3.1.2. Alpha Amino Nitrogen (AAN):


The silages were shown different changing pattern of AAN during storage

(shown in Table 4.12, 4.15, 4.18). The AAN content of sulphuric acid treatment A1

was 25.27 mg-N 100 g -1

initially which increased up to 42.78 mg-N 100g-1

at the end

of 24th

day. Treatment A2 and A3 had 14.80, 11.89 mg-N100g-1

AAN initially which

increased up to 47.71 and 45.24 mg-N100g-1

at the end 30th

day. Finally no changes

in AAN of treatment A2 and A3 were observed from day 30 to 60th

day storage and

last recorded AAN was 47.71 and 45.24 mg-N 100g-1

(Shown in Table 4.12 and Fig.

4.6).

The results of Two-way ANOVA test (Table 4.13) indicated that there was

significant difference in AAN among the treatments of A1, A2 and A3 respectively

(p<0.05).

Table 4.4 pH Changes in different treatment of sulphuric acid silage during

storage

Days Treatments

A1 A2 A3

0 2.97 1.94 1.33

6 3.35 2.12 1.47

12 3.76 2.35 1.65

18 4.16 2.45 1.77

24 4.62 2.66 1.88

30 - 2.66 1.92

36 - 2.66 1.92

42 - 2.66 1.92

48 - 2.66 1.92

54 - 2.66 1.92

60 - 2.66 1.92

Note: A1, A2 and A3 indicate Sulphuric acid silage 2.5 %, 3.5 % and 4.5%

respectively

Table 4.5 ANOVA for pH changes in different treatment of Sulphuric acid silage

F cal. <F crit. therefore, treatments are significantly different at p > 0.05

Source of

Variation

Sum of

Square

Degree of

Freedom

Mean of

square F-value P-value F crit.

Rows 10.26766 10 1.026766 0.675664 0.734094 2.347878

Columns 4.263521 2 2.13176 1.402806 0.269081 3.492828

Error 30.39281 20 1.51964

Total 44.92398 32

Table 4.6 pH Changes in different treatment of Formic acid silage during storage

Days Treatments

B1 B2 B3

0 3.43 3.14 2.73

6 3.75 3.26 3.05

12 4.04 3.36 3.13

18 4.44 3.55 3.25

24 4.64 3.65 3.41

30 - 3.65 3.41

36 - 3.65 3.41

42 - 3.65 3.41

48 - 3.65 3.41

54 - 3.65 3.41

60 - 3.65 3.41

Note: B1, B2 and B3 indicate Formic acid silage 2.5 %, 3.5 % and 4.5% respectively

Table 4.7 ANOVA for pH Changes in different treatment of Formic acid silage.

F cal. > F crit. therefore, treatments are significant difference at p < 0.05

Table 4.8 SNK Test for pH Changes in different treatment of Formic acid silage

NSD: Not Significantly Different SD: Significantly Different

Source of

Variation

Sum of

Square

Degree of

Freedom

Mean of


Rows 12.35657 10 1.235657 0.718262 0.698642 2.347878

Columns 18.21907 2 9.109536 5.295188 0.014271 3.492828

Error 34.40685 20 1.720342

Total 64.98249 32

Comparison Difference SE q p q crit. Conclusion

3 Vs 2 0.259091 0.395468 0.65515 3 3.593 NSD

3 Vs 1 1.689697 0.395468 4.272654 2 2.960 SD

2 Vs 1 1.430606 0.395468 3.617504 2 2.960 SD

Treatment B1 B2 B3

Average 1.845152 3.534848 3.275758

Rank 1 3 2

Table 4.9 pH changes in different treatment of Biological silage during storage.

Days Treatments

C1 C2 C3

0 6.85 6.56 6.75

6 5.06 4.45 4.14

12 5.24 4.27 4.04

18 - 4.45 4.25

24 - 4.56 4.30

30 - 4.65 4.30

36 - - 4.30

42 - - 4.30

48 - - 4.30

54 - - 4.30

60 - - 4.30

Note: C1, C2 and C3 indicate Biological silage 5 %, 10 % and 15% molasses

respectively

Table 4.10 ANOVA for pH changes in different treatment of Biological silage

F cal. > F crit. therefore, treatments are significantly different at p < 0.05

Table 4.11 SNK Test for pH changes in different treatment of Biological silage.


Source of

Variation

Sum of

Square

Degree of

Freedom

Mean of


Rows 92.1325 10 9.21325 3.412756 0.00936 2.347878

Columns 47.97904 2 23.98952 8.886154 0.001732 3.492828

Error 53.99302 20 2.699651

Total 194.1046 32

Treatment C1 C2 C3

Average 1.559091 2.630909 4.478485

Rank 1 2 3


3 Vs 2 1.847576 0.495402 3.72945 3 3.593 SD

3 Vs 1 2.919394 0.495402 5.892983 2 2.960 SD

2 Vs 1 1.071818 0.495402 2.163534 2 2.960 NSD

Fig.4.3 pH Changes in different treatment of Sulphuric acid silage during

storage

Fig 4.4 pH Changes in different treatment of Formic acid silage during storage.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 6 12 18 24 30 36 42 48 54 60

pH

Days

Sulphuric acid

2.50% 3.50% 4.50%

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 6 12 18 24 30 36 42 48 54 60

pH

Days

Formic acid

2.50% 3.50% 4.50%

Fig.4.5 pH Changes in different treatment of Biological silage during storage

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0 6 12 18 24 30 36 42 48 54 60

pH

Days

Biological silage

5% 10% 15%

It was confirmed by SNK test (Table 4.14). Further SNK test indicated that AAN of

treatment A1 was significantly different from A2 and A3. Treatment A2 and A3 were

not significantly different.

4.3.1.2.2 Formic Acid:

The AAN content of Formic acid treatment B1 was 26.97 mg-N 100 g -1

initially which increased up to 48.49 mg-N 100g-1

at the end of 18th

day. The AAN

content of treatment B2 and B3 were 16.31, 14.76 mg-N100g-1

initially which

increased up to 52.15 and 49.02 mg-N 100g -1

at the end 24th

day. Finally no changes

in AAN of treatment B2 and B3 were observed from day 24 to 60th

day storage and

last recorded AAN was 52.15 and 49.02 mg-N 100g -1

(Table 4.15 and Fig.4.7).


significant difference in AAN among the treatments (p<0.05). It was confirmed by

SNK test (Table 4.17).Further SNK test indicated that AAN of treatment B1 was

significantly different from B2 and B3. Treatment B2 and B3 were not significantly

different.

4.3.1.2.3. Biological silage

The AAN content of Biological silage with molasses treatment C1and C2 were

16.61, 14.28 mg-N 100 g -1

initially which increased up to 21.97and 34.92 mg-N

100g-1

at the end of 12th

and 24th

day respectively. The AAN content in treatment C3

was 13.34 mg-N100g-1

initially which increased up to 37.35 mg-N100g-1

at the end

24th

day. Finally no changes in AAN of treatment C3 was observed from day 24 to

60th

day storage and last recorded AAN was 37.35 mg-N 100g-1

(Shown in Table 4.18

and Fig.4.8)


significant difference in AAN among the treatments (p<0.05). It was confirmed

Table4.12AAN (mg-N100g-1

) changes in different treatment of sulphuric acid silage

Days Treatments

A1 A2 A3

0 25.27 14.80 11.89

6 34.40 21.95 17.19

12 45.26 27.66 23.89

18 47.74 36.51 30.33

24 42.78 44.42 38.77

30 - 47.71 45.24

36 - 47.71 45.24

42 - 47.71 45.24

48 - 47.71 45.24

54 - 47.71 45.24

60 - 47.71 45.24

Note: A1, A2, and A3 indicate Sulphuric acid silage 2.5 %, 3.5 % and 4.5% respectively

Table 4.13 ANOVA for AAN changes in different treatment of sulphuric acid silage


Table 4.14 SNK Test for AAN changes in different treatment of sulphuric acid silage


Source of

Variation

Sum of

Square

Degree of

freedom

Mean of


Rows 1209.931 10 120.9931 0.376591 0.942649 2.347878

Columns 2922.275 2 1461.137 4.547792 0.023552 3.492828

Error 6425.701 20 321.2851

Total 10557.91 32

Treatment A1 A2 A3

Average 17.7682 39.2339 35.7748

Rank 1 3 2


3 Vs 2 3.45909 5.40442 0.64005 3 3.593 NSD

3 Vs 1 21.4658 5.40442 3.97189 2 2.96 SD

2 Vs 1 18.0067 5.40442 3.33184 2 2.96 SD

Table 4.15 AAN (mg-N100g-1

) changes in different treatment of formic acid silage

Days Treatments

B1 B2 B3

0 26.97 16.31 14.76

6 35.50 25.49 26.50

12 44.39 34.47 36.10

18 48.49 45.21 46.33

24 - 52.15 49.02

30 - 52.15 49.02

36 - 52.15 49.02

42 - 52.15 49.02

48 - 52.15 49.02

54 - 52.15 49.02

60 - 52.15 49.02

Note: B1, B2 and B3 indicate Formic acid silage 2.5 %, 3.5 % and 4.5% respectively

Table 4.16 ANOVA for AAN changes in different treatment of formic acid silage


Table 4.17 SNK Test for AAN changes in different treatment of formic acid silage


Source of

Variation

Sum of

Square

Degree of

Freedom

Mean of

square F-value P-value F-crit.

Rows 1248.34 10 124.834 0.422387 0.918909 2.347878

Columns 6275.137 2 3137.569 10.61624 0.000721 3.492828

Error 5910.887 20 295.5444

Total 13434.36 32

Treatment B1 B2 B3

Average 14.12242 44.23061 42.43636

Rank 1 3 2


3 Vs 2 1.794242 5.183403 0.346151 3 3.593 NSD

3 Vs 1 30.10818 5.183403 5.808574 2 2.960 SD

2 Vs 1 28.31394 5.183403 5.462423 2 2.960 SD

Table 4.18 AAN (mg-N100g-1

) changes in different treatment of biological silage

Days

Treatments

C1 C2 C3

0 16.61 14.28 13.34

6 18.29 18.21 20.27

12 21.97 23.20 26.00

18 - 29.53 32.64

24 - 34.92 37.35

30 - 34.92 37.35

36 - 0.00 37.35

42 - 0.00 37.35

48 - 0.00 37.35

54 - 0.00 37.35

60 - 0.00 37.35

Note: C1, C2 and C3 indicate Biological silage 5 %, 10 % and 15% molasses respectively.

Table 4.19 ANOVA for AAN changes in different treatment of biological silage


Table 4.20 SNK Test for AAN changes in different treatment of biological silage


Source of

Variation

Sum of

Square

Degree of

Freedom

Mean of


Rows 815.0191 10 81.50191 0.561186 0.825752 2.347878

Columns 4157.293 2 2078.646 14.31263 0.000139 3.492828

Error 2904.632 20 145.2316

Total 7876.944 32

Treatment C1 C2 C3

Average 5.169394 14.09576 32.15242

Rank 1 2 3

Comparison Difference SE q P q crit. Conclusion

3 Vs 2 18.05667 3.633576 4.969393 3 3.593 SD

3 Vs 1 26.98303 3.633576 7.426026 2 2.960 SD

2 Vs 1 8.926364 3.633576 2.456633 2 2.960 NSD

Fig 4.6 AAN changes in different treatment of Sulphuric acid silage during

storage

Fig 4.7 AAN changes in different treatment of Formic acid silage during

storage.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

0 6 12 18 24 30 36 42 48 54 60

AA

N m

g-N

/100

-1

Days

Sulphuric acid

2.50% 3.50% 4.50%

0

10

20

30

40

50

60

0 6 12 18 24 30 36 42 48 54 60

AA

Nm

g-N

100g -

1

Days

Formic acid

2.50% 3.50% 4.50%

Fig 4.8 AAN changes in different treatments of Biological silage during storage

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

0 6 12 18 24 30 36 42 48 54 60

AA

N m

g-N

100g

-1

Days

Biological silage

5% 10% 15%

SNK test (Table 4.20) Further SNK test indicate that AAN of treatment C3

was significantly different from C1 and C2. Treatment C1 and C2 were not

significantly different.

4.3.1.3 Total Volatile Base Nitrogen (TVB-N):


The sulphuric acid treatment A1 shown 16.85 mg-N 100 g -1

TVB-N initially

which increased up to 38.22 mg-N100g -1

at the end of 24th

day. The TVB-N content

in treatment A2 and A3 were 16.99 and 17.17 mg-N 100g -1

initially which increased

up to 27.07 and 26.29 mg-N100g -1

at the end 24th

day. Finally no changes in TVB-N

of treatment A2 and A3 were observed from day 24 to 60th

day storage. (Shown in

Table 4.21 and Fig.4.9)


significant difference in TVB-N among the treatments (p<0.05). It was confirmed by

SNK test (Table 4.23). Further, SNK test indicated that TVB-N of treatment A1 was

significantly different from A2 and A3. Treatment A2 and A3 were not significantly

different.

4.3.1.3.2 Formic acid:

In case of Formic acid, treatment B1 shown 19.79 mg-N100g -1

initially

which increased up to 39.67 mg-N100g -1

at the end of 24th

day. The TVB-N content

in treatment B2 and B3 were 17.13 and 16.99 mg-N100g -1

initially which increased

up to 27.16 and 25.25 mg-N 100g -1

at the end 18th

day. Finally no changes in TVB-N

of treatment B2 and B3 were observed from day 18 to 60th

day storage (shown in

Table 4.24 and Fig.4.10).


significant difference in TVB-N among the treatments (p<0.05). It was confirmed by

SNK test (Table 4.26) Further SNK test indicated that TVB-N of treatment B1 was

significantly different from B2 and B3. Treatment B1 and B2 were not significantly

different.

4.3.1.3.3 Biological silage:

Biological silage, treatment C1 had initial TVB-N value of 22.73 mg-N100g-1

which increased up to 57.35 mg-N100g-1

at the end of 12th

day. Then sample was

corrupted. Similar case was observed in treatment C2, TVB-N increase initially from

22.17 mg-N100g-1

to 39.43 mg-N100g-1

at the end of 30th day. The TVB-N content in

treatment C3 was 19.83 mg-N100g-1

initially which increased up to 33.55 mg-N100g-

1 at the end 18

th day. Finally no changes in TVB-N of treatment C3 were observed

from day 18 to 60th

day storage (Shown in Table 4.27 and Fig. 4.11).


significant difference in TVB-N among the treatments C1, C2, C3 (p<0.05). It was

confirmed by SNK test (Table 4.29). Further SNK test indicated that TVB-N of

treatment C1 was significantly different from C2 and C3. Treatment C2 and C3 were

not significantly different.

Table 4.21TVB-N (mg-N100g-1

) changes in different treatment of sulphuric acid silage

Days Treatments

A1 A2 A3

0 16.85 16.99 17.17

6 22.73 19.97 20.39

12 29.96 23.67 24.55

18 35.42 25.34 25.62

24 38.22 27.07 26.29

30 - 27.07 26.29

36 - 27.07 26.29

42 - 27.07 26.29

48 - 27.07 26.29

54 - 27.07 26.29

60 - 27.07 26.29

Note: A1, A2 and A3 indicate Sulphuric acid silage 2.5 %, 3.5 % and 4.5% respectively

Table 4.22 ANOVA for TVB-N changes in different treatment of Sulphuric acid silage

F cal. > F crit. therefore, treatments are significantly different at p < 0.05.

Table 4.23 SNK Test for TVB-N changes in different treatment of Sulphuric acid silage


Source of

Variation

Sum of

Square

Degree of

freedom

Mean of


Rows 765.0177 10 76.50177 0.765149 0.659541 2.347878

Columns 1031.441 2 515.7204 5.158086 0.015616 3.492828

Error 1999.658 20 99.9829

Total 3796.117 32

Treatment A1 A2 A3

Average 13.01576 25.04 24.70364

Rank 1 3 2


3 Vs 2 0.336364 3.014856 0.111569 3 3.593 NSD

3 Vs 1 12.02424 3.014856 3.988331 2 2.960 SD

2 Vs 1 11.68788 3.014856 3.876762 2 2.960 SD

Table 4.24 TVB-N Changes in different treatment of Formic acid silage.

Days Treatments

B1 B2 B3

0 19.79 17.13 16.99

6 25.57 19.83 19.69

12 31.17 25.29 24.13

18 35.18 27.16 25.25

24 39.67 27.16 25.25

30 - 27.16 25.25

36 - 27.16 25.25

42 - 27.16 25.25

48 - 27.16 25.25

54 - 27.16 25.25

60 - 27.16 25.25

Note: B1, B2 and B3 indicate Formic acid silage 2.5 %, 3.5 % and 4.5% respectively.

Table4.25 ANOVA for TVB-N Changes in different treatment of Formic acid silage


Table 4.26 SNK Test for TVB-N Changes in different treatment of Formic acid silage


Source of

Variation

Sum of

Square

Degree of

freedom

Mean of


Rows 839.8711 10 83.98711 0.796329 0.633709 2.347878

Columns 882.2881 2 441.1441 4.182733 0.030367 3.492828

Error 2109.358 20 105.4679

Total 3831.517 32

Treatment B1 B2 B3

Average 13.76152 25.41212 23.88909

Rank 1 3 2


3 Vs 2 1.52303 3.096448 0.491864 3 3.593 NSD

3 Vs 1 11.65061 3.096448 3.762571 2 2.960 SD

2 Vs 1 10.12758 3.096448 3.270707 2 2.960 SD

Table 4.27 TVB-N (mg-N100g-1

) Changes in different treatment of Biological silage

Days Treatments

C1 C2 C3

0 22.73 22.17 19.83

6 42.75 25.39 26.74

12 57.35 32.62 29.77

18 - 34.43 33.55

24 - 36.77 33.55

30 - 39.43 33.55

36 - 0.00 33.55

42 - 0.00 33.55

48 - 0.00 33.55

54 - 0.00 33.55

60 - 0.00 33.55

Note: C1, C2, C3 indicate Biological silage 5 %, 10 % and 15% molasses respectively

Table 4.28 ANOVA for TVB-N changes in different treatment of Biological silage


Table 4.29 SNK Test for TVB-N Changes in different treatment of Biological silage


Source of

Variation

Sum of

Square

Degree of

freedom

Mean of


Rows 2881.361 10 288.1361 1.264232 0.313076 2.347878

Columns 2351.114 2 1175.557 5.157897 0.015617 3.492828

Error 4558.281 20 227.914

Total 9790.756 32

Treatment C1 C2 C3

Average 11.16606 17.34636 31.34303

Rank 1 2 3


3 Vs 2 13.99667 4.293068 3.260294 3 3.593 NSD

3 Vs 1 20.17697 4.293068 4.432684 2 2.960 SD

2 Vs 1 6.180303 4.293068 1.357752 2 2.960 NSD

Fig 4.9 TVB-N changes in different treatment of Sulphuric acid silage during

storage

Fig 4.10 TVB-N changes in different treatment of Formic acid silage during

storage

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

0 6 12 18 24 30 36 42 48 54 60

TV

B-N

(m

gN

-10

0g

-1)

Days

Sulphuric acid

2.50% 3.50% 4.50%

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

0 6 12 18 24 30 36 42 48 54 60

TV

B-N

(m

g-N

100g

-1)

Days

Formic acid

2.50% 3.50% 4.50%

Fig 4.11 TVB-N changes in different treatment of Biological silage during

storage

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

0 6 12 18 24 30 36 42 48 54 60

TV

B-N

(m

gN

-100g-1

)

Days

Biological silage

5% 10% 15%

4.3.2 Microbiological aspect:

4.3.2.1 Total Plate Count (TPC):

The changes in TPC during fermentation period are given in (Table 4.30and

Fig. 4.12). Treatment C1 had initial TPC was 3.16 x 106

cfu/g. Treatment C2 shown

decrease in TPC from 2.74 x 106 to 8.20 x 10

4 at the end of 20

th days after that there

was increase in TPC. The last measured TPC was 2.40 x 105 at the end of 30

th day.

Treatment C3 silage had shown gradual decreased with initial TPC of 4.50 x 106 cfu/g

which decreased up to 8.20 x 103 cfu/g at the end of 60

th day.

The results of Two- way ANOVA (Table 4.31) indicated that there was

significant difference in TPC among the treatments (p<0.05). It was confirmed by

SNK test (Table 4.32).

4.3.2.2 Lactic Acid Bacteria (LAB):

The changes in LAB during fermentation period are given in Table 4.33 and

Fig 4.13. Treatment C1 silage had 2.73 x106 cfu/g LAB initially. Treatment C2 silage

shown increase in LAB from 4.70 x106 cfu/g to 5.50x10

8 cfu/g

at the end of 15

th day,

After that there was decrease in LAB count. The last measured count of LAB was

8.40x 106

cfu/g. Treatment C3 silage had shown gradual increasing with initial LAB

of 2.24 x 106 cfu/g which increased up to 3.67 x 10

9 cfu/g at the end of 20

th day and

then decreasing. At the end of 60th

day last measured LAB was 5.10 X107

cfu/g.


significant difference in LAB among the treatments (p<0.05) and it was confirmed by

SNK test (Table 4.35). Further SNK test indicate that treatment C1, C2 C3 were

significantly different

Table 4.30 TPC (Cfu/g) changes in different treatment of Biological silage

Days Treatments

C1 C2 C3

0 3.16 ×106

(6.50) 2.74×106

(6.44) 4.50×106

(6.66)

5 8.54×105

(5.93) 5.46×105

(5.73) 7.10×104

(4.85)

10 3.31×105

(5.52) 1.62×105

(5.21) 2.69×104

(4.43)

15 5.31×105

(5.73) 1.24×105

(5.09) 2.16×104

(4.33)

20 - 8.20×104

(4.92) 1.43×104

(4.16)

25 - 1.52×105

(5.18) 1.28×104

(4.11)

30 - 2.40×105

(5.38) 1.10×104

(4.04)

35 - - 1.00×104

(4.00)

40 - - 8.80×103

(3.95)

45 - - 8.60×103

(3.95)

50 - - 8.50×103

(3.92)

55 - - 8.30×103

(3.92)

60 - - 8.20×103

(3.91)

Value in bracket indicates log conversion of cfu/g

Note: C1,C2, C3 indicate Biological silage with 5 %, 10 % and 15% molasses respectively

Table 4.31 ANOVA for TPC changes in different treatment of Biological silage


Table 4.32 SNK Test for TPC changes in different treatment of Biological silage.


Source of

Variation

Sum of

Square

Degree of

freedom

Mean of


Rows 132.4732 12 11.03943 3.899781 0.002211 2.18338

Columns 41.07621 2 20.5381 7.255275 0.003432 3.402826

Error 67.93877 24 2.830782

Total 241.4881 38

Treatment C1 C2 C3

Average 1.816769 2.919385 4.324538

Rank 1 2 3


3 Vs 2 1.405154 0.46664 3.011219 3 3.532 NSD

3 Vs 1 2.507769 0.46664 5.374104 2 2.919 SD

2 Vs 1 1.102615 0.46664 2.362885 2 2.919 SD

Table 4.33 LAB Changes in different treatment of Biological silage during storage

Days Treatments

C1 C2 C3

0 2.73×106

(6.44) 4.70×106 (6.67) 2.24×10

6 (6.35)

5 4.94×107(7.69) 6.80×10

7 (7.83) 4.30×10

8 (8.63)

10 7.69×107(7.89) 5.30×10

8 (8.72) 3.36×10

9 (9.53)

15 5.20×106(6.72) 5.50×10

8 (8.74) 3.67×10

9 (9.56)

20 - 4.40×107 (7.64) 3.10×10

9 (9.49)

25 - 3.32×107 (7.52) 2.31×10

8 (8.36)

30 - 8.40×106 (6.92) 1.10×10

8 (8.04)

35 - - 8.60×107 (7.93)

40 - - 8.40×107 (7.92)

45 - - 8.00×107 (7.90)

50 - - 7.50×107 (7.88)

55 - - 6.30×107 (7.80)

60 - - 5.10×107 (7.71)

Value in bracket indicates log conversion of cfu/g

Note: C1,C2, C3 indicate Biological silage with 5 %, 10 % and 15% molasses respectively

Table 4.34 ANOVA for LAB Changes in different treatment of Biological silage


Table 4.35 SNK Test for LAB Changes in different treatment of Biological silage


Source of

Variation

Sum of

Square

Degree of

freedom

Mean of


Rows 212.5667 12 17.71389 3.080179 0.009155 2.18338

Columns 246.1265 2 123.0633 21.39885 4.63E-06 3.402826

Error 138.0223 24 5.750929

Total 596.7155 38

Treatment C1 C2 C3

Average 2.210231 4.158154 8.239231

Rank 1 2 3


3 Vs 2 4.081077 0.665116 6.135888 3 3.532 SD

3 Vs 1 6.029 0.665116 9.064586 2 2.919 SD

2 Vs 1 1.947923 0.665116 2.928697 2 2.919 SD

Fig 4.12 TPC changes in different treatment of Biological silage during storage

Fig 4.13 LAB changes in different treatment of Biological silage during storage

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 5 10 15 20 25 30 35 40 45 50 55 60

Log v

alu

e

Days

Biological silage

5% 10% 15%

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0 5 10 15 20 25 30 35 40 45 50 55 60

Log v

alu

e

Days

Biological silage

5% 10% 15%

4.3.2 Proximate composition of the different types of Fish silages after 60 days.

At the end day of storage study, proximate compositions of the different

methods of silages were analysed that are shown in Table 4.36 and Fig.4.14. A higher

moisture level was found in acid silage i.e. 75.24 ± 0.16, 75.54 ± 0.10, 75.32 ± 0.06,

75.49 ± 0.10 % in treatments A2, A3, B2, B3 (Sulphuric acid 3.5, Sulphuric acid 4.5,

Formic acid 3.5, Formic acid 4.5%) respectively. Moisture level of treatment C3

(biological silage 15%) was 73.62 ± 0.20 %. The acid silage treatments A2, A3, B2,

B3 (Sulphuric acid 3.5, Sulphuric acid 4.5, Formic acid 3.5, Formic acid 4.5%) had

the lowest value of crude protein i.e. 13.11 ± 0.16, 12.91 ± 0.12, 13.03 ± 0.11, 12.82 ±

0.07 %. Treatment C3 (biological silage 15%) had highest value of crude protein

content i.e. 14.72 ± 0.12 % compared to the acid silages. The treatment C3 (biological

silage 15%) shown lower value of fat and higher value of ash content i.e. 4.54 ± 0.06

and 5.89 ± 0.10 % respectively.

After completed storage study of different methods of silages, on the basis of

protein content I select biological method 15% molasses for preparation of fish silage

powder. The result of one-way ANOVA (Table 4.36a) shown that there was

significant difference among all the treatment. SNK test also revealed that treatment

C3 (Biological silage 15%) was significantly different from treatment A2, A3, B2, B3

(Sulphuric acid 3.5, Sulphuric acid 4.5, Formic acid 3.5, Formic acid 4.5%)

respectively.

Table 4.36 Proximate composition of the different types of Fish silages after 60 days.

Note: A2, A3, B2, B3, C3 indicate sulphuric acid 3.5%, Sulphuric acid 4.5%, Formic

acid 3.5%, Formic acid 4.5%, Biological silage 15% molasses respectively

Table 4.36a One-way ANOVA for protein content of different methods of silages.

Source of

Variation

Sum of

square

Degree of

freedom

Mean

Square F value P-value F crit.

Between

Groups 7.565093 4 1.891273 41.27615 3.4706 3.47805

Within

Groups 0.4582 10 0.04582

Total 8.023293 14


Table 4.36b SNK test for protein content of different methods of silages.

Treatment A2 A3 B2 B3 C3

Average 13.11333 12.91 13.03333 12.82 14.72667

Rank 4 2 3 1 5

Note: A2, A3, B2, B3 and C3 indicate Sulphuric acid 3.5%, Sulphuric acid 4.5%,

Formic acid 3.5%, Formic acid 4.5% and biological silage 15% respectively

Treatments

(Silage)

Moisture (%) Protein (%) Lipid (%) Ash (%)

A2 75.24 ± 0.16 13.11 ± 0.16 5.06 ± 0.10 5.12 ± 0.11

A3 75.54 ± 0.10 12.91 ± 0.12 5.24 ± 0.07 4.84 ± 0.16

B2 75.32 ± 0.06 13.03 ± 0.11 5.02 ± 0.15 5.03 ± 0.13

B3 75.49 ± 0.10 12.82 ± 0.07 5.46 ± 0.12 4.72 ± 0.15

C3 73.62 ± 0.20 14.72 ± 0.12 4.54 ± 0.06 5.89 ± 0.10



5 Vs 4 1.613333 0.123585 13.05441 5 4.756 SD

5 Vs 3 1.693333 0.123585 13.70173 4 4.415 SD

5 Vs 2 1.816667 0.123585 14.6997 3 3.949 SD

5 Vs 1 1.906667 0.123585 15.42794 2 3.199

SD

4 Vs 3 0.08 0.123585 0.647326 4 4.415 NSD

4 Vs 2 0.203333 0.123585 1.645287 3 3.949 NSD

4 Vs 1 0.293333 0.123585 2.373529 2 3.199 NSD

3 Vs 2 0.123333 0.123585 0.997961 3 3.949 NSD

3 Vs 1 0.213333 0.123585 1.726203 2 3.199 NSD

2 Vs 1 0.09 0.123585 0.728242 2 3.199 NSD

Fig.4.14 Proximate composition of the different types of Fish silages after 60

days.

0

10

20

30

40

50

60

70

80

%

Proximate composition(%)

Moisture

Protein

Fat

Ash

4.4. Fish silage powder:

4.4.1 Drying rate of powder fish silage containing different concentration of rice

bran.

The initial moisture content of T1, T2, T3, T4 and T5 (10 %, 20%, 30%, 40%,

50% rice bran respectively) were 67.63, 56.36, 53.83, 50.91% respectively. Although

the three mixture i.e. T3, T4, T5 (30%, 40%, 50% rice bran) appeared to reach

equilibrium moisture content below 15% after 48 hours. The last recorded moisture

content was 13.23, 11.88 and 9.84 % respectively. Two mixtures T1 and T2 (10% and

20% rice bran) taken 72 hours to reach a moisture content below 15 %(Shown in

Table 4.37, Fig.4.15).Two-way ANOVA also revealed that there was significance

difference among all the treatment T1, T2, T3, T4 and T5 respectively shown in Table

(4.38).SNK test also revealed that treatments were significantly different from each

other.

4.4.2 Proximate composition of powder fish silage made with different quantity

of rice bran

Proximate composition of powder fish silage made with different quantity of

rice bran is shown in (Table 4.40 and Fig 4.16).Powder fish silage made with 10 %

rice bran by weight of liquid silage contained 13.74 ± 0.12 % moisture, 29.50 ± 0.22

% protein, 16.28 ± 0.11 % fat, 14.21 ± 0.12 % ash. Powder fish silage made with 20

% rice bran by weight of liquid silage contained 12.54 ± 0.30% moisture, 28.56 ±

0.12% protein, 15.71 ± 0.14% fat, and 14.55 ± 0.11 % ash. Powder fish silage made

with 30 % rice bran by weight of liquid silage contained 10.91 ± 0.13 % moisture,

27.66 ± 0.10 % protein, 14.45 ± 0.11 % fat, and 15.27 ± 0.13 % ash. Powder fish

silage made with 40 % rice bran by weight of liquid silage contained 10.18 ± 0.05 %

moisture, 26.53 ± 0.18% protein, 13.73 ± 0.17 % fat, and 15.65 ± 0.15 % ash. Powder

fish silage made with 50 % rice bran by weight of liquid silage contained 9.18 ± 0.02

% moisture, 25.73 ± 0.08 % protein, 12.60 ± 0.10 % fat and 15.99 ± 0.12% ash.

Table 4.37 Drying rate of powder fish silage containing different concentration of

rice bran.

Hours Treatment

T1 T2 T3 T4 T5

0 67.63 63.63 56.36 53.83 50.91

6 61.83 58.92 52.87 48.27 44.76

12 57.00 53.41 47.38 43.71 39.61

18 54.17 49.9 43.99 39.15 35.46

24 51.34 45.39 38.6 35.15 31.22

30 45.51 40.88 32.4 29.59 26.16

36 40.68 35.37 26.21 24.03 19.31

42 35.8 30.86 20.84 18.47 15.16

48 30.41 25.76 13.23 11.88 9.84

54 25.37 21.81 - - -

60 21.16 17.62 - - -

66 16.03 14.25 - - -

72 15.42 13.92 - - -

Note: T1, T2, T3, T4 and T5 indicate Rice bran 10%, Rice bran 20%, Rice bran 30%,

Rice bran 40% and Rice bran 50% respectively

Table 4.38 ANOVA for Drying rate of powder fish silage containing different


Source of

Variation

Sum of

Square

Degree of

Freedom

Mean of


Rows 21342.01 12 1778.501 345.9544 2.7842 1.960121

Columns 3717.35 4 929.3376 180.775 2.6928 2.565241

Error 246.761 48 5.140853

Total 25306.12 64

F cal > F crit therefore, treatments are significantly different at p < 0.05

Table 4.39 SNK Test for drying rate of powder fish silage containing different


Note: T1, T2, T3, T4 and T5 indicate Rice bran 10%, Rice bran 20%, Rice bran

30%, Rice bran 40% and Rice bran 50% respectively


Treatment T1 T2 T3 T4 T5

Average 40.18077 36.28615 25.52923 23.39077 20.95615

Rank 5 4 3 2 1


5vs4 3.8946 0.6288 6.1933 5 4.039 SD

5vs3 14.6515 0.6288 23.2990 4 3.737 SD

5vs2 16.7900 0.6288 26.6996 3 3.399 SD

5vs1 19.2246 0.6288 30.5711 2 2.829 SD

4vs3 10.7569 0.6288 17.1058 4 3.737 SD

4vs2 12.8954 0.6288 20.5064 3 3.399 SD

4vs1 15.3300 0.6288 24.3779 2 2.829 SD

3vs2 2.1385 0.6288 3.4006 3 3.399 SD

3vs1 4.5731 0.6288 7.2721 2 2.829 SD

2vs1 2.4346 0.6288 3.8715 2 2.829 SD

Fig.4.15 Drying rate of powder fish silage containing different concentration of rice bran.

0

10

20

30

40

50

60

70

0 6 12 18 24 30 36 42 48 54 60 66

Mois

ture

%

Hours

Drying rate of powder fish silage containing different concentration of rice bran.

Rice bran 10%

Rice bran 20%

Rice bran 30%

Rice bran 40%

Ricebran 50%

Table 4.40 Proximate composition of powder fish silage made with different


(Mean ± standard error)

Fig 4.16 Proximate composition of powder fish silage made with different

quantity of rice bran.

0

5

10

15

20

25

30

Rice bran

10%

Rice bran

20%

Rice bran

30%

Rice Bran

40%

Rice Bran

50%

%

Proximate composition (%)

Protein

Fat

Moisture

Ash

Rice bran

(%)

Moisture (%) Protein (%) Fat (%) Ash (%)

10 13.74 ± 0.12 29.50 ± 0.22 16.28 ± 0.11 14.21 ± 0.12

20 12.54 ± 0.30 28.56 ± 0.12 15.71 ± 0.14 14.55 ± 0.11

30 10.91 ± 0.13 27.66 ± 0.10 14.45 ± 0.11 15.27 ± 0.13

40 10.18 ± 0.05 26.53 ± 0.18 13.73 ± 0.17 15.65 ± 0.15

50 9.18 ± 0.02 25.73 ± 0.08 12.60 ± 0.10 15.99 ± 0.12

Table 4.40.1a One-way ANOVA for protein content of powder fish silage made


Source of

Variation

Sum of

square

Degree of

freedom

Mean


Between

Groups 27.55136 4 6.88784 96.82999 5.93E-08 3.47805

Within

Groups 0.711333 10 0.071133

Total 28.26269 14


Table 4.40.1b SNK for protein content of powder fish silage made with different



Average 29.50333 28.56667 27.66667 26.53333 25.73333

Rank 5 4 3 2 1




5 Vs 4 0.936667 0.153984 6.082878 5 4.756 SD

5 Vs 3 1.836667 0.153984 11.92764 4 4.415 SD

5 Vs 2 2.97 0.153984 19.2877 3 3.949 SD

5 Vs 1 3.77 0.153984 24.48304 2 3.199 SD

4 Vs 3 0.9 0.153984 5.844758 4 4.415 SD

4 Vs 2 2.033333 0.153984 13.20482 3 3.949 SD

4 Vs 1 2.833333 0.153984 18.40017 2 3.199 SD

3 Vs 2 1.133333 0.153984 7.360066 3 3.949 SD

3 Vs 1 1.933333 0.153984 12.55541 2 3.199 SD

2 Vs 1 0.8 0.153984 5.195341 2 3.199 SD

SD: Significantly Different NSD: Not Significantly different

Table 4.40.2a One-way ANOVA for fat content of powder fish silage made with


Source of

Variation

Sum of

square

Degree of

freedom

Mean


Between

Groups 26.43971 4 6.609927 123.2123 1.8408 3.47805

Within Groups 0.536467 10 0.053647

Total 26.97617 14


Table 4.40.2b SNK for Fat content of powder fish silage made with different quantity

of rice bran.


Average 16.28333 15.71667 14.45 13.73667 12.60667

Rank 5 4 3 2 1




5 Vs 4 0.566667 0.133724 4.23757 5 4.756 NSD

5 Vs 3 1.833333 0.133724 13.70979 4 4.415 SD

5 Vs 2 2.546667 0.133724 19.04414 3 3.949 SD

5 Vs 1 3.676667 0.133724 27.49435 2 3.199 SD

4 Vs 3 1.266667 0.133724 9.472216 4 4.415 SD

4 Vs 2 1.98 0.133724 14.80657 3 3.949 SD

4 Vs 1 3.11 0.133724 23.25678 2 3.199 SD

3 Vs 2 0.713333 0.133724 5.334353 3 3.949 SD

3 Vs 1 1.843333 0.133724 13.78457 2 3.199 SD

2 Vs 1 1.13 0.133724 8.450214 2 3.199 SD


Table 4.40.3a One-way ANOVA for moisture content powder fish silage made


Source of

Variation

Sum of

square

Degree of

freedom

Mean


Between

Groups 40.27616 4 10.06904 126.2417 1.63E-08 3.47805

Within

Groups 0.7976 10 0.07976

Total 41.07376 14


Table 4.40.3b SNK for Moisture content of powder fish silage made with different



Average 13.74667 12.54667 10.91 10.18667 9.18

Rank 5 4 3 2 1



Comparison Difference SE q P q-crit. Conclusion

5 Vs 4

1.2 0.163054 7.359517 5 4.756 SD

5 Vs 3 2.836667 0.163054 17.39708 4 4.415 SD

5 Vs 2 3.56 0.163054 21.83323 3 3.949 SD

5 Vs 1 4.566667 0.163054 28.00705 2 3.199 SD

4 Vs 3 1.636667 0.163054 10.03756 4 4.415 SD

4 Vs 2 2.36 0.163054 14.47372 3 3.949 SD

4 Vs 1 3.366667 0.163054 20.64753 2 3.199 SD

3 Vs 2 0.723333 0.163054 4.436153 3 3.949 SD

3 Vs 1 1.73 0.163054 10.60997 2 3.199 SD

2 Vs 1 1.006667 0.163054 6.173817 2 3.199 SD


Table 4.40.4a One-way ANOVA for Ash content of powder fish silage made with


Source of

Variation

Sum of

square

Degree of

freedom

Mean


Between

Groups 6.642507 4 1.660627 35.11829 7.35E-06 3.47805

Within Groups 0.472867 10 0.047287

Total 7.115373 14

F cal > F crit therefore, treatments are significantly different at p < 0.05

Table 4.40.4b SNK test for Ash content of powder fish silage made with different



Average 14.21 14.55 15.27667 15.65 15.99

Rank 1 2 3 4 5


Rice bran 40% and Rice bran 50% respectively.


5 Vs 4 0.34 0.125548 2.708134 5 4.756 NSD

5 Vs 3 0.713333 0.125548 5.681772 4 4.415 SD

5 Vs 2 1.44 0.125548 11.46975 3 3.949 SD

5 Vs 1 1.78 0.125548 14.17788 2 3.199 SD

4 Vs 3 0.373333 0.125548 2.973638 4 4.415 NSD

4 Vs 2 1.1 0.125548 8.761611 3 3.949 SD

4 Vs 1 1.44 0.125548 11.46975 2 3.199 SD

3 Vs 2 0.726667 0.125548 5.787973 3 3.949 SD

3 Vs 1 1.066667 0.125548 8.496108 2 3.199 SD

2 Vs 1 0.34 0.125548 2.708134 2 3.199 NSD


Fig 4.17 Protein content of powder fish silage made with different quantity of

rice bran

Fig 4.18 Fat content of powder fish silage made with different quantity of rice

bran

23

24

25

26

27

28

29

30

Rice bran

10%

Rice bran

20%

Rice bran

30%

Rice bran

40%

Rice bran

50%

%

Protein (%)

0

2

4

6

8

10

12

14

16

18

Rice bran

10%

Rice bran

20%

Rice bran

30%

Rice bran

40%

Rice bran

50%

%

Fat (%)

Fig.4.19 Moisture content of powder fish silage made with different quantity of

rice bran

Fig.4.20 Ash content of powder fish silage made with different quantity of rice bran

0

2

4

6

8

10

12

14

Rice bran

10%

Rice bran

20%

Rice bran

30%

Rice bran

40%

Rice bran

50%

%

Moisture (%)

13

13.5

14

14.5

15

15.5

16

Rice bran

10%

Rice bran

20%

Rice bran

30%

Rice bran

40%

Rice bran

50%

%

Ash (%)

One-way ANOVA (shown in Table 4.40.1a, 4.40.2a, 4.40.3c and 4.40.4d) revealed

that all the treatments were significantly different. Rice bran 30 % obtained as good

quality powder so that it was carried for next experiment i.e. storage study.

4.5 Storage study of powder silage at ambient temperature.

4.5.1 Biochemical and microbial changes in 30 % Rice bran packed powder fish

silage during storage.

4.5.1.1. Changes in moisture:

The moisture content of packed powder silage increased from 10.91 ± 0.14 to

11.15 ± 0.10 % during the storage period from 0 to 90 days (shown in Table 4.41 and

Fig.4.21).

4.5.1.2. Changes in protein:

The crude protein content of packed powder silage decreased from 27.66 ±

0.10 to 27.04 ± 0.06 % during the storage period from 0 to 90 days (shown in Table

4.41.Fig.4.21).

4.5.1.3. Changes in Fat:

The Fat content of packed powder silage decreased from 14.45 ± 0.11 to

13.42± 0.10 % during the storage period from 0 to 90 days (shown in Table 4.41and

Fig.4.21).

4.5.1.4. Changes in ash:

The ash content of packed powder silage decreased from 15.27 ± 0.11 to 14.6

± 0.09 % during the storage period from 0 to 90 days (shown in Table 4.41and

Fig.4.21).

4.5.1.5. Changes in Fiber:

The fiber content of packed powder silage decreased from 9.89 ± 0.06 to 9.64 ±

0.07 % during the storage period from 0 to 90 days (shown in Table 4.41and

Fig.4.21).

4.5.1.6. Changes in carbohydrate:

The carbohydrate content of packed powder silage increased from 21.82 ± 0.07

to 23.14 ± 0.11 % during the storage period from 0 to 90 days (shown in Table 4.41

and Fig.4.21).

4.5.1.7. Changes in pH:

The pH of packed powder silage increased from 6.45 ± 0.05 to 6.59 ± 0.14 %

during the storage period from 0 to 90 days (shown in Table 4.41 and Fig.4.21).

4.5.1.8. Changes in TPC:

The TPC of packed powder silage increasing from 2.10 × 104 to 2.36× 10

4

cfu/g during the storage period from 0 to 90 days (shown in Table 4.41 and Fig.4.21)

Table 4.41 Biochemical and microbial changes in 30 % Rice bran packed powder silage during storage.

Days

Crude

Protein

(%)

Fat

(%)

Moisture

(%)

Ash

(%)

Fiber

(%)

Carbohydrate

(%)

pH

TPC (cfu/g)

(Log value)

0 27.66 ± 0.10 14.45± 0.11 10.91± 0.14 15.27±0.11 9.89±0.06 21.82 ± 0.07 6.45±0.05 2.10×10

4

(4.32)

30 27.31 ± 0.11 14.16±0.07 10.99± 0.10 15.07±0.08 9.84±0.06 22.21 ± 0.08 6.48±0.09 2.15×10

4

(4.33)

60 27.16 ± 0.33 13.83±0.08 11.06 ±0.18 14.87±0.06 9.75±0.13 22.64 ± 0.13 6.53± 0.10 2.25×10

4

(4.35)

90 27.04 ± 0.06 13.42± 0.10 11.15± 0.10 14.6 ± 0.09 9.64±0.07 23.14 ± 0.11 6.59±0.14 2.36×10

4

(4.37)

(Mean ± standard error)

Fig 4.21 Biochemical and microbial changes in 30 % Rice bran packed powder silage during storage.

0

5

10

15

20

25

30%

Biochemical and microbial changes in 30 % Rice bran packed powder silage during storage.

0 Day

30 Days

60 Days

90 Days

5.0 DISCUSSION

Fish market waste can successfully converted into powder fish silage by using

locally available cheap sources of molasses and curd for lactic acid fermentation. Rice

bran was used as co-drying material for drying liquid fish silage. This process is safe,

economical and eco-friendly as compared to fish meal. Various Biochemical and

microbiological quality changes in different treatments of silages and powder silage

during storage have been discussed in this chapter

5.1 Proximate composition of fish waste:

Proximate composition of fish waste is shown in Table 4.1 and Fig 4.1. The

moisture, crude protein, fat and ash content of fish waste were 77.09, 15.20, 4.03,

3.30 and 0.38 % respectively. Similar results were found by Palekar (2009) depicted

moisture content 79.47 % protein 15.78 %, lipid 2.13% and 2.32 % ash in fish waste.

Rahmi et al. (2008) reported similar result that Fish silage made from fish wastes

contains 13.62% protein, 8.8% lipid, 17.35% ash. Abowei and Tawari (2011) reported

that silage made from herring offal contained 13.5%, 8.7%, 75.4% and 2.6 % of crude

protein, lipid, moisture, ash respectively and silage made from white fish offal

contained 15.0, 0.5%, 78.9% and 4.2% of crude protein, lipid, moisture, ash

respectively. Gullu et al. (2015) depicted similar value of silage mixture contained

moisture 64.04 %, crude protein 16.84 %, lipid 10.81 % and ash 3.95 % respectively.

White fish offal contains 80 % water, 15 % protein, 4.5 % ash and 0.5 % fat analysed

by Tatterson and Windser (1974). Majumdar et al. (2014) reported similar result,

proximate analysis of freshwater fish viscera contained 68.42 % moisture, 15.59 %

protein, 12.81 % fat and 2.97% ash. Hasan and heath (1987) observed the proximate

composition of white perch fish waste contained moisture, protein, fat, and ash of

73.19%, 15.99%, 4.57% and 6.25% respectively. Similar results of moisture, ash,

protein, and lipid content of raw material (pony fish) were found to as 76.9%, 3.5%,

16.4% and 1.9%, respectively depicted by the Ozyurt et al. (2015).

5.2 Biochemical and microbiological analysis of fish waste.

Before starting experiment biochemical quality parameters and initial

microbial count of fish waste was analysed (shown in Table 4.2). The initial pH of

fish waste was 6.8. The fish waste had 10.64 mg-N100 g-1

of AAN which indicate

good quality of raw material. TVB-N and TPC of fish waste were 18.64 mg-N100g-1

and 5.1× 106 cfu/g respectively.

Similar results were depicted by Palekar (2009) reported pH, TVB-N, α amino

nitrogen (AAN) and TPC of pink pearch is 7.16, 18.68 mg-N100g-1

, 32.11 mg-

N100g-1

,6.7×106

cfu/g respectively. Similar results observed by Deepak et al. (2002).

They reported pH, TVB-N, α amino nitrogen (AAN) and TPC of Squilla were 8.5,

29.40 mg-N100g-1

, 34.99 mg-N100g-1

and 5.7 ×106

cfu/g prior to ensilation.

5.3 Proximate composition of rice bran:

Proximate composition of rice bran is shown in Table 4.3 and Fig 4.2. The

moisture, crude protein, lipid and ash content of rice bran were 9.45, 16.05, 13.42 and

10.44 respectively. Similarly Tao et al. (1993) analysed the proximate composition of

rice bran from the long grain and medium grain showed similar results. They reported

protein, lipid, ash, moisture, crude fibre and NFE content were 16.07 %, 19.20 %,

9.23 %, 11.20 %, 8.49 %, and 47.01 % in rice bran from long grain and 16.20 %,

21.97 %, 9.46 %, 10.83 %, 8.41 % and 44.07 % in rice bran from medium grain on

dry weight basis. Satter et al. (2014) analysed rice bran variety BRRI-28 that

contained 6.54 to 9.48 % moisture, 7.24 to 10.63 % ash, 12.26 to 14.01 % proteins,

23.53 to 27.86 % fats, 2.5 to 10.10 % fibre, 42.19 to 45.74 % carbohydrate.

Rice bran contained moisture, protein, fat, ash and carbohydrate were 4.30 and

5.40, 17.50 and 19.25, 13.10 and 17.20, 4.92 and 4.64, 52.33 and 48.55 g/100g

respectively. Similar results were given by the Bhosale and Vijayalakshmi (2015).

Hossain and Alam (2015) depicted slightly different results of rice bran. Rice bran

contained an average 9.32 % protein, 17.94% lipid, 18.67% ash and 9.65 % moisture

and 44.42 % NFE.

5.4 Biochemical and microbiological changes during storage.

5.4.1 pH:

5.4.1.1 Sulphuric acid:

In present study the pH of different treatment of sulphuric acid silage A1, A2,

A3 (sulphuric acid 2.5%, 3.5% and 4.5% respectively) were 2.97, 1.94, and 1.33

initially (shown in Table 4.4and Fig 4.3). In treatment A1 (sulphuric acid 2.5%), pH

was reached up to 4.62 at the end of 24th

day. Then sample was corrupted due to

increase in pH. It may be inadequate amount of acid used to prevent the activity of

putrefactive bacteria. In Treatments A2 and A3 (Sulphuric acid 3.5% and 4.5%) pH

was stable at 2.66 and 1.92 at the end of 30th

day respectively. Similar trends were

observed by Mousavi et al. (2013). They reported when 98% sulphuric acid with

weight percentage 2.5% used sample got musty after 20 days. Sulphuric acid with

weight 3.5% and 4.5% used pH of sample reached a stable level 2.58 and 1.94

respectively after 40 days. In the present study, on the basis of pH, sulphuric acid 3.5

% silage gets good. Sulphuric acid 4.5% had a low pH value and necessary

neutralizing it prior feeding to animals, so production cost increased.

5.4.1.2 Formic acid:

In present study the initial pH of different treatments of Formic acid silages

viz. B1, B2, B3 (Formic acid 2.5%, 3.5% and 4.5%) were 3.43, 3.14, and 2.73

respectively shown in Table 4.6 and Fig 4.4. In, Treatment B1 (Formic acid 2.5%),

pH was increased from 3.43 to 4.64 at the end of 24th

day. Then sample was corrupted

because amount of acid which used was incapable for prevent the activity of

putrefactive bacteria. pH in treatments B2 and B3 (Formic acid 3.5% and 4.5%) were

stable at 3.65 and 3.41 at the end of 24th

day respectively. Mousavi et al. (2013)

reported that when 98% Formic acid with weight percentage 2.5% used sample got

musty after 6 days at pH 3.61. Formic acid with weight 3.5% and 4.5% used pH of

sample reached a stable level 3.88 and 3.61 respectively after 56 and 66 days

respectively. Formic acid 3.5 % silage gets good.


The initial pH of Biological silage prepared using different percentages of

molasses viz. C1, C2 and C3 (Biological silage 5%, 10 % and 15%) were 6.85, 6.56

and 6.75 respectively (shown in Table 4.9 and Fig 4.5). In treatment C1 (Biological

silage 5%), pH was decreased from 6.85 to 5.24 at the end of 12th

day. Then sample

was corrupted. When fish: molasses ratio of 100:5 gave silage stable only up to few

days reported Kompiang et al. (1979). In, treatments C3 (Biological silage 15%) pH

were decrease and then increase and stable at 4.30 at the end of 24th

days respectively.

Treatment C2 (Biological silage 10%) got putrefied at the end of 30th

day. The last

measured pH was 4.65. Production of organic acid helps to reduction of pH value in

fermented silage and prevents the growth of spoilage organism. If sufficient

carbohydrate is not present in the medium, required levels of acid will not be

produced, as results of putrefying bacteria increased. Neethiselvan et al. (2002)

depicted similar results, pH of silage reduced below 4.5 and stabilized throughout the

storage study. Palekar (2009) also reported pH of silage was 7.28 initially, which

reached up to 4.42 at the end of 90th

day of storage. During storage, the pH value of

the silage remained stable below 4.5, which is recommended pH value for preserved

fish silage reported by Epse and Lied (1999).The slight fluctuation in pH during the

storage period in all silages was probably caused by the dissolving of fish skin, bones

and scales (Ozyurt et al.,2015).

5.4.2 Alpha Amino Nitrogen (AAN):


The AAN content of different treatment of sulphuric acid silage viz. A1, A2

and A3 (sulphuric acid 2.5%, 3.5% and 4.5%) were 25.27, 14.80, 11.89 mg-N 100g-

1initially and it reached up to 47.71 and 45.24 mg-N100 g

-1 at the end of 30

th day in

treatments A2 and A3 (sulphuric acid 3.5 and 4.5%) respectively (shown in Table

4.12 and Fig 4.6). There was no increase in AAN even after 30th

day of storage.

Treatment A3 (Sulphuric acid 4.5 %) showed slow rate of autolysis compared to

treatments A1 and A2 (sulphuric acid 2.5 and 3.5%). The rate of liquefaction of

proteins during silage production is different when organic and mineral acids are

used. This is due to difference in the pH produced by these acids. At pH 3 the rate of

autolysis and yield of soluble matter was less (Raa and Gildberg, 1982). High

concentration of acid is found inhibitory to proteolysis. Raghunath and Mc Curdy

(1990) reported that endo- and exopeptidases were active at pH 3.0, the silage quickly

breaking down the protein nitrogen to amino nitrogen. But in pH 2 silage, only acid

endopeptidase and a weak exopeptidase activity were detected, thus slowing the

autolysis. Stone and hardy (1986) reported that acid stabilised silage (sulphur 2.45%)

of pacific whiting, no increase in levels of amino nitrogen was noticed even after 42

days of storage indicating the absence of autolysis.


The AAN content of different treatments of Formic acid silage viz B1, B2 and

B3 (Formic acid 2.5%, 3.5% and 4.5%) were 26.97, 16.31, 14.76 mg-N 100g-1

initially

and it reached up to 52.15 and 49.02 mg-N100 g -1

at the end 24th

day in treatments B2

and B3 (Formic acid 3.5 and 4.5%) respectively shown in Table 4.15 and Fig 4.7.

There was no increase in AAN even after 24th

day of storage. Babu et al. (2005)

reported maximum alpha amino nitrogen value (as % of TN) was lower in 2.5% Acid

silage (21.21% of TN) than in 3% Acid Silage (24.33% of TN) and 2% Acid silage

(27.30% of TN). The similar results observed by Haaland and Njaa (1989). When,

acid addition was too low (1.4%) pH increase during storage, resulting in much higher

production of ammonia. Palekar (2009) reported AAN of formic acid was 39.76 mg-N

100g -1

which increased up to 272.69 mg-N 100g -1

at the end of 90th

day.


Different treatments of Biological silage viz.C1, C2 and C3 (Biological silage

5%, 10 % and 15% ) contained initial AAN were 16.61, 14.28 and 13.34 mg-N 100g-1

which increased up to 34.92 and 37.35 mg-N100 g -1

at the end 24th

day in treatments

C2 and C3 (biological 10% and 15 %) respectively shown in Table 4.18 and Fig.4.8.

There was no increase in AAN even after 24th

day of storage in treatments C2 and C3

(biological silage 10 % and 15%) respectively. Biological method produced lower

protein solubilisation value compared to acidified silage. Dapkeevicius et al. (1998)

reported similar results that preserving by biological methods offered lower protein

solubilisation values compared to acidified silage. Babu et al. (2005) observed AAN

values in FS were constant at 21% of TN by 15th day in both the concentrations (10

% FS and 12% FS) from an initial concentration of 11% of TN. Ozyurt et al.(2015)

depicted that AAN was 0.07g/100g initially increased up to 0.69 g/100g at the end of

60 days. Palekar (2009) observed that AAN in biological silage was 36.67 mg N100g

-1 which increase up to 157.00 mg N100g

-1 at the end of 90

th day of storage.

5.4.3 Total Volatile base Nitrogen (TVB-N):


TVB-N content of Sulphuric acid treatments A1, A2 and A3 (Sulphuric acid

2.5, 3.5 and 4.5%) were 16.85, 16.99 and 17.17 mg N100g -1

initialy which increased

up to 27.07, 26.29 mg N100g -1

in treatments A2 and A3 (Sulphuric acid 3.5 and

4.5%) respectively (shown in Table 4.21 and Fig 4.9).


TVB-N content of Formic acid treatments B1, B2, B3 (Formic acid 2.5, 3.5

and 4.5%) were 19.79, 17.13, 16.99 mg-N100g -1

initially which increased up to

27.16, 25.25 mg-N100g -1

in treatments B2 and B3 (Formic acid 3.5 and 4.5%)

respectively. The similar results were obtained by Haaland and Naaja (1989).

According to Ahmed and Mahendrakar (1996a) reported during fermentation of fresh

water fish viscera, the TVB-N values increased to 8% of the total nitrogen from initial

1.3%. Tanuja et al. (2014) showed that TVB-N level in both the treatments (with and

without antioxidant) were well below the limit of acceptability (35- 40 mg %) except

on the 90th day of storage. Palekar (2009) reported similar value of TVB-N in formic

acid silage increased from 18.22 mg-N 100 g-1

to 57.67 mg -N100g-1

at the end of 90th

day.


In case of biological silage TVB-N value of treatments C1, C2 and C3

(Biological silage 5%, 10%, 15%) were 22.73, 22.17, 19.83 mg N100g-1

initially and

which increased up to 57.35, 39.43, 33.55 mg N100g-1 at the end of 12th

,30th

,60th

day respectively. Faid et al. (1996) showed that TVN increased in trial 1 from 71.26

mg N100g -1

to reach 95.03 mg N100g -1

after1 day and remain constant around 132

mg N100g -1

after 15 days of fermentation at 22º C. The initial TVB-N of CS (Curd

silage) was 20.13 mg-N 100g-1

which increased up to 133.28 mg -N 100 g-1 at the

end of 90th

day reported by Palekar (2009).

5.4.4 Total Plate Count (TPC):

In, biological silage treatments C1, C2, C3 (Biological silage 5, 10, 15%)

shown TPC 3.16×106, 2.74×10

6, 4.50×10

6 cfu/g initially. The study showed that

decrease in TPC during storage. In Treatment C2 (Biological silage 10%) TPC was

decreased and then increased after 24 days. If sufficient carbohydrate is not present in

the medium, required levels of acid will not be produced, as results of putrefying

bacteria increased. The last measured TPC in C3 (Biological silage 15%) was 8.20

×103

cfu/g at the end of 60th

day (shown in Table 4.30 and 4.12). Ozyurt et al. (2015)

observed slightly similar result. They reported initial bacterial load was 4.09 log cfu/g

which decreased up to 5.77 cfu/g at the end of 60 days of storage. Palekar (2009) were

reported similar results. Curd silage shown the initial TPC was 8.70 ×106

cfu/g which

decreased to 4.26 ×103 cfu/g at the end of 90

th day.

Bello et al. (1993) studied

bacterial fish silage produced from several fish species mixed with molasses, fruits

sorbate (pineapple and papaya) and starter culture of lactobacillus plantarum. It was

found that the bacterial silage showed only few aerobic mesophilic organisms due to

the low pH values and the development of LAB. Rahmi et al. (2008) reported an

increased in lactic acid bacteria from 3.2 ×106 cfu/g to 4.8×10

9 cfu/g, Standard plate

count decreased.

5.4.5 Lactic Acid Bacteria (LAB):

In biological silage C1, C2 and C3 (Biological silage 5, 10, 15%) initial LAB

count were 2.73×106, 4.70×10

6, 2.24×10

6 cfu/g respectively. A sharp increase in LAB

count was observed after fermentation shown in (Table 4.33 and Fig. 4.13).The last

measured LAB in treatment C3 (Biological silage 15%) was 5.10 ×107

cfu/g at the

end of 60th

day. During storage study there was increase in LAB than observed

initially during storage. But at a certain stage LAB count was decreased due to

depletion in carbohydrate sources. Similar results were shown by Palekar (2009)

reported Curd silage shown the initial LAB was 1.07 ×106

cfu/g which decreased to

1.20 ×107 cfu/g at the end of 90

th day. Ozyurt et al. (2015) reported slightly similar

result. The initial LAB count was 2.82 log cfu/g which reached up to 7.59 cfu/g at day

14. Ahmed and Mahendrakar (1996b) depicted that lactobacilli begin to increase in

number and reach 109/g within 12 h. Zahar et al. (2002) observed in LAB count of

ensiled sardine waste (Head, viscera and frames) in sugar cane molasses 60:40(w/w)

in closed jar at 24°C increasing at starting and then reduction in LAB on logarithmic

cycle was reported.

5.4.6 Changes in proximate composition:

5.4.6.1 Protein:

In present study found that significant decrease in crude protein content of

silages with respect to increase in experimental duration as well as increase in

concentration of acid level. During the initial day of the experiment, the crude protein

content recorded was 15.20%. But at the end of the experiment, a fall in protein level

was noticed with considerable level of 13.11%, 12.91% in treatments A2, A3 (3.5 and

4.5 % sulphuric acid) ensilages and 13.03 %, 12.82 % in B2 and B3 (3.5 and 4.5 %

formic acid ensilages) respectively. In biological silage, treatment C3 (15 %

molasses) showed protein levels fall down from 15.20 % to 14.72 % at the end of 60

days. Reduction of protein content in the ensilage may be due to break down of

protein (FAO, 2007). Vidotti et al. (2003) observed a reduction in crude protein level

in combined (2% each of formic acid and sulfuric acid) fermented silage of tilapia

filleting residue when compared to non-fermented tilapia filleting residue.

Ramasubburayan et al. (2013) reported initial crude protein content were 40.62, 40.45

and 40.38 in 2, 2.5 and 3% formic acid ensilages, respectively. But at the end of the

experiment, a fall in protein level was noticed with considerable level of 38.40, 36.66

and 36.06 % in 2, 2.5 and 3% formic acid ensilages, respectively on dry matter basis.

Palekar (2009) reported similar results, that protein content was 15.78 % initially and

it was decreased up to 13.57, 15.03 and 15.55% in formic acid silage, lactobacillus

plantarum silage and curd silage respectively after 90 days of storage.

5.4.6.2 Fat:

The fat content in the present study revealed that at the beginning of the

experiment, it was an average 4.03 % in concentrations of sulphuric acid, formic acid,

and biological silages. But when the experimental days prolonged, the lipid content

increased at the end of the experiment, the increase in lipid content were 5.06 %, 5.24

% in treatments A2 and A3 (3.5 and 4.5 % sulphuric acid silages), 5.02 %, 5.46 % in

B2 and B3 (3.5 and 4.5 % formic acid silages) and 4.54 in C3 (biological silage with

15% of molasses) respectively. In the present study the continuous increase in lipid

content during storage period may be due to release of fats from raw material used.

Dapkevicius et al. (1998) reported 3.6% increase in lipid content from 11.3 to 14.9%

from initial to final stage (15 days) of storage of 3% formic acid ensilage of blue

whiting. The lipid content was between 8.08 and 8.27 % in 2, 2.5,3 % concentrations

of formic acid silages but at the end of the experiment, the increase in lipid content

increased between up to 10.66 and 12.24 respectively in the 2, 2.5, 3 % concentrations

of formic acid silages on dry matter basis reported by Ramasubburayan et al. (2013).

Babu et al. (2005) observed that fat content was lower in fermented silage (1.0 to

1.14%) than acid silage (3.67 to 5.13%). Palekar (2009) depicted that fat content in

formic acid silage, Lactobacillus plantarum silage and curd silage after 90 days of

storage was 3.38, 2.41 and 2.67 % respectively.

5.4.6.3 Moisture:

In present study found that significant decrease in moisture content of silages.

The initial moisture content recorded was 77.09 %. But at the end of the experiment, a

fall in moisture level was noticed with considerable level of 75.24 %, 75.54% in

treatments A2 and A3 (3.5 and 4.5 % sulphuric acid ensilages); 75.32%, 75.49% in

B2 and B3 (3.5 and 4.5 % formic acid ensilages) and 73.62 in C3 (biological silage 15

% molasses) respectively. Ozurt et al. (2015) observed moisture content was

decreased in all silages (Formic acid, Formic acid + Sulfuric acid, and streptococcus

thermophiles) except Lactobacillus plantarum. Fermented silage showed lesser

amount of moisture than acid silage. Palekar (2009) reported moisture content in

formic acid silage, Lactobacillus plantarum silage and curd silage after 90 days of

storage was 78.61, 77.62 and 75.91 % respectively. The reason for this may be

addition of lactic acid bacteria source such as curd might have increased the solid

matter and decreased the moisture level.

5.4.6.4 Ash:

Result of ash content in the present experiment showed decreasing rate with

respect to increase in concentrations of acid. During the initial day of the experiment,

the average ash content recorded was 3.30 %. But at the end of the experiment,

increase in ash level was noticed with considerable level of 5.12, 4.84 % in A2 and

A3 (3.5 and 4.5 % sulphuric acid silages); 5.03, 4.72 % in B2 and B3 (3.5 and 4.5 %

formic acid silages) and 5.89 % in C3 (biological silage with 15 % molasses)

respectively. Ozurt et al. (2015) reported ash content was increased in all silages

(Formic acid, Formic acid + Sulfuric acid, Lactobacillus plantarum and streptococcus

thermophiles). Neethiselven et al. (2002) depicted ash in curd, Lactobacillus

plantarum and FC silages 4.39, 3.37 and 3.35% respectively. Palekar (2009) reported

similar results that ash content in formic acid silage, Lactobacillus plantarum silage

and curd silage after 90 days of storage was 4.31, 4.62 and 4.85 % respectively. Babu

et al. (2005) observed that ash content was higher in fermented silage 5.16 to 5.68%

than acid silage (4.1 to 4.34%).

In present study found that biological silage had higher protein content

compare to inorganic and organic acid silage. So that biological silage was used for

preparation of fish silage powder. Fermented silage would be a good potential source

for animals in arid regions as it contained high protein (Al-Abri et al., 2014, Pagarkar

et al., 2006, Palekar, 2009). In all three methods of fish silage production (mineral

acid, organic acid and biological method), the optimum amount of sulphuric acid,

formic acid and molasses were determined 3.5%, 3.5% and 15% respectively.

5.5 Fish silage powder:

Fish silage is a liquid product. It can be difficult to storing and transporting,

but on other hand, it has good nutritive quality, which might be sufficient for animal

feeding. Liquid silage cannot be dried directly in sun because, some nutrients will be

drain out. The silage was therefore mixed with rice bran. Liquid silage was

neutralized by using 1.5% sodium carbonate (pH 6.45) and mixed with rice bran

before drying.

The current study demonstrated that fermented silage was successfully dried

with rice bran in solar tunnel drier. The moisture levels of co-dried mixtures were

reduced to less than 10% within 2-3 days. Solar tunnel dryer had temperature range

between 35°C to 50°C. In present study treatment T1 and T2 (10 and 20 % rice bran)

took 72 hours to lower down the moisture content below 15 %. Three mixtures i.e.

T3, T4 and T5 (30%, 40% 50 % rice bran) reach to equilibrium moisture content after

48 hours. Similar trend was observed by Al-Abri et al. (2014), Goddard and Al-

Yahyai (2001) and Goddard and Perret (2005).

5.5.1 Proximate composition of powder fish silage made with different quantity

of rice bran

In the present study the protein content of 10% , 20%, 30 %, 40 % and 50%

rice bran were 29.50%, 28.56%, 27.66%, 26.53% and 25.73% respectively. Fat

content of 10%, 20%, 30 %, 40 % and 50% rice bran were 16.28%, 15.71%, 14.45%,

13.73% and 12.60% respectively. Moisture and ash content of 10%, 20%, 30 %, 40 %

and 50% rice bran were 13.74%, 12.54%, 10.91%, 10.18%, 9.18 % and 14.21%,

14.55%, 15.27%, 15.65%, 15.99% respectively. Similar trends were reported by

Hossain and Alam (2015), protein content of 20 %, 30 %, 40 % and 50% rice bran

were 21.75, 20.84, 19.87 and 18.73 % respectively. Fat content of 20%, 30 %, 40 %

and 50% rice bran were 34.71, 33.73, 32.88 and 30.74 respectively. Moisture and ash

content of 20%, 30 %, 40 % and 50% rice bran were 11.68, 10.83, 10.17, 9.66 % and

13.36, 14.05, 14.28, 14.55 % respectively.

With increasing the quantity of rice bran, the levels of protein, lipid, ash and

moisture were decreased. Nutrient content of 40 and 50% rice bran were low

compared to 10, 20 and 30 % rice bran similar observation were reported by Hossain

and Alam (2015). At 30% rice bran it was found that the protein content was enhanced to

about 10 % and 20 %and other nutrient contents were also comparatively acceptable for

animal feeding.

It was possible to increase protein content more by decreasing the quantity of rice

bran. The mixture of 10 % and 20 % rice bran might be difficult to dry due to the lesser

content of dry matter and there would be a probability of mould attack. The product with

lower rice bran will not be stable for long time use. Therefore, considering all the

limitation, powder fish silage with 30 % rice bran was found to be better and carried for

further storage study. Similar observation was discussed by Hossain and Alam ( 2015).

5.5.2 Storage study of powder silage at ambient temperature

5.5.2.1 Biochemical and microbial changes in 30 % Rice bran packaged fish

silage powder during storage

5.5.2.1.1 Changes in Moisture:

In the present study the moisture content of powder fish silage sample was found

to be increasing in trends during through the storage study (0 to 90 days) in the range of

10.91 to 11.15 %. Similar trend were observed by Hossain and Alam (2015) reported

moisture content of 30% rice bran was increased from 10.83 to 10.98 % at the end of

4 months.

5.5.2.1.2 Changes in Protein:

In the present study the protein content of powder fish silage sample was found

to be decreasing in trends during through the storage study (0 to 90 days) in the range of

27.66 to 27.04 %.%. Hossain and Alam (2015) reported protein content of 30% rice

bran was decreasing from 20.84 to 20.30 % at the end of 4 months.

5.5.2.1.3 Changes in Fat:

In the present study the fat content of powder fish silage sample was found to be

decreasing in trends during through the storage study (0 to 90 days) in the range of 14.45

to 13.42 %. Hossain and Alam (2015) reported lipid content of 30% rice bran was

decreasing from 33.73 to 32.41 % at the end of 4 months.

5.5.2.1.4 Changes in Ash:

In the present study the ash content of powder fish silage sample was found to be

decreasing in trends during through the storage study (0 to 90 days) in the range of 15.27

to 14.60 %. Hossain and Alam (2015) reported ash content of 30% rice bran was

decreasing from 14.05 to13.49 % at the end of 4 months.

5.5.2.1.5 Changes in Fiber:

In the present study the fiber content of powder fish silage sample was found to

be decreasing in trends during through the storage study (0 to 90 days) in the range of

9.89 to 9.64 %. Hossain and Alam (2015) reported fiber content of 30% rice bran was

decreasing from 6.61 to 6.32 % at the end of 4 months.

5.5.2.1.6 Changes in Carbohydrate:

In the present study the carbohydrate content of powder fish silage sample was

found to be increasing in trends during through the storage study (0 to 90 days) in the

range of 21.82 to 23.14 %. Hossain and Alam (2015) reported carbohydrate content of

30% rice bran was increasing from 13.94 to 16.50 % at the end of 4 months.

5.5.2.1.7 Changes in pH:

In the present study the pH of powder fish silage sample was found to be

increasing in trends during through the storage study (0 to 90 days) in the range of 6.45 to

6.59.Hossain and Alam (2015) reported pH of 30% rice bran was increasing from 6.54

to 6.76 at the end of 4 months.

5.5.2.1.8 Changes in TPC:

In the present study the TPC content of powder fish silage sample was found to

be increasing in trends during through the storage study (0 to 90 days) in the range of

2.10×104 to 2.36 ×104 cfu/g.

6.0 SUMMARY

Fish is one of the most important sources of animal protein available

worldwide and has been widely accepted as a good source of protein and other

elements for the maintenance of healthy body. Utilized fishes were sold locally as

fresh fish and remaining used for preparation of value added products or preparation

of fish meal. Processing of seafood generates large amount of fish waste which

includes heads, detoriated fillets, scales, skins, gut, roes etc. These wastes are

potential source of pollution. So that fish waste was successfully transfer eco-friendly

into silage and prepared aqua feed to reduce the cost of aqua feed production.

In the present study an attempt was made to study the different methods of

silage preparation, fish silage powder using fish market waste and biochemical,

microbiological changes during storage. The important findings are summarised as:

6.1 Proximate analysis of fish waste:

The proximate composition of fish waste was observed to be moisture 77.09 ±

0.14 %, crude protein 15.20 ± 0.15 %, fat 4.03 ± 0.07 % and ash 3.30 ± 0.11 %.

6.2 Biochemical and microbiological analysis of fish waste:

The pH, α- amino nitrogen, TVB-N and TPC of fish waste were 6.8 ±0.49,

10.64 ± 0.13 mg-N100 g -1

,18.64 ± 0.09 mg-N100g -1

, and 5.1× 106 cfu/g respectively.

6.3 Proximate composition of rice bran.

The proximate composition of rice bran was observed to be moisture 9.45±

0.19 %, crude protein 16.05 ± 0.08 %, fat 13.42 ± 0.15 % and ash 10.44 ±0.14 %.

6.4 Different methods of silage preparation and their qualities

Fish market waste was used for preparation of silage. Fish silage was prepared

by three different methods viz. Inorganic acid (sulphuric acid), organic acid (Formic

acid) and biological method. Inorganic and organic acid silage were prepared by using

weight percentages i.e. 2.5, 3.5, 4.5% respectively. Biological silage also prepared by

using weight percentage of 5, 10, 15% molasses and curd used as lactic acid bacteria

source for fermentation. These silages were stored at 27-30 °C and analysed various

changes in pH, AAN, TVB-N, TPC, and LAB during 60 days of storage.

In sulphuric acid silage rate of autolysis was slow compared to formic acid

and biological silage due to low pH. The rate of AAN and TVB-N were gradually

increased and then stable at a point. The last measured AAN and TVB-N in sulphuric

acid 3.5%, 4.5% at the end of 60 days were 47.71, 45.24 and 27.07, 26.29 mg-N100g

1 respectively. Sulphuric acid 2.5% concentration silage gets corrupted at the end of

24 days. In sulphuric acid, 3.5% concentration gets good.

In formic acid silage 3.5%, 4.5% last measured AAN and TVB-N were 52.15,

49.02 and 27.16, 25.25 mg-N100g-1

respectively at the end of 60 days. In formic acid

silage 3.5% concentration get good. Formic acid 2.5% concentration silage gets

corrupted at the end of 24 days.

In biological method 5%, 10% molasses silage gets corrupted at the end of

12th

and 30th

respectively. The last measured AAN and TVB-N in 15 % molasses was

37.25 and 33.55 mg-N100g-1

respectively at the end of 60 days. TPC was gradually

decreased from 4.50×106 to 8.20×10

3 cfu/g. LAB was increased and then decreased

from 2.24×106 to 5.10×10

7 cfu/g at the end of 60 days.

In present study found that biological silage had higher protein content

compare to inorganic and organic acid silage. So that biological silage was used for

preparation of fish silage powder. Fish silage is a liquid product, difficult to storing

and transporting. So preparation of fish silage powder, liquid silage was neutralized

by using 1.5% sodium carbonate (pH 6.45) and mixed with rice bran before drying.

The mixtures were dried in solar tunnel drier within 2-3 days.

6.5 Proximate composition of powder fish silage made with different quantity of

rice bran

The proximate composition of powder fish silage made with different quantity

of rice bran 10, 20, 30, 40 and 50% contained moisture content as 13.74 ± 0.12, 12.54

± 0.30, 10.91 ± 0.13, 10.18 ± 0.05, 9.18 ± 0.02 % respectively; crude protein content

as 29.50 ± 0.22, 28.56 ± 0.12, 27.66 ± 0.10, 26.53 ± 0.18, 25.73 ± 0.08 %

respectively; fat content as 16.28 ± 0.11, 15.71 ± 0.14, 14.45 ± 0.11, 13.73 ± 0.17,

12.60 ± 0.10% respectively; ash content 14.21 ± 0.12, 14.55 ± 0.11, 15.27 ± 0.13,

15.65 ± 0.15, 15.99 ± 0.12 % respectively. Considering all the limitation, powder fish

silage with 30 % rice bran was found to be better and carried for further storage study.

6.6 Storage study of powder fish silage with 30% rice bran at ambient

temperature.

Moisture content was increased in the range from10.91 ± 0.14 to 11.15 ± 0.10

%, whereas the protein content was decreased in the range from 27.66 ± 0.10 to 27.04

± 0.06 %, fat content decreased in the range from 14.45 ± 0.11 to 13.42 ± 0.10 %, ash

content decreased in the range from 15.27 ± 0.11 to 14.60 ± 0.09 %, fiber content

decreased in the range from 9.89 ± 0.06 to 9.64 ± 0.07 %, Carbohydrate content

increased in the range from 21.82 ± 0.07 to 23.14 ± 0.11 %, pH increased in the range

from 6.45 ± 0.05 to 6.59 ± 0.14 and TPC increased in the range from 2.10×104 to 2.36

×104 cfu/g during the storage period from 0 to 90 days.

6.7 Conclusion

It can be concluded that fish market waste is suitable for preparation of fish

silage powder. The optimum amount of sulphuric acid, formic acid and molasses for

preparation of silage were determined as 3.5%, 3.5% and 15% respectively. Powder

fish silage could be stored up to more than 3 months at ambient temperature without

the loss of major nutrient components.

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