production of protein concentrate by enzymatic hydrolysis of shrimp (l. vannamei) head

PRODUCTION OF PROTEIN CONCENTRATE BY ENZYMATIC HYDROLYSIS OF SHRIMP (L. vannamei) HEAD

By

JUDITH SALIM

A Bachelor’s Thesis

Submitted to the Faculty of

LIFE SCIENCE

Department of FOOD TECHNOLOGY

in partial fulfillment of the

requirements for

BACHELOR’S DEGREE

IN

FOOD TECHNOLOGY

Swiss German University

EduTown BSDCity Tangerang 15339

INDONESIA

July 2011

Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head of 100

Judith Salim

STATEMENT BY THE AUTHOR

I hereby declare that this submission is my own work and to the best of my

knowledge, contains no material previously published or written by another person,

nor material which to a substantial extent has been accepted for the award of any

other degree or diploma at any educational institution, except where due

acknowledgement is made in the thesis.

_______________________________________ ________________

Judith Salim Date

Approved by:

________________________________________ __________________

Dr. Singgih Wibowo, MS (Advisor) Date

________________________________________ __________________

Ir. Murniyati (Co-Advisor) Date

______________________________________ _________________

Chairman of the Examination Steering Committee Date


Judith Salim

ABSTRACT

PRODUCTION OF SHRIMP PROTEIN CONCENTRATE BY ENZYMATIC HYDROLYSIS OF SHRIMP (L. vannamei) HEAD

By

Judith Salim

SWISS GERMAN UNIVERISTY

Bumi Serpong Damai

Dr. Singgih Wibowo, MS, Major Advisor

Shrimp head is often considered as waste and not as by-products from shrimp

processing. However, the head itself takes up 29% of the shrimp and contains many

nutrients. One of the most abundant nutrients is protein. To improve the digestibility

and palatability of shrimp head, enzymatic hydrolysis was done. There were two types

of enzyme that were used, which were pure and crude papain. The treatments were

enzyme concentration (10%, 20%, and 30%) and temperature (45oC, 50oC, 55oC,

60oC). The highest protein content was produced by pure papain at 50oC incubation

temperature. The concentration did not seem to have a significant effect on soluble

protein content. However, the sensory level of acceptance of products hydrolyzed

with pure enzyme was lower than those hydrolyzed by crude papain. On the other

hand, hydrolysis by crude papain produced products with lower water content and

higher ash content of minerals compared to hydrolysis products of pure papain.


Judith Salim

DEDICATION

I dedicate this thesis to God because He still loves me that He gave me these

obstacles, my lovely family who supports me all the way, and mankind.


Judith Salim

ACKNOWLEDGMENTS

First of all, I want to thank God the Almighty for His guidance and blessing during

thesis work that I can complete this thesis on time. There are so many people involved

in the making of this thesis. I would like to thank all people that had helped me

through this process. I would like to express my gratitude and appreciation to:

1. Dr. Singgih Wibowo, MS as Thesis Advisor, for his great help, assistance,

guidance and advice to complete this thesis.

2. Ir. Murniyati as Thesis Co-Advisor, for her great help, assistance, guidance,

and advice to complete this thesis.

3. Prof. (ris.) Hari Eko Irianto, Ir. PhD., who allows me to do my thesis work at

Research Center for Marine and Fisheries Product Processing and

Biotechnology (Balai Besar Riset Pengolahan Produk dan Bioteknologi

Kelautan dan Perikanan).

4. Research Center for Marine and Fisheries Product Processing and

Biotechnology which gives me facilities and financial support during this

completion of thesis.

5. Ms. Nurhayati, Mrs. Hasta, Mr. Yayat, Mr. Tazwir, Mrs. Fateha, Mrs. Rury,

Ms. Wiwi, and all laboratories personnels who give me a lot of information

and help me to complete my thesis.

6. My parents Karel and Iim, my sisters Stephanie and Vania, and all of my

families who have support me during my work on this thesis. I cannot thank

you enough.

7. Mr. Tabligh Permana that has provided me with knowledge in laboratory and

gave his advices through my laboratory and report works.

8. All of my classmates at SGU that has helped me finish this thesis by

supporting me when I was down and helping me when I needed a hand.

9. Debby Ardi, Seruni Marshella, and Sheila Ariani for their continuous support

and helping me a lot in this thesis work.

10. Some other people that I cannot mention one by one, thank you for your

contribution in completing this thesis.


Judith Salim

I realize that this thesis is far from perfect. Therefore, any comments and critics

will be welcomed in order to improve this thesis report. I hope that all my hard

works can give my benefits and contribution for academic purpose especially

Food Technology Faculty, readers and the world.

Jakarta, July 2011

Judith Salim


Judith Salim

TABLE OF CONTENTS

STATEMENT BY THE AUTHOR ............................................................................... 2 ABSTRACT ................................................................................................................... 3 DEDICATION ............................................................................................................... 4 ACKNOWLEDGMENTS ............................................................................................. 5 CHAPTER 1 – INTRODUCTION .............................................................................. 12

1.1. Background ................................................................................................. 12 1.2. Research Problem ........................................................................................ 13 1.3. Research Objectives .................................................................................... 14 1.4. Hypothesis ................................................................................................... 14 1.5. Research Scope ............................................................................................ 15

CHAPTER 2 – LITERATURE REVIEW ................................................................... 16 2.1. Shrimp ......................................................................................................... 16

2.1.1. Litopenaeus vannamei ........................................................................... 17

2.1.2. Utilization of shrimp waste .................................................................... 19

2.2. Protein ......................................................................................................... 20 2.3. Enzyme ........................................................................................................ 22

2.3.1. Protease .................................................................................................. 24

2.3.2. Papain ..................................................................................................... 25

2.4. Protein hydrolysis ........................................................................................ 27 2.4.1. Chemical hydrolysis ............................................................................... 27

2.4.2. Enzymatic hydrolysis ............................................................................. 27

2.5. Protein hydrolysate ...................................................................................... 28 2.5.1. Shrimp protein hydrolysate .................................................................... 29

2.5.2 Quality Standards for Fish Protein Hydrolysate .................................... 31

2.6. Zero Waste Concept .................................................................................... 33 CHAPTER 3 – METHODOLOGY ............................................................................. 35

3.1. Time and Venue .......................................................................................... 35 3.2. Materials ...................................................................................................... 35

3.2.1. Raw Materials ........................................................................................ 35

3.2.2. Chemicals ............................................................................................... 35

3.3. Equipments .................................................................................................. 36 3.4. Procedures ................................................................................................... 36

3.4.1. Examination of raw material .................................................................. 36

3.4.2. Assay of enzyme papain (food grade and pure) ..................................... 38

3.4.3. Hydrolysis of shrimp head waste (Limam, 2008) .................................. 38

3.4.4. Analysis of proximate composition and yield of hydrolysate ............... 39

3.4.5. Sensory evaluation ................................................................................. 41

3.5. Experimental Design ................................................................................... 41


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3.6. Data Analysis .............................................................................................. 42 CHAPTER 4 – RESULT & DISCUSSION................................................................. 43

4.1. Proximate composition of shrimp head (L.vannamei) ................................ 43 4.2. Determination of enzyme activity ............................................................... 43 4.3. Effect of incubation time and filtration after centrifugation to protein concentration ............................................................................................................ 44 4.4. Effect of pH to protein concentration .......................................................... 45 4.5. Effect of centrifugation and filtration using muslin cloth to protein content 45 4.6. Analyses of yield and proximate compositions to treatments type of papain, concentration of papain, and temperature of incubation .......................................... 46

4.6.1. Yield ....................................................................................................... 48

4.6.2. Water Content ........................................................................................ 50

4.6.3. Ash Content ........................................................................................... 52

4.6.4. Protein Content ...................................................................................... 55

4.6.5. Fat Content ............................................................................................. 61

4.7. Sensory Evaluation ...................................................................................... 63 CHAPTER 5 - CONCLUSIONS AND RECOMMENDATIONS .............................. 66

5.1. Conclusion ................................................................................................... 66 5.2. Recommendation ......................................................................................... 66

LIST OF REFERENCES ............................................................................................. 67 APPENDICES ............................................................................................................. 72 CURRICULUM VITAE .............................................................................................. 99


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LIST OF TABLES

Table 2.1 Indispensable (essential) amino acid requirement……………..………….. 20

Table 2.2. Comparison of crude papain and pure papain……………………………. 23

Table 4.1 Proximate composition of L. vannamei head and P. monodon head…..… 41

Table 4.2 Data summary of yield and proximate analyses………………………….. 45

Table 4.3 Recovered protein of each treatment…………….……………………….. 56


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LIST OF FIGURES

Figure 2.1Proximate composition of raw mixed shrimps…………...……………….. 14

Figure 2.2 Litopenaeus vannamei……………………………………………………. 15

Figure 2.3 Anatomy of L. vannamei………………………………………………… 16

Figure 2.4 Hydrolysis mechanism of papain…………………………………………. 24

Figure 2.5 Material flows today……………………………………………………… 31

Figure 2.6 Improved material flows………………………………………………….. 31

Figure 4.1 Graph of concentration versus yield………………………………………. 46

Figure 4.2 Graph of temperature versus yield……………………………………….. 47

Figure 4.3 Graph of concentration versus water content…………………………….. 48

Figure 4.4 Graph of temperature versus water content……………………………….. 49

Figure 4.5 Graph of concentration versus ash content…………….………………… 51

Figure 4.6 Graph of temperature versus ash content………………………………… 52

Figure 4.7 Comparison of ash of hydrolysate………………………………………. 52

Figure 4.8 Graph of concentration versus protein content…………………………… 53

Figure 4.9 Graph of temperature versus protein content…………………………….. 54

Figure 4.10 Graph of concentration versus recovered protein………………………. 56

Figure 4. 11 Graph of temperature versus recovered protein………………………… 57

Figure 4.12 Graph of concentration versus protein content (dry basis)……………… 58

Figure 4.13 Graph of temperature versus protein content (dry basis)……………….. 59

Figure 4.14 Result of centrifugation of hydrolysate from pure papain……………… 61

Figure 4.15 Result of centrifugation of hydrolysate from crude papain……….. 61

Figure 4.16 Condition of sensory evaluation…..……………………………………. 62

Figure 4.17 Result of hedonic test…………………………………………………… 78


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LIST OF APPENDICES

Appendix 1 Standard curve of Lowry………………………………………… 72

Appendix 2 Standard Curve for Enzyme Activity……………………………. 72

Appendix 3 Statistical analysis of yield………………………………………. 73

Appendix 4 Statistical analysis of water content……………………………… 77

Appendix 5 Statistical analysis of ash content………………………………… 81

Appendix 6 Statistical analysis of protein content…………………………….. 84

Appendix 7 Statistical analysis of recovered protein………………………….. 85

Appendix 8 Two-way ANOVA of appearance in hedonic test………………... 89

Appendix 9 Two-way ANOVA of color in hedonic test……………………… 90

Appendix 10 Two-way ANOVA of smell in hedonic test………………………. 90

Appendix 11 t-test between samples for smell………………………………….. 91

Appendix 12 Two-way ANOVA of taste in hedonic test……………………….. 95

Appendix 13 t-test between samples for taste…………………………………… 96


Judith Salim

CHAPTER 1 – INTRODUCTION

1.1. Background

Wastes mostly become an issue to the environment. Some usually produce

terrible smell; others may contain toxic substances in it. There are many ways

to overcome this problem. One of the solutions is to use the waste as raw

material to make other products. This utilization of waste will increase the

value of the product and also solve the environmental problems.

Litopenaeus vannamei, also known as white leg shrimp or Pacific white

shrimp, is produced widely in Indonesia, although it is not originated from

Asia. It was first introduced to Philippines in 1978. Indonesia began to

produce this shrimp later than other Asian countries, which was in 2001. There

is a significant increase in the production from 5000 tons in 2002 (Briggs et

al., 2004) to approximately 198 kilo tons in 2009 (Ministry of Marine Affairs

and Fisheries, 2009).

In L. vannamei processing (IQF shrimp or block frozen shrimp), the shell and

head are usually thrown away, producing a terrible odor. The head itself took

about 36-49% of total weight of the shrimp (Purwaningsih, 2000 in Sulastri,

2009). By turning the shrimp head to protein concentrate, it may increase the

value of the waste and producing an alternative nutraceutical for human. The

protein concentrate that exists in Indonesia comes from various sources, such

as milk, soy bean, sesame, and also fish. However, these sources are able to be

sold in their unprocessed condition, whereas the waste cannot.

To obtain such protein concentrate from the shrimp head, enzymatic

hydrolysis of the head will be needed. Shrimp head contains chitin, which bind

to the protein and makes it not digestible by human digestion system. Both of

them can be separated by hydrolyzing the shrimp head by either enzymes or

chemicals. This study will use enzyme instead of chemical because enzyme is


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specific and not toxic, consume less energy, and produce less byproducts. It is

more environmental friendly than chemicals because some chemicals can

produce toxic waste which can harm the environment.

1.2. Research Problem

Conducting hydrolysis using enzyme must be done correctly. The first

consideration is the type of enzyme. Enzyme is divided to 6 classes but the

one that is used to hydrolyze substance is hydrolase. Hydrolase can hydrolyze

large molecules to smaller ones. In this case, protein is the molecules that will

be degraded to small peptides and amino acids. Therefore, the type of

hydrolase enzyme that will be used is protease.

The consideration of enzyme selection depends on price, availability,

concentration needed, and condition of hydrolysis. Past researches about

shrimp hydrolysate indicates that serine protease, such as trypsin (Limam et

al., 2008), chymotrypsin (Simpson et al., 1997), and Alcalase © (Mizani et al.,

2007) were used to hydrolyze the shrimp head. However, these enzymes are

not available widely in Indonesia and it might cause a problem during the

research. Instead papain enzyme, which is a cysteine protease, is used as

hydrolase enzyme for protein hydrolysate. Papain enzyme has been used to

hydrolyze both shrimp (Valdez-Pena et al., 2010) and fish (Hosomi et al.,

2010). However, the hydrolysis of shrimp did by Valdez-Pena only a

comparison of hydrolytic activity between crude enzymes and not find a way

to optimize amount of the protein hydrolysate produced.

There are two types of papain enzyme that will be used in this experiment.

The first one is crude papain, which is sold widely in supermarket as meat

tenderizer. This enzyme also contains other additives like salt and sugar.

Crude papain doesn’t undergo purification process. Hence, it has lower

enzyme activity than pure papain. The second one is pure papain. This is a

product of purification. Pure papain does not contain any additives. It also has

higher enzyme activity. However, the crude papain is cheaper and it exists

widely. Therefore, the ability of pure papain and crude papain to hydrolyze


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should be monitored whether both enzymes, which concentration has been

equalized, produce insignificantly different protein concentrate.

Papain works optimally at neutral pH but it catalyzes reaction at relatively

high temperature (50-60oC). If the temperature is too high, it will result in

denaturation of protein and causing the protein to lose its beneficial contents.

Temperature should be examined carefully in order to avoid denaturation of

protein. Concentration of enzyme added will also affect the catalytic activity.

The higher the enzyme activity, the higher the rate of reaction will be.

However, it also has a maximum rate of reaction, so another addition after that

will not affect the rate anymore. Therefore, it is important to know how much

papain should be added to the concentration.

Another common problem in seafood hydrolysates is bitterness. This

bitterness is caused by some amino acids and small peptides (Belitz et al.,

2009). The protein that is produced for human consumption should not have

bitter taste because it is somehow unlikable. Descriptive sensory evaluation

must be conducted in order to examine the taste and the odor of the protein

concentrate.

1.3. Research Objectives

The objectives of this research are

To determine which papain enzyme produce protein concentrate with

better protein content.

To find the effective concentration of papain enzyme for hydrolysis.

To find the optimum condition for papain enzyme.

To determine which protein concentrate has more acceptable sensory

score.

1.4. Hypothesis

1. Crude papain can hydrolyze shrimp head as good as pure papain, in

terms of protein content.

2. The effective concentration of papain enzyme for hydrolysis is 10%.

3. The optimum condition for papain enzyme to hydrolyze is at 45 – 60oC


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4. Protein concentrate from hydrolysis of crude papain will have a better

sensory score than the pure papain.

1.5. Research Scope

This study was targeted to hydrolyze the waste from shrimp production

(shrimp head) to produce nutritive protein concentrate. Protein concentrate

was aimed to be consumable product for human. Since the separation of

quality in shrimp protein concentrate has not existed, the quality reference

would be from fish protein concentrate that was regulated from FAO.

Hydrolysis using enzyme was done so that the product was environmentally

friendly and safe for human consumption. Enzyme that was used for this

hydrolysis was papain because it was available widely in Indonesia, has a

good hydrolysis capability, and was available at low price. The optimum

condition and concentration for this hydrolysis was experimented, so that

hydrolysate contained high yield of protein.


CHAPTER 2 – LITERATURE REVIEW

2.1. Shrimp

Shrimp is known as a crustacean. Sometimes it is falsely identified as prawn.

Although by taxonomy division both types are the same from its kingdom to

its sub-ordo. The main difference between shrimp and prawn lies in its family

and below. Shrimp comes from the family Penaeidae, whereas prawn comes

from the family Caridea (Wickins and Lee, 2002).

Besides its fine texture and delicious taste, shrimp is also nutritious. Shrimp is

known to contain high content of protein. Shrimp, in general, contains 20.31 g

of protein from 100 g. Its moisture content is 75.86 g and it has 1.2 g of ash

content.

Figure 2.1 Proximate Composition of Raw Mixed Shrimp Source: USDA National Nutrient Database for Standard Reference, Release 23 (2010)


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2.1.1. Litopenaeus vannamei Litopenaeus vannamei (L. vannamei) is originated in Mexico and spread

around Latin America. This broodstock of this shrip was then imported to

Indonesia. L. vannamei is also known as Pacific white shrimp or whiteleg

shrimp. This shrimp is also included in the family of Penaidae. Adult shrimp is

a part of marine animal, but during their early stages of development

(juvenile), they move to the estuaries and after reaching adult phase they move

back to the ocean (Wickins and Lee, 2002). The taxonomy of L. vannamei can

be seen below (Boone, 1931 in Sulastri, 1999).

Kingdom : Animalia

Subkingdom : Metozoa

Phylum : Arthropoda

Subphylum : Crustacea

Class : Malacostraca

Subclass : Eumalacosteraca

Superordo : Euracida

Ordo : Decapoda

Subordo : Denderobrachiata

Family : Penaeidae

Genus : Litopeneus

Species : Litopenaus vannamei

Figure 2.2 Litopenaeus vannamei Source: http://sysbio.iis.sinica.edu.tw/page/ (2011)


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L. vannamei’s body is made of two branches (Biromous), which are exopodite

and endopodite. Vannamei’s anatomy can be divided to two parts:

Head (thorax)

Its head contains antenula, antenna, mandibular, and two pair maxillae. It also

has three pairs of maxilliped (organ which is used to take up food), 5 pairs of

pereipodes (this is used to walk). Pereiopodes are segmented. It is divided to

type, with clamp or without. The fourth and fifth pereipodes don’t have

clamps but the first until third pereipodes have clamps.

Stomach (abdomen)

The abdomen is made of six segments. At this part, there are five pairs of

pleopods (this organ is used to swim) and a pair of uropodes (this is similar to

tail) which is fan-like shaped.

Figure 2.3 Anatomy of L. vannamei Source: Bondad-Reantoso et al. (2001)

Due to the several advantages, Indonesia started to culture this shrimp in 2000.

The originally cultured shrimp species in Indonesia is Penaeus monodon and

Penaeus merguiensis. However, there was an outbreak of WSSV and YHV in

Penaeus monodon (Bondad-Reantoso et al., 2001). This was the main reason

for importing the broodstock for L. vannamei. The shrimp that is produced in

Indonesia is imported from Hawaii (Yap et al. 2005). The production of the


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shrimp itself is still increasing. For example, the shrimp production in 2002

was only 5000 tons but in 2003, it increased to 20000 tons (Briggs et al.,

2004). Within the big production of this shrimp, there will be great amount of

waste too. The waste of shrimp (shell, head, or tail) from the industrial shrimp

factory is usually thrown away after production. They bury the waste with soil

to remove its smell and through time the waste will become compost.

2.1.2. Utilization of shrimp waste In industrial frozen shrimp processing there are many types of shape, like head

on or headless, peeled shrimp is also popular. Peeled shrimp is usually

headless and contains no shell. The yield that lies on the meat itself is 57%,

the yield from head is 33%, and 10% from the shell (Sulastri, 2009). It can be

concluded that the yield from waste is almost half of the total mass.

There are some alternatives to process this usually discarded waste. It can be

used as protein feed for fish meal (Nwanna et al., 2004) or animal feed

because of the high content of amino acids. Aside from utilization as feed,

shrimp waste can also be used as flavoring agent (Teerasuntonwat and

Raksakulthai, 1995). In Indonesia, processed shrimp waste known as terasi is

very popular (Abun, 2009). It is actually fermented shrimp waste or small

shrimp, and it is usually used quite widely as flavoring in Indonesia despite the

fact that it produces a terrible smell because of the ammonia produced.

Surprisingly, not only Indonesia uses this fermented shrimp as flavoring, other

neighboring countries such as Kamboja, The Philippines, and Japan.

Other use of shrimp waste is as source of pigment. Shrimp waste contains

carotenoids and it can be extracted by using organic solvents and solvent

mixtures (Sachindra et al., 2006). Aside from pigment, the commonly

utilization of shrimp waste these days is to produce chitin. The shell and

carapace of shrimp contain high content of chitin and it is therefore extracted.

There are two main processes in producing chitin; the first one is

deproteinization and followed by demineralization. Deproteinization can be

done enzymatically and chemically. Chemically, the shell can be hydrolyzed


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using sodium hydroxide (NaOH). Enzymatically, hydrolysis can be done by

wide range of protease enzyme. For demineralization process, H2SO4 can be

used so that the mineral will be extracted from the shell (Abun, 2000). The

extraction of chitin can also be optimized by combining it with production of

protein hydrolysate (Synowiecki et al., 2000). The firstly demineralized shell

was hydrolysed using Alcalase and NaOH. It was inactivated using HCl and

precipitated using centrifugation. From this point forward, the processing of

chitin and protein hydrolysate was separated. The chitin was stored in the shell

and it was dried. Therefore, crude chitin was produced. The protein was

solubilized from the hydrolysis, so the protein will exist in the solution. The

solution will be lyophilized and the product will be in the form of protein

powder.

2.2. Protein

Protein is one of macromolecules and it is also the most abundant. It is also a

very important constituent of our body and a great source of nutrient and

energy. Protein is constructed from its monomer which is amino acid. These

amino acids are linked covalently using peptide bond.

The functions of protein are for growth, maintenance, and repair of cells by

their action as:

enzymes that catalyze metabolic reactions

structural proteins that maintain the shape of cell

hormones that regulates cell activities

antibodies that provide defense mechanism

contractile proteins, transport proteins, toxins, and components of

intracellular structures

energy source (4kcal)

(Yeung and Laquatra, 2003)

Amino acid is what makes up the protein. Amino acid consists of R groups,

carboxyl, and amino group that bond to carbon atom. R groups are the

determining molecules that differentiate each amino acid. In general, R groups


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are divided to 5 groups. The hydrophobic ones are the nonpolar aliphatic

(glycine, alanine, proline, valine, leucine, isoleucine, methionine) and

aromatic groups (phenylalanine, tyrosine, and tryptophan). Hydrophobic

means that it doesn’t like water, the opposite is hydrophilic which means

water loving. The hydrophilic R groups are the polar uncharged (serine,

threonine, cysteine, asparagine, and glutamine), positively charged (lysine,

histidine, and arginine) and negatively charged (aspartic acid and glutamic

acid). (Lehninger et al., 2005)

The 20 common amino acids in our body are divided to 2 main groups based

on its ability to be synthesized in the body: essential amino acids and non-

essential amino acids. Essential amino acids are those amino acids that human

requires, whereas non-essential amino acid can be produced in the body. There

are 9 essential amino acids: histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, threonine, tryptophan, and valine. Alanine, arginine,

asparagine, aspartic acid, cysteine, glycine, cysteine, glutamic acid, proline,

tyrosine, serine, glutamine are the non-essential amino acids that can be

synthesized in human body (Yeung and Laquatra, 2003).

Protein is varied from the simple form to the complex form. The simplest form

is primary structure which is sequence of amino acids in peptide chain.

Secondary structure is determined by hydrogen bond between amino acids

within the polypeptide chain (alpha-helix of beta-conformation).The tertiary

structure is more complex because it is the 3D conformation of the protein.

Based on the shape, tertiary structure is divided to fibrous and globular

protein. The most complex form of protein are quaternary structure which is a

joined of two or more polypeptide subunits.

In order to be digested in body, large protein molecules must be degraded by

proteolytic enzyme in gastrointestinal tract. The product will be peptides

(small chain of protein) and amino acids that are easier to be absorbed. The

enzymes of gastrointestinal tract are pepsin and trypsin. The amino acid

requirements for human can be seen below.


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Table 2.1 Indispensable (essential) amino acid requirement

Source: WHO (2007)

2.3. Enzyme

Enzyme is a component that can catalyze a reaction. Most of enzymes are

protein in their tertiary structure. Enzyme can increase the rate of reaction by

lowering its activation energy (Ea). Some enzymes do not need any chemical

groups for activity other than the amino acid residues. However, there are also

some enzymes that need additional components. These components are called

cofactor. It can either be inorganic ions or complex, which is usually called

coenzyme (Bugg, 2004).

To catalyze a reaction, enzyme needs substrate. Substrate is molecule that

bound to the active site of enzyme. When substrate is bound to enzyme,

substrate will alter its structure and becoming the product of the reaction.

Afterwards, the enzyme will release itself from the product and it can be used

again for other substrates. The simple reaction of enzyme and substrates can

be described below.


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Where E is enzyme, S is substrate, P is product, ES is enzyme and substrate

complex, and EP is enzyme and product complex. Most of enzyme reactions

are reversible. It means that the product can be turned back to its original state

by the same enzyme. However, some reactions are irreversible or it will need

other enzyme to change back (Lehninger et al., 2005).

Enzyme works specifically. It means that only the right substrates can bind to

the enzyme and start the reaction. The mechanism of enzyme and substrate

binding has been explained in many ways. Two of the famous mechanisms are

lock and key mechanism and induced fit mechanism. Lock and key

mechanism stated that enzyme and substrate is comparable to lock and key. If

a key doesn’t fit to its lock, the lock will not be opened. Same with the enzyme

and substrate, if it doesn’t fit, it will not react. However, there was also

another opinion. In induced fit mechanism, it is said that the enzyme can alter

themselves a little to fit the substrate. It means that the shape of enzyme can

change.

To work at their optimum condition, enzyme is affected by several factors.

The factors that affect the enzymes are temperature, pH, salts, and organic

solvents, and concentration. Enzymes usually work within range. Outside of

their optimum range, this enzyme will not work effectively. Temperature is

one of the crucial factors of enzyme’s work. Some enzymes will work

optimally at low temperature and room temperature, but there are some that

work optimally at high temperature. However, since enzyme is protein, it can

denature. If the temperature is too high, the enzyme will lose its catalytic

activity due to the denaturation.

The acidity also affects the work of enzyme. There are three classification of

enzymes based on its optimum pH: acid, neutral, and base (Rahman, 2003). It

is important to know which pH is suitable for the enzyme. Salts can either be a

cofactor or an inhibitor. Cofactor, as explained earlier, can activate the works

of enzyme. However, the inhibitor can inhibit the reaction. There are two main

types of inhibitor: reversible and irreversible. Reversible inhibitor is further


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classified to three types, which are competitive inhibitor, uncompetitive

inhibitor, and mixed inhibitor. Salt is most probably categorized as

uncompetitive inhibitor which binds on the non-active site of enzyme.

Based on reaction types, enzyme is classified to six main classes:

oxidoreductase, transferase, lyase, hydrolase, isomerase, and ligase. The most

important enzyme for hydrolysis reaction is hydrolase. Hydrolysis is a process

of hydration to the molecules. Hydrolase usually degrades large molecules to

smaller molecules. There are many types of hydrolase, for example protease,

amylase, and lipase.

2.3.1. Protease Protease is classified as hydrolase. It is an enzyme that hydrolyzed peptide

bonds. Protease can also be called peptidase. Peptidase is really important for

survival and it makes up about 2 % of genes in all organisms (Polaina and

MacCabe, 2007). Protease is used in many industrial process, for example in

cheese processing, rennet or ficin is used as clotting agent, it can also be used

as clarifying agent for beer, tenderize meat, and hair removal in leather

processing.

Protease can be classified based on its catalytic type. The catalytic type is

related to the group responsible for catalysis of peptide bond hydrolysis. There

are six specific catalytic types: serine protease, threonine protease, cysteine

protease, aspartic protease, glutamic protease, and metallo protease (Polaina

and MacCabe, 2007).

Since protease exists in all organisms, protease can also be extracted from

some organisms like animals, plants, and microorganisms. Animal proteases

are trypsin, pepsin, and rennin. Proteases from plant source are ficin,

bromelain, and papain. Nowadays, many industrial enzymes are extracted

from microorganism. Especially those commercially produced ones, like

Alcalase, Neutrase, and Protamex.


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2.3.2. Papain Papain (EC 3.4.22.2) is part of cysteine protease. Cysteine protease is divided

to 2 main classes: exopeptidase and endopeptidase. Papain is included in

endopeptidase. It means that papain will cleave bonds that are distant from N

and C termini. Papain is drived from Carica papaya. The crude dried latex

from papaya usually contains 4 cysteine proteases (papain, chymopapain,

caricain, and glycyl endopeptidase) and other enzymes.

Papaya with high quality usually produces the highest quality and activity

comes from tropical areas. This is very suitable to be applied to Indonesia.

There are some methods that can be used to purified crude papain. Water

extraction with reducing and chelating agents, salt precipitation, and solvent

extraction are usually done. To produce pure papain, some additional process

may be added. It is usually done by affinity chromatography methods. There

are some significant difference between crude papain and pure papain.

Table 2.2 Comparison of crude papain and pure papain Characteristics Crude Papain Pure Papain

Color Brown to white White

Smell Not preferred More preferred

Non dissolve material Up to 30% Maximum 0.05%

Water content Up to 18% Maximum 6%

Ash content Up to 14% Maximum 5%

Proteolytic activity (U/g) 70-500 70-1000

Source: Muchtadi et al. (1992) in Rahman (2003)

If dissolved in water, the crude papain will leave brownish particles around

them, but not with pure papain. Pure papain is pure white and it will be sticky

if it comes in contact with water. Since crude papain contains additives,

sometimes the materials cannot be dissolved completely. Therefore there will

be some loss of activity.

Papain is the model enzyme of cysteine protease. Its mechanism has been well

studied. Papain’s enzymatic activity is utilized by catalytic dyad formed by


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cysteine and histidine residues, which in pH between 3.5 and 8.0 forms an ion-

pair. Asparagine is used for orientation of imidazolium ring or histidine in

catalytic cleft. To be able to catalyze, thiol group of enzyme should be

reduced. Hence, the protease require reducing and acidic environment to be

active. Formation of S-acyl enzyme is a basic step in hydrolysis. Afterwards,

water molecule reacts with intermediate, N-terminal fragment is released, and

free cysteine protease molecule can begin a new cycle. (Polaina and MacCabe,

2007)

Figure 2.4 Hydrolytic mechanism of papain Source: Polaina and MacCabe (2007)

Papain has a broad specificity and is able to split many peptide bonds. is said

to be active at 50-57oC (Grzonka et al., 2007). Heating at 75oC for 30 minutes

will decrease the enzyme activity up to 20%. The optimum pH for papain is

between 5.0 and 7.0 and its isoelectric point is at 8.75. Papain is quite stable

and active if stored at 4oC.


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2.4. Protein hydrolysis

Hydrolysis is a reaction when H2O becomes H+ (hydrogen cation) and OH-

(hydroxide anion) (Wijayanti, 2009). Protein hydrolysis will degrade large

protein to small peptides. There are many purposes that can be achieved

through hydrolysis. It can increase the nutritive value, prevent damage,

provide texture, increase and decrease solubility, and increase foaming and

coagulations, add emulsion capacity, and eliminate toxin (Rahman, 2003).

Protein can be hydrolyzed by using chemical and enzyme.

2.4.1. Chemical hydrolysis Chemical hydrolysis can be done in acid condition or base condition. It is easy

to be done and relatively inexpensive. However, it is usually hard to control

and the products can produce different chemical composition and functional

properties. Chemical hydrolysis is usually done at extreme temperatures and

pH. The downsides are the product will have reduced nutritional qualities and

poor functionality.

Acid hydrolysis is more common than alkaline hydrolysis. The process is

harsh and hard to control. Vegetables protein is preferred for this kind of

process. Acid hydrolysis has some disadvantage, tryptophan, asparagine,

glutamine, and some other amino acids are destroyed. The product of acid

hydrolysis is not suitable for human consumption and usually is used as

fertilizer.

Opposite to acid hydrolysis, alkaline hydrolysis use alkali reactants like

sodium hydroxide to hydrolyze protein. However its product usually has a

poor functionality and can affect nutritive value of hydrolysate. Alkaline

hydrolysis is almost exclusively used for determination of tryptophan. Because

of formations of lysinoalanine, ornithinoalanine, and lanthionine, toxic

substances can be produced (Binsan, 2007).

2.4.2. Enzymatic hydrolysis Enzymatic protein hydrolysis can be done by using proteolytic enzyme. This

method is more preferable because it can improve the functionality and avoid


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the destruction of product. Hydrolysis using non-specific protease like papain

can increase the solubility of hydrophobic protein (Rahman, 2003). However,

this hydrolysis might produce bitter-tasting peptides which can affect its

sensory properties. The bitterness is due to hydrophobicity as well as

molecular configuration. Bitter amino acids are generally within L-series. The

bitterest amino acids are L-tryptophan and L-tyrosine but D-tryptophan named

as the sweetest one (Belitz et al., 2009).

Nowadays, enzymatic hydrolysis is more preferable for the making of protein

concentrate. It is also safer for human and animal consumption because

enzyme is naturally exists in body. There are some protease enzymes that are

usually used for producing protein concentrate, like papain, bromelain,

trypsin, chymotrypsin, Alcalase, Neutrase, Protamex. In order for enzymatic

hydrolysis to succeed, the optimum condition for each enzyme should be taken

into account.

2.5. Protein hydrolysate

In order to be healthy and grow well, human needs to consume protein in

adequate amount. However, consuming food is sometimes not enough. There

are some nutrients that are harder to find than others. Nowadays, human can

also consume supplements and nutraceuticals. This compound is designed to

fulfill human needs of nutrients. Protein hydrolysate can be consumed in the

form of powder and liquid.

The same goes for protein. Although almost all foods contain protein, not all

food contains the required essential amino acids in adequate amount. In human

diet, they do not usually consider or count the required amount of nutrients

they need. By consuming protein hydrolysate, this problem might be solved.

Protein hydrolysate is the product of protein extraction from its raw material.

The raw material can vary from plants, animals, and even microbes. The

example of protein hydrolysate from plant is soy protein isolate. Nowadays,

the most popular one is protein concentrate from milk. However, milk is


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relatively expensive in some countries, so researchers tried to find alternatives.

One of the alternatives is by harnessing the waste of seafood production.

Fish and shrimp production usually produce waste. The waste can be from

scales, shells, head, tail, or even the small fishes that are not acceptable

according to standards. These wastes can be used for producing protein

hydrolysate.

2.5.1. Shrimp protein hydrolysate Shrimp is known to contain high content of protein. Whole shrimp is predicted

to contain protein from 18% to 21%. Therefore, the use of shrimp as protein

hydrolysate has been researched frequently. The hydrolysate itself can be

produced from several parts of the shrimp, from meat (Simpson et al., 1998),

shell (Synowiecki et al., 2000), or head (Limam et al., 2008). However, the

meat of shrimp already has high value. Therefore many researches are focused

on the utilization of waste (shell and head).

There are many ways to produce shrimp protein hydrolysate, for example by

adding proteolytic enzymes (Ruttanapornvareesakul et al., 2005) or chemicals

like sodium hydroxide (Abun, 2006), biological process by fermentation

(Junianto et al., 2009) and also physical treatment (Adrizal et al., 1999) to the

sample can also extract the protein.

The focus of today’s researches is in the enzymatic hydrolysis because it

produces less undesirable by-products and also increases its functional and

nutritional value (Kristinsson and Rasco, 2000). There are some key processes

in making shrimp protein hydrolysate by enzyme.

1. Mincing

Mincing is meant to downsize the size of sample. The size needs to be

smaller because enzyme will work more effective if the smaller is given in

the smaller size and in larger amount rather than larger size but small

amount. Mincing can be done manually or automatically. Since mincing

manually spends more energy and time, machines are usually used to


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mince. The machines that are usually used are blender, food processor, and

meat grinder. Blender and food processor are used if the sample is not

produced in large scale. For large scale production, it is better to use meat

grinder for industrial process.

2. Incubation

Incubation is the most crucial part of the hydrolysis process. The factors

that need to be considered are time, temperature, pH, and salt addition

because enzyme is used. It is important to set the condition precisely for

each enzyme to get the best result because some enzymes work in acidic

condition, others in base condition.

Temperature should also be considered as crucial, because some enzymes

can only work in low temperature, and the effect of denaturation if the

temperature is too high should also be considered because denaturation of

enzyme can affect its functionality. Time will not affect the hydrolysis

process but it will affect the end result. If the time is too short, the process

will not be effective and if it is too long it will not be efficient.

Researchers stated that the optimum time for incubation was between 1 to

5 hours. Although there are some enzymes that don’t need salt, there are

also those that need it. Salt can act as the cofactor of the enzyme, so that it

will activate the work of enzyme. Salt addition to improve the activity of

enzyme had been done by Mizani and Aminlari (2007)

3. Inactivation of enzyme

The purpose of inactivation is to stop the enzyme catalyzed reaction. If the

reaction is not stopped, enzyme will continue to degrade the product which

can lead to microbial contamination. The other purpose is to pasteurize the

product. Note that pasteurization is only for inactivation that uses

temperature. Pasteurization is done to kill the contaminant in product and

also increase the shelf life of product.

Enzyme can be inactivated by several ways; the easiest is to raise the

temperature. Raising temperature to certain level can cause the enzyme to

denature and lose its functionality. Some enzymes can withstand high


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temperature, so the temperature should be considered carefully. In

contrary, heating the product can also cause the protein of product to

denature, so it is better to inactivate the enzyme in short time. The other

way is by adding chemicals that induce denaturation. Acid is usually added

to put the sample in extreme pH (Synowiecki et al., 2000), so that

denaturation occurs.

4. Filtration

Filtration is an optional process. Some researches stated that after enzyme

inactivation, they went straight to centrifugation. Others eliminated the

centrifugation process and did filtration instead (Valdez-Pena et al., 2010).

The purpose of filtration is to separate the large pieces of the shrimp head

and the small molecules, soluble solid, and liquid. Filtration may be done

using sieve, muslin or cheese cloth, and vacuum filter.

5. Centrifugation

Centrifugation is a process of separation based on the density of product.

In hydrolysis of shrimp head, the protein will be soluble in the distilled

water. To remove the impurities of hydrolysate, centrifugation is one of

the most efficient ways. The impurities can come from the shrimp head

that are not soluble, sand and minerals from the shrimp head, and also the

fat. According to Synowiecki et al. (2000), centrifugation will remove

chitin residue from hydrolysate. Centrifugation process may reduce the

yield of dried product but increase the quality and purity of the

hydrolysate.

2.5.2 Quality Standards for Fish Protein Hydrolysate Since shrimp protein concentrate hasn’t had any quality standards, the quality

standards of shrimp protein hydrolysate will be compared to those regulations

from fish protein concentrate. FAO has divided quality of fish protein

concentrate to three types (Windsor, 2001).

Type A: This product is virtually odorless and tasteless powder. The

maximum total fat content is 0.75%


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Type B: This product has no specific limit of odor and flavor, but it still has

fishy flavor. The maximum fat content of this product is 3%

Type C: this product will not be consumed by human. It is usually used as fish

meal and is produced under satisfactorily hygienic conditions.

The reason why fat is one of the requirement of quality is because of the

rancid taste that fat will produce during oxidation. In dehydrated fish protein,

the protein content of the product can reach up to 65% and up to 80% in type

A fish protein concentrate.

The standards of fish protein concentrate (fish protein isolates as referred by

FDA) also exist in FDA (Food and Drug Administration). It is written in

section 172.340 noted as fish protein isolate. If fish protein isolate is about to

be used as food additive as food supplement, it should follow several

prescribed conditions and specifications of product (FDA, 2010):

1. Additive shall consist dried fish protein prepared from edible portions of

fish after removal of head, fins, tails, bones, scales, viscera, and intestinal

content.

2. Additive shall be derived from bony fish that are recognized by qualified

scientists as safe for human consumption and can be processed as

prescribed to meet the required specifications.

3. Only wholesome fresh fish suitable for human consumption may be used.

It shall be handled expeditiously under sanitary conditions (GMP).

4. Additive shall be prepared by extraction with hexane and food-grade

ethanol to remove fat and moisture. Solvent residues shall be reduced by

drying.

5. Protein content (Kjedahl method), shall not be less than 90% by weight of

final product.

6. Moisture content shall not be more than 10% by weight of final product.

7. Fat content shall not exceed 0.5% by weight of final product.

8. Solvent residues in final product shall not be more than 5 ppm (parts per

million) of hexane and 3.5 percent ethanol by weight.


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2.6. Zero Waste Concept

Waste can cause great loss of value and resources. By identifying and

harnessing waste, mankind can save money, save the world by making

sustainable resources. Zero waste is a concept, in which all part of resources

used in processing will not produce any waste. Waste is recalled as

inefficiency in zero waste concepts.

Instead of throwing the waste away, waste can be considered as potential

resource to be harnessed. Waste is not a burden anymore but it is an

opportunity. It will reduce costs for discarding waste and increase profits at

the same time. On the positive side, the application of zero waste concepts can

solve the environmental issue and also provide sustainability of resources.

This concept is applicable to various kind of organization from community,

business, and school, industry wide and even at home.

Figure 2.4 below showed the material flows in today’s industry. The raw

materials are processed and used and discarded after its life ended. There are

recovery and disposal. The recovered materials will be used as compost and

the rest of the waste will be disposed in land fill, which cause terrible smell

and also exploit space, or incinerated causing pollution to the air.

Figure 2.5 Material flows today Source: http://www.zerowaste.org/case.htm (2011)

This concept of material flows is what the zero waste concept tries to avoid. A

zero waste society will not disposed and incinerated the waste. Instead, it will


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be used as reusable and recycled material. The rest of the waste will be used as

compost.

Figure 2.6 Improved material flows Source: http://www.zerowaste.org/case.htm (2011)

The example of zero waste concept can be taken from frozen shrimp

processing. In frozen shrimp processing, it is usually only the meat that was

used while head and shell are usually discarded or used as compost. This head

and shell can be reused completely to produce another product. The head and

shell can be hydrolyzed and producing shrimp protein concentrate. This

process will produce waste as undissolved materials. However this materials

can be used to produce chitin. Both protein concentrate and chitin are valuable

and can even have higher price than the shrimp itself. If all industry can apply

this concept to their production process, they can gain a lot more profits and

also protect the environment.

However, it is hard to apply this concept to all industries. Considering that to

do research about how to harness the waste is time and money consuming.

Other things that will be disadvantages to this concept is that processing of

waste itself will need other materials that are not related to the industry which

will produce other costs. The industry is usually already feel fine about

discarding their waste and do not have any intention to protect the

environment. This awareness of environmental problem should come from the

company itself and maybe by support of environmentalists.


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CHAPTER 3 – METHODOLOGY

3.1. Time and Venue

Research was conducted within 4 months from March 2011 until June 2011.

This was done in Research Center for Marine and Fisheries Product

Processing and Biotechnology (Jakarta, Indonesia) and Swiss Germany

University (BSD, Tangerang). The research used more than one laboratory,

which are Chemistry Laboratory, Biotechnology Laboratory, Sensory

Laboratory, and Product Processing Laboratory.

3.2. Materials

3.2.1. Raw Materials Raw material for this research was shrimp head of Litopenaeus vannamei. L.

vannamei for this research was taken from a shrimp processing company in

Ancol, Jakarta. The shrimps were taken right after the production to retain its

freshness. The shrimps were then stored in freezer at -20oC.

3.2.2. Chemicals Crude papain

Pure papain

Ethyl ether

N-hexane

H2SO4

HCl

Casein 1%

Buffer pH 7 and pH 8 (KH2PO4 and K2HPO4)

TCA (Trichloro acetic)

Folin Ciocalteau

Biuret reagent (Cu2SO4, Na-K Tartarate, NaOH, Na2SO3)

Na2CO3

Na2SO4

BSA (Bovine Serum Albumin)


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Tyrosine

3.3. Equipments

1. Analytical Mass Balance

2. Oven

3. Furnace

4. Soxhlet

5. Kjeltec 2300 FOSS

6. Water Bath Shaker

7. Hot Plate

8. Spectrophotometer

9. Distillation set

10. Micropipette

11. Centrifuge

12. Crucibles

13. Dessicator

14. Round bottom flasks

15. Erlenemeyer flasks

16. Beaker glasses

3.4. Procedures

3.4.1. Examination of raw material Raw material was examined using proximate analysis. Proximate analysis

included moisture content, ash content, fat content, and protein content. Before

the proximate analysis, the sample was ground first using blender. The method

used in this analysis followed SNI (Standar Nasional Indonesia).

1. Water content ( SNI 01-2354.2-2006)

Crucibles were already prepared in oven overnight at 100oC and were put

into dessicator for 15 minutes. The crucible was weighed afterward and 2

g of sample was put into the crucible. Crucible with sample was put into

the oven (100oC) and left overnight. It was moved to dessicator for 15

minutes and was weighed once more. The moisture content was measured

by this equation

Water content =


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This experiment was replicated 2 times.

2. Ash content (SNI 01-2354.1-2006)

The dried crucible and sample from previous experiment was put into

furnace (550oC) and was left there for 8 hours or until the color turned to

white. After that, the temperature is lowered to 40oC and the crucible was

put in the dessicator for 30 minutes. If the sample was not white, it should

be put in the furnace again, but first it was given distilled water and dried

on hot plate, then was dried again. The experiment was done until ash was

white and weight was constant. To calculate the ash content, equation

below was used.

% Total ash =

This experiment was replicated 2 times.

3. Lipid content (SNI 01-2354.3-2006)

Sample was put into an extraction thimble. Extraction thimble is made

from filter paper. If sample was wet, sodium sulfate (Na2SO4) should be

added twice the weight of sample to dry it. Sample was later put into the

extraction soxhlet. Round flask for extraction was weighed and boiling

stone should be added inside. Soxhlet was assembled and 150 ml ether

was added. The extraction was done at 60oC for 8 hours. Fat and ether was

evaporated. Round bottom flask with fat was put in oven at 105oC for 2

hours to eliminate ether and vapor. Flask was weighed again. This analysis

was done twice. The equation for lipid content is displayed below.

% fat =

A= weight of empty round bottom flask (g), B= weight of sample (sodium

hydroxide was excluded) (g), C= weight of round bottom flask + fat (g)

4. Protein content (SNI 01-2354.4-2006)

1 g of sample was weighed and added with 0.2 N HCl. Sample destruction

used H2SO4 with catalyst K2SO4. Using 250 ml flask, H2SO4 used was 10-

15 ml. Destruction was done at 410oC for 2 hours until the solution was

clear. This is indication of perfect destruction. Afterward, the sample was

distilled and titrated using Kjeltec 2300 (FOSS).


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3.4.2. Assay of enzyme papain (food grade and pure) Determining enzyme activity was separated to two main parts. The first was

determining enzyme activity. This was done by combining 250 μl casein 1%

as substrate, 250 μl Buffer pH 7, and 250μl enzyme of interest in an Eppendorf

tube. The incubation time was 20 minutes at 50oC. After incubation 750 μl

TCA was added to stop reaction. The tube was centrifuged 8000 rpm at 4oC

for 10 minutes. 300 μl supernatant from tube was taken and mixed with

1000μl Na2CO3 and 200μl Folin Ciocalteau. Sample was put into

spectrophotometer to measure absorbance at 578 nm. Standard curve was

generated from tyrosine sample to determine protein activity. Volume activity

was determined after this experiment.

3.4.3. Hydrolysis of shrimp head (Limam, 2008)

Initially, research was done to find the optimum time for incubation (3, 4, and

5 hours), pH that was suitable for research (pH 7, pH 8, and distilled water).

These experiments were combined with the effect of centrifugation and


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filtration to protein content and the effect of filtration after hydrolysate was

recovered from centrifugation. The enzyme used in this experiment was only

the crude papain at 10% concentration. The incubation was done at 50oC in

water bath shaker with occasional stirring.

The protein content on early experiments was analyzed and conclusion was

made. The optimum time for incubation was 4 hours and distilled water was

used instead of buffer. Both filtration and centrifugation were done but the

hydrolysate was not filtered once again.

The method of making shrimp head protein concentrate followed Limam et al.

(2008) with slight modification. Shrimp head was ground using blender. 100 g

of the shrimp head was taken for each hydrolysis. Then, it was added with

distilled water at ratio 1:1 (w/v) that was already added with enzyme (the

enzyme used in this experiment was two types of papain: pure papain and

crude papain). This solution is homogenized using glass rod. Incubation period

was 4 hours and during this period, it was occasionally stirred. Incubation was

followed by inactivation of enzyme at 90oC for 5 minutes. After the solution

had cooled down, the solution was filtered using muslin cloth. The waste from

the unfiltered material was thrown and the liquid part was processed once

again. Solution was centrifuged at 9000 rpm for 15 minutes at 4oC and the

supernatant were taken as the protein hydrolysate. The treatments for this

experiment were type of enzyme (crude and pure papain), concentration of

enzyme (10, 20, and 30%), and temperature of incubation (45, 50, 55, and

60oC).

3.4.4. Analysis of proximate composition and yield of hydrolysate Proximate analyses that will be done are water, ash, protein, and fat. Fat

analysis was done to two samples from each enzyme that had the highest

protein concentration. The proximate analysis was done with the same method

as analysis of raw material except the protein and fat content. Soluble protein

was determined using Lowry assay and fat content was analyzed using batch

solvent extraction method.


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1. Fat content

Round bottom flask was put into oven at 105oC for one night. After that, it

was put into dessicator for 15 minutes and weighed. The round bottom

flask was put into the oven again for an hour and afterwards it was put into

dessicator for 15 minutes and was weighed. The weight of the first and the

second measurement should not exceed 0.004 g.

15 g of liquid sample was taken and put into separator funnel. 15 ml of n-

hexane was added to the separator funnel. The solution was homogenized

by shaking the separator funnel manually. The solution was left to settle

and produced two separate parts. The n-hexane were put into the round

bottom flask. Sample was added with n-hexane again and the process was

repeated three times. The round bottom flask containing n-hexane was

distilled using distillation set at 60oC. After all n-hexane evaporated, the

round bottom flask containing fat was put into oven for two hours.

Afterwards, it was put into dessicator for 15 minutes and was weighed.

The fat content was calculated by the equation below,

where A was weight of empty round bottom flask, B was weight of round

bottom flask plus fat, and C was the initial weight of sample.

2. Lowry Assay (Lowry, 1951)

In Lowry assay, 0.6 ml protein sample will be mixed with 3 ml Biuret

solution. This solution was vortexed. After ten minutes, this solution was

added with 0.15ml Folin-Ciocalteau (1:2 v/v). The solution was left for 30

minutes and afterwards the absorbance of sample was measured at 650 nm.

To know the concentration of protein in sample, standard curve should be

made using BSA (bovine serum albumin) solution with concentration 0.1,

0.2, 0.4, 0.6, 0.8, 1.0 mg/ml. The procedure of absorbance was the same as

the protein sample.


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Other than the protein content, recovered protein from the process was also

calculated. Recovered protein is percentage of soluble protein in

hydrolysate from total protein in raw material.

Yield was calculated based on the initial mass of the mixture and the mass

after centrifugation. It means that the weight of water and enzyme were taken

into account. Yield was calculated by the equation below.

3.4.5. Sensory evaluation The samples that will be examined were the two samples from each enzyme

that has the highest protein content. The type of sensory test that will be used

is hedonic test to know the preferred product between the shrimp head

hydrolyzed with pure and crude papain. The result was obtained from three

replications. The parameters that were be used are taste, appearance, color,

and smell. The scoring were from level 1 (very dislike) to 7 (very like). The

hedonic test used 7 trained panelists and 6 semi-trained panelists.

3.5. Experimental Design

Experimental Design that was used on this research was Completely

Randomized Factorial Design with three factorials as the treatments.

Replication was done twice. The model of the experimental design was as

following.

Yij = μ + Ai + Bj +Ck+ ABij + ACik + BCjk + ABCijk + Eijk

Yij : Observation value

μ : Mean value

Ai : Influence of Treatment A, where i=1 and 2

Bj : Influence of Treatment B, where j=1, 2, 3

Ck : Influence of Treatment C, where k= 1, 2, 3, and 4

ABij : Influence of interaction between Treatment A and Treatment B

ACik : Influence of interaction between Treatment A and Treatment C


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BCjk : Influence of interaction between Treatment B and Treatment C

ABCijk: Influence of interaction between Treatment A, B, and C

Eijk : Influence of error

There were 3 factors used as treatments on this research. They were called as

Treatment A, Treatment B and Treatment C. Treatment A covered the type of

enzyme used for hydrolysis, which were crude papain and pure papain.

Treatment B covered various concentration of enzyme papain used in

hydrolysis, which were 10%, 20%, 30%. Treatment C covered the temperature

used as hydrolysis temperature, which were 45oC, 50oC, 55oC, and 60oC.

A. Type of enzyme variation

A1 = crude papain

A2 = pure papain

B. Concentration variation

B1 = 10%

B2 = 20%

B3 = 30%

C. Temperature Variation

C1 = 45oC

C2 = 50oC

C3 = 55oC

C4 = 60oC

3.6. Data Analysis

Data analysis for proximate composition and yield of products were done

using OpenStat with three-way ANOVA (Analysis of Variance) with 95% of

confidence level. Post hoc analysis was done using Tukey HSD (Honestly

Significant Difference). Sensory test was analyzed using two-way ANOVA in

Microsoft Excel Data Analysis with 95% of confidence level, and t-test

between samples for mean was done to the parameters that showed significant

difference.


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CHAPTER 4 – RESULT & DISCUSSION

4.1. Proximate composition of shrimp head (L.vannamei)

The raw material was analyzed for its water, ash, protein, and fat content. The

table below showed the result of the analysis.

Table 4.1 Proximate composition of L. vannamei head and P. monodon head

Parameter L.vannamei P. monodona

Water (%) 79.138 + 1.008 78.5

Ash (%) 4.416 + 0.547 5

Fat (%) 2.123 + 0.173 3.1

Protein (%) 11.599 + 0.518 13.6 afrom Teerasuntonwat and Raksakulthai (1995)

The objective of finding the proximate composition of raw material was to

compare it to the proximate composition of products. More specifically, the

protein content of raw material was used to determine the recovered protein

from the result. Water content of whole shrimp is 75.86 %. It has 1.2 % ash

content and about 20.31% protein. The fat content is 1.73%. Compared to the

literature, the water, ash and fat content were higher.

Protein content in shrimp head is lower than the protein of reference. It ought

to be noted that the composition in the literature was for whole shrimp. If

compared to other research on shrimp head with different species, this

proximate composition was not really different. It can be concluded that

shrimp head contain less protein than whole shrimp but more fat, ash, and

water.

4.2. Determination of enzyme activity

The enzyme activities of both pure and crude papain were determined. To fit

the standard curve, the enzyme should be diluted first. The pure papain was

diluted 1000 times and the crude papain was diluted 5 times. The enzyme

activity of pure papain was 2375.78 U/g and for crude papain was 21.2 U/g.


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This determination of enzyme activity was very important because both

enzymes were used to hydrolyze the same thing.

The enzyme activity of crude papain was much lower than the pure papain.

Thus, it was not possible to compare the hydrolytic capability of both enzymes

by its concentration. Therefore, the approximate enzyme activity was the one

that was being equalized. Since the treatment of the experiment also required

enzyme concentration, concentration of crude papain was made as the

reference. The enzyme concentration 10, 20, and 30% was derived to mass

unit as 10 g, 20 g, and 30 g. By equalizing the activity of both enzymes, the

pure papain added as 10% was 0.89g, 20% was 0.178g, and 30% was 0.267g.

4.3. Effect of incubation time and filtration after centrifugation to protein concentration

From the experiment, statistical analysis showed that there were no significant

difference between incubation time 3, 4, and 5 hours. However, from the

graph, there was an increase of protein concentration at 4 hours incubation

time. So, it was decided that the appropriate time for incubation was 4 hours.

The decrease of protein content after 4 hours was the result of ununiformed

stirring, quality of raw material was not the same, or capability of enzyme to

hydrolyze was not the same (Wijayanti, 2009).

There were three phases after centrifugation of hydrolysate: the solid part at

the bottom, which contained insoluble material and solid waste, the middle

part which contained the hydrolysate, and the third phase which stuck to the

wall of the centrifuge tube and floated around the hydrolysate. To get rid of

this flocculent, another process of filtration using filter paper was done. To

compare this process, the hydrolysate which were not filtrated, were also

prepared. It seems that there was a significant difference in protein content

between the filtration and non- filtration. The products which were not filtered

contained more protein than the one that was filtered. It means that the

flocculent were also a part of the protein, which means it was important.

Therefore, the filtration process after centrifugation process was eliminated.


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4.4. Effect of pH to protein concentration

When working with enzyme, pH is one of the factors to be considered in order

to obtain the best result. Each enzyme has its own optimum pH. Papain is

considered to be active in broad range of pH. However, it is optimum between

the pH 6-8. Therefore, the experiment was to determine whether or not, there

was a significant influence of using pH buffer to maintain the pH or not.

In the experiment, the crude papain was dissolved in three different solvent,

which are buffer pH 7, buffer pH 8, and distilled water. The pH of the shrimp

head with distilled water was controlled using pH paper every hour during

incubation. The pH was 8 and it was stable during the incubation. After the

process, the protein content was analyzed. Apparently from the result it can be

concluded that there was no significant difference between the use of buffer

and distilled water. Hence, the main research used the distilled water as

solvent.

4.5. Effect of centrifugation and filtration using muslin cloth to protein content

In this experiment, two key processes were examined. Some of the products of

incubation were filtered using muslin cloth and others were centrifuged. In the

result, it showed that there was no significant difference in protein content

between filtration and centrifugation.

However, both products had a significant difference in appearance. The

product that was only filtered was filled with many flocculent and it was

darker, hazier and there were some precipitations. In comparison, the

centrifuged product was lighter, had less flocculent, was also clearer and smell

less fishy than another. In the end, the combination of filtration and

centrifugation were done in the main research.


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4.6. Analyses of yield and proximate compositions to treatments type of papain, concentration of papain, and temperature of incubation

There were a total of 24 treatments for the shrimp protein hydrolysate. Each

treatment was analyzed for its yield, water content, ash content, and protein

content. The data summary can be viewed on Table 4.1. C indicated the

concentraton of enzyme (%) and T indicated the temperature of incubation

(oC). CP stood for crude papain and PP stood for pure papain. The data

displayed was average of each treatment plus minus its standard deviation.


WATER CONTENT (%)

ASH CONTENT (%)

PROTEIN CONTENT (%)

YIELD (%)

C T CP PP CP PP CP PP CP PP

10

45 87.28 ± 0.375

92.83 ± 0.073

3.71 ± 0.032

0.46 ± 0.014

5.07 ± 0.947

6.62 ± 0.021

72.30 ± 0.199

77.17 ± 0.001

50 88.28 ± 0.375

91.53 ± 0.127

3.79 ± 0.057

0.46 ± 0.003

5.54 ± 0.166

7.91 ± 0.021

73.54 ± 0.259

71.07 ± 0.198

55 87.31 ± 0.082

91.32 ± 0.158

3.75 ± 0.022

0.54 ± 0.002

6.24 ± 0.041

7.95 ± 0.075

74.27 ± 0.015

77.58 ± 0.322

60 88.71 ± 0.090

90.83 ± 0.093

3.60 ± 0.054

0.54± 0.016

6.28 ± 0.476

8.42 ± 0.393

68.68 ± 1.485

67.82 ± 0.476

20

45 84.21 ± 0.229

92.56 ± 0.035

6.22 ± 0.639

0.46 ± 0.003

5.02 ± 0.062

6.74 ± 0.041

72.45 ± 0.177

77.29 ± 0.269

50 84.22 ± 0.715

91.32 ± 0.370

6.47 ± 0.426

0.47 ± 0.011

5.87 ± 0.021

8.10 ± 0.269

70.27 ± 4.110

71.00 ± 0.601

55 83.94 ± 0.394

91.20 ± 0.049

6.74 ± 0.070

0.54 ± 0.001

6.71 ± 0.994

7.82 ± 0.162

83.11 ± 0.411

78.09 ± 0.215

60 83.63 ± 0.856

91.39 ± 0.030

6.57 ± 0.204

0.55 ± 0.003

6.48 ± 0.884

7.95 ± 0.104

70.29 ± 0.058

67.97 ± 0.212

30

45 81.90 ± 0.294

92.46 ± 0.051

8.92 ± 0.190

0.46 ± 0.008

5.10 ± 0.166

6.94 ± 0.207

71.99 ± 0.267

77.36 ± 0.252

50 81.47 ± 0.208

90.90 ± 0.035

9.25 ± 0.401

0.47 ± 0.005

6.59 ± 0.746

7.98 ± 0.352

73.21 ± 0.038

71.93 ± 0.021

55 80.72 ± 0.386

91.17 ± 0.009

9.21 ± 0.214

0.55 ± 0.001

5.89 ± 0.083

7.95 ± 0.122

84.10 ± 0.397

78.07 ± 0.197

60 80.57 ± 0.509

90.79 ± 0.030

9.39 ± 0.079

0.55 ± 0.005

6.69 ± 1.139

8.57 ± 0.104

83.75 ± 0.802

67.53 ± 0.019

Table 4.2 Data summary of yield and proximate analyses


4.6.1. Yield Yield was determined to know how much product that can be recovered after the

process. Yield is important to predict the outcome a product. It can be used to

determine the expected result from raw material. Based on statistical analysis

(Appendix 3), it was known that there were significant difference of yield between

type of enzyme, concentration of enzyme, and temperature. There were also

interactions between all treatments.

Figure 4.1 Graph of concentration versus yield

Figure 4.1. projected the relationship of papain concentration (both crude and pure

papain) with its yield. In the graph, the yield increased as the papain concentration

increased. However, based on statistical analysis (Appendix 3) there were significant

differences of yield only in papain concentration 10% and 30%, and 20% and 30%.

That means that the yield of product when the crude papain 30% was the highest.

In the graph, it can be seen that the yield increased as crude papain concentration

increased. However, the same did not happen to the pure papain. The yield of product

added with pure papain did not increase significantly based on the graph. Statistical

analysis also showed that yield was not affected by concentration of pure papain. In

deciding which type of enzyme produced products with better yield, statistical


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analysis was also done. Apparently, there was no significant difference between the

yield from pure papain and crude papain.

The increase of yield for products hydrolyzed with crude enzyme might happen

because the amount of enzyme added was much higher than the pure papain. The

papain was dissolved into water and when the hydrolysate was filtered, the papain

came out along with the water. Therefore, the yield of product will be higher too.

Figure 4.2 Graph of temperature versus yield

The graph above (Figure 4.2) showed the relationship between temperature and yield

from products hydrolyzed by crude and pure papain. Statistical analysis (Appendix 3)

stated that yield of products were influenced by temperature. Previewing the graph, it

can be seen that the yield for pure papain at 50oC and 60oC were lower than the yield

at 45oC and 55oC. The same went for the crude papain. Although statistical analysis

showed that temperature had effect on yield, result of graph temperature versus yield

was irrelevant compared to any literature. It did not show any significant increment or

decrement. The factors that may affect this result was the filtration using muslin cloth.

The filtration using muslin cloth was done manually. Therefore, the work done in the

products may not be uniform. This may cause the fluctuation of the data. According

to Yulistianti (2009), liquid flavor (liquid concentrate in this case) is volatile and


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chemically unstable against air, light, humidity, and temperature on storage, this may

be the factor why the yield was unstable as well.

4.6.2. Water Content The water content of the product was analyzed. Since the protein concentrate was in

its liquid phase, the water content was definitely higher than 80%. The data that was

obtained from the analysis of water content can be seen below.

Based on statistical analysis using three way analysis of variance (ANOVA), it was

known that there was significant difference between the water content of product

hydrolyzed by crude papain and pure papain (Appendix 4). There were also

significant differences in water content among concentration of enzyme and

incubation temperature. There were significance interactions between type of enzyme

and its concentration, type of enzyme and incubation temperature, concentration of

enzyme and incubation temperature, and type of enzyme with its concentration and

incubation temperature.

Figure 4.3 Graph of concentration versus water content

Since the early result showed that there was significant difference in water content

between the concentrations, statistical analysis showed that water content hydrolyzed

with 10% enzyme concentration was different to 20% and 30%, and 20% enzyme

concentration was also different from the 30%. This difference was easier to see in the


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graph (Fig 4.3). The water content of products decreased as the concentration of

enzyme increase.

Figure 4.4 Graph of temperature versus water content

Besides the effect of concentration, temperature also gave a significant effect to water

content. Therefore, the difference needed to be pointed out. From statistical analysis

(Appendix 4), it was known that water content with products incubated at 45oC were

difference to those incubated at 50oC, 55oC, and 60oC. However, the difference of

water content between 50oC, 55oC, and 60oC was insignificant.

From the result, it showed that the water content of products hydrolyzed with crude

enzyme at several concentrations was significantly different. When crude enzyme was

applied at 10% concentration, the water content was different from 20% and 30%

enzyme concentration. The products with 20% and 30% enzyme concentration also

showed different water content. However, the products hydrolyzed with pure enzyme

didn’t show any significant difference among its concentration. To equalize the

enzyme activity of crude papain, the pure papain added to the solution was

approximately hundred times less than the crude papain. Therefore, more crude

papain dissolved to the solution which automatically decreased the water content. The

pure papain added to equalize the crude papain were 0.089 g, 0.178 g, and 0.267 g,

which didn’t affect the water content significantly. Within each incubation

temperature, the average water content from each concentration showed no significant


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difference. It means that temperature did not affect the enzyme concentration effect

on water content.

Crude papain contains additives like sugar and salt. Both materials are soluble in

water. Because of the solubility of these materials, the free bound water in the

products will also decrease (Anonymous, 2011). This phenomenon contributed to the

decrease of water content.

The increase of water soluble material can either be adventageous or disadventageous.

It is adventegous if the soluble material that are dissolved are the protein part. This

process is considered not successful if most of the soluble material is the salt and

sugar, not the protein. The difference between the water content of hydrolysate that

was hydrolyzed with pure papain and crude papain can be seen clearly. The water

contents of the hydrolysates from crude papain were under 90% while the water

contents of hydrolysates from pure papain were over 90%. The pure enzyme added to

the solution was hundred times less than crude enzyme added. Moreover, the pure

papain contains only enzyme and no additives, which means the only dissolved

material from the powder was the enzyme. If the process were to be continued to

drying, the products hydrolyzed with crude enzyme will probably contain high sugar

and salt concentration.

4.6.3. Ash Content Ash content was calculated based on the residue of excessive heating at 550oC. Ash

content gives a quick glance of mineral trace in the product. Mineral was not volatile

and it can withstand high temperature. Therefore, when it is burned at high

temperature, other organic materials will evaporate which leave the ash behind

Statistical analysis (Appendix 5) showed that there was significant difference between

the ash content of products hydrolyzed by crude papain and pure papain. The

difference of ash content among the concentration of enzymes was also significant.

However, temperature did not have significant impact to the ash content. The

significant interaction was only between the type of enzyme and concentration. The


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interaction between type of enzyme and temperature, concentration and temperature,

and the three of them were insignificant.

Figure 4.5 displayed the relationship between ash content and concentration of

enzyme for both types of enzyme. The ash content of products hydrolyzed by crude

papain increased as the concentration increased. However, it can be seen that the ash

content of pure papain products did not increase or decrease as the concentration

increase. It made an almost linear line. The difference between ash content produced

by the pure and crude papain was also significant, as it can be seen on the graph. The

range of ash content for crude papain was between 3 to 9 % and the range of ash

content for pure papain was only 0.4-0.5%.

Figure 4.5 Graph of Concentration versus ash content

To be specific of the difference, the statistical difference among the concentration was

divided per type of enzyme. Results showed that hydrolysis using crude papain at

several concentrations will produce significantly different ash content. As the

concentration of crude enzyme got higher, the ash content also increased. The ash

content of 10% concentration was significantly different to the 20% and 30%. The

products with 20% enzyme concentration were also different from the 30%. However,

the ash content of products from pure papain did not show significant difference

among the concentrations. At 10, 20, and 30% concentration, there were significant


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differences between the ash content of products from pure and crude papain

hydrolysis, it was the same conclusion as the interpretation of the graph.

Figure 4.6 Graph of temperature versus ash content

The difference between the ash from crude and pure papain hydrolysate was not only

in percent but also in appearance. Products from pure papain produced totally white

ash and it takes shorter time to ash it. In comparison, the products from crude papain

produce white ash with some trace of black particles. It also took longer time to

produce the ash. This result was most probably due to the content of crude papain.

Figure 4.7 Comparison of ash of hydrolysate

Crude papain contains not only papain but also salt and sugar. The proportion of salt

and sugar was not clearly described but both were soluble in distilled water. Most of

minerals that are water soluble are usually in the form of salts (Traverso, 2004).

Crude papain hydrolysate

Pure papain hydrolysate


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Hence, salt is also considered as mineral, so it will not evaporate when put into the

furnace. This explained the high ash content in products of crude papain hydrolysis.

4.6.4. Protein Content Protein content was the essential part of this product. The higher the yield of protein

was the better. However, it is also important not to waste sources and energy even if

the yield is high. Therefore, statistical analysis to protein data should be done.

Based on the statistical analysis (Appendix 6), it can be concluded that there was

difference between the types of enzyme used. The temperature also gave significantly

different protein result. However, there was no interaction between all the treatments.

Figure 4.8 Graph of concentration versus protein content

Figure 4.8 showed the relationship between papain concentration and protein

concentration. It was said that concentration apparently did not have any significant

effect to the protein content. The correlation can be seen by looking at the graph. The

protein content tend to be constant even though the concentration of enzyme

increased. It also means that 10% enzyme concentration is already sufficient to

produce a good protein hydrolysate. The protein content produced by both enzymes

was significantly different. This graph indicated that the average content of protein of


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products hydrolyzed by crude enzyme was 6%, while the others were approximatelly

8%.

Figure 4.9 Graph of temperature versus protein content

Temperature was shown to have significant effect on protein. Figure 4.9 represented

the effect of temperature to protein content. For both types of papain, there were slight

increases of protein content as the temperature got higher. Therefore, the point of

difference should be located. It was known that when incubation temperature was

45oC, it produced significantly different protein results from those that are incubated

at 50, 55, and 60oC. However, the protein result between the incubation temperature

of 50, 55, and 60oC showed no significant difference.

From the experiment, results showed that pure papain produced higher protein

content. This result was expected because even though the activity has been

equalized, crude papain still contained high amount of additives. These additives

might affect the work of the enzyme itself so that the hydrolysis will be disrupted. The

other problem with using crude papain was that it was not totally soluble in the water.

The amount of enzyme that was added was 10, 20, and 30% of the water weight.

During the experiment, there were some particles that could not dissolve to the

distilled water. This may be the cause of ineffective hydrolysis. The particles that


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could not dissolve were not able to hydrolyze and cause the protein unable to dissolve

in water.

Result showed that when temperature was 45oC, it could not produce protein with

higher result, which means that the rate of hydrolysis was not as good as those

incubated at 50, 55, and 60oC. It was consistent to the literature that said that papain is

optimum between temperature 50-60oC. Therefore, it was also predicted that at 45oC,

soluble protein will be less than at 50-60oC. With consideration of denaturation and

energy use, it could be concluded that the effective incubation temperature for

hydrolysis of shrimp head waste by both papain was 50oC.

Concentration of enzyme was said to have no significant difference in the protein

content. It can be concluded that 10% enzyme concentration was adequate to be used

for hydrolysis of L. vannamei hydrolysis. For crude papain, 10% was probably

adequate because when the concentration was higher than 10%, there were more

insoluble particles, which made it inefficient. When concentration of enzyme added

reaches a certain point, the increase of soluble protein in hydrolysate will not increase

significantly or even does not increase at all. This may be the reason why the

concentration did not provide higher protein result for the pure papain.

Shrimp head hydrolysis was a process to degrade the long protein chain in shrimp

head to small peptides and amino acids. It also takes out and degrades protein that is

stored in chitin. Chitin is a part of the shell and head. The digestibility of protein will

decrease if it is stored inside chitin (Adrizal et al., 1999). To know how effective the

process of protein hydrolysis went, recovered protein should be calculated. The data

below showed the recovered protein from the head.


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Table 4.3 Recovered protein of each treatment Concentration (%)

Temperature (oC) Crude Papain Pure Papain

10

45 52.10 ± 1.038 87.22 ± 0.273

50 114.30 ±3.421 89.02 ± 0.547

55 75.23 ± 0.499 90.71 ± 2.706

60 73.31 ± 5.559 97.04 ± 0.254

20

45 63.49 ± 0.785 96.75 ± 3.216

50 104.79 ± 0.370 96.50 ± 4.257

55 77.67 ± 11.511 102.90 ± 0.968

60 73.68 ± 10.053 106.88 ± 2.217

30

45 55.83 ± 1.815 104.49 ± 1.608

50 108.78 ± 12.307 99.32 ± 4.641

55 66.37 ± 0.934 93.36 ± 1.216

60 75.51 ± 12.851 100.04 ± 1.209

The protein recovered was the ratio of soluble protein in hydrolysate and protein in

raw material (shrimp head). Statistical analysis using three-way ANOVA showed that

there were significant differences among type of enzyme, concentration, and

incubation temperature to the protein recovered. However, the significant interactions

were only from type of enzyme and concentration of enzyme and type of enzyme and

incubation temperature.

Figure 4.10 Graph of concentration versus recovered protein

Figure 4.10 showed the relationship between papain concentration and recovered

protein. From the graph, recovered protein from products hydrolyzed with pure


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papain was higher than the crude papain. The difference of protein recovered between

the enzyme concentrations laid between 10% and 20% concentration. However,

protein recovered from 10% and 30% and 20% and 30% showed no significant

difference. Among incubation temperatures, protein recovered was different

significantly. The differences were between all of the temperature, except protein

recovered between 55oC and 60oC.

In graph temperature versus recovered protein, the relationships were not linear. The

highest recovered protein seemed to come from 50oC incubation temperature.

Therefore, it supported the analysis of protein content conclusion that 50oC was the

best temperature to incubate the head of L.vannamei.

Figure 4.11 Graph of temperature versus recovered protein

If the effect of enzyme concentration were to look at separately based on the type of

enzyme, they would have shown that there was no significant difference on protein

recovered between the enzyme concentration on both crude and pure enzyme. Protein

recovered gave rough view of whether the enzyme hydrolyzed properly. Most of the

protein recovered results were more than 50%. It was considered as effective because

it has hydrolyze the protein from chitin and obtained half of the protein content in the

shrimp head. There were also some data of protein recovered that exceeds 100%. This


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showed a very effective hydrolysis. It was possible because in determining the protein

content in raw material was not all uniform. Some shrimp heads might contain higher

protein content than the result.

Other than recovered protein, the other important determination of which enzymes

produced better result was done by comparing the dry basis of both products.

Comparing dry basis gave a rough indication of the percentage of protein in product

in solid part.

Figure 4.12 Graph of concentration versus protein content (dry basis)

This graph (Figure 4.12) showed that concentration of enzyme affected the dry basis

protein content. Although in wet basis calculation, there seemed to be no difference

between protein content hydrolyzed with different concentrations, the difference can

be seen in this graph. The protein content decreased as the papain concentration

increased. This may happen because of the proportion of high ash content. As said

earlier, the process of hydrolysis can be considered a success if the protein content

was higher than the ash content. However, in these products, the ash content seemed

to be higher.

In comparison, products hydrolyzed with pure papain did not really show any changes

in protein content. The difference between protein content of products hydrolyzed by


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crude and pure papain was different significantly. The protein content of crude papain

hydrolysis was about 30-50% but it is up to 90% in pure papain hydrolysate.

Protein content for each enzyme was insignifantly different based on the graph of

temperature versus protein (Figure 4.13). The line was almost linear and that means

that temperature has no effect in protein content. Statistical analysis also supported

this prediction. Once again, the protein content of the of pure and crude papain was

significantly different.

Figure 4.13 Graph of temperature versus protein content (dry basis)

4.6.5. Fat Content Fat content was determined for two products from each enzyme that produced the best

protein result according to statistical analysis. Since the statistical analysis done to

protein content showed that concentration gave no significant difference to protein

content, the enzyme concentration used for this fat analysis was 10%. The

temperature that was chosen was 50oC because based on the statistical analysis; it

showed that from 50oC onwards there were no significant changes in protein content.

The fat content from the sample that was hydrolyzed with pure papain was 0.068%

and the fat content from sample hydrolyzed with crude papain was 0.16%. Both

results showed that the hydrolysates had lower fat content than the raw material

because raw material contains about 2.1% fat. The result of fat in the hydrolysate was


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lower because this calculation was calculated from wet basis. The dry basis

calculation showed that the raw materials contained about 10.18% fat content. The

products however, contained 0.8% for pure papain hydrolysate and 1.4% for crude

papain hydrolysate. That means not all the fat that existed in the shrimp head entered

the hydrolysate. It may be because the fat was not filtered through the muslin cloth.

There was also one possibility why fat did not enter the hydrolysate. Earlier in this

chapter, it was said that centrifugation phase left out three phases: the solid part, the

liquid part, and the floating solid part, which usually remained on the wall of tube.

The floating part is most probably contained the fat. However, because in preliminary

research filtering the floating particles cause significance changes in protein content,

it was not filtered in the main research.

The fat content of product hydrolyzed with pure papain was lower rather than the one

hydrolyzed with crude papain. During centrifugation of product hydrolyzed by crude

papain, there were a lot of floating particles on the top but not in the product of pure

papain hydrolysis. Product of pure papain hydrolysis also had three phases, but the

floating particles of crude papain hydrolysis existed in the middle of the supernatant

and the solid waste.

Figure 4.14 Result of centrifugation of hydrolysate from pure papain

Liquid phase

Solid phase


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Figure 4.15 Result of centrifugation of hydrolysate from crude papain

The crude papain contains salt and when salt is dissolved in water, the water will

become salt water and will have higher density than water. The salt water density is

1.025 g/cm3 while water’s density is 1 g/cm3 (Chang, 2000). The principle of

centrifugation is separation based specific gravity. If the floating particles did not

float in the protein concentrate hydrolyzed by pure enzyme, it means that the floating

particles were supposed to be in the bottom but due to the higher density of salt water,

the particles identified as fat will be floating.

4.7. Sensory Evaluation

Hedonic test was done to two products which had the highest protein content. Since

sensory analysis requires the sample to be fresh, new batch of hydrolysates were

made. Although there were only two products, they were made into three replicates.

The reason in doing this was to compare the homogeneity of panelists score. The

samples were displayed in small transparent glass with lid. The lid was applied to

prevent the smell to evaporate. The room used for this test was a special room for

sensory purpose only. It was bright and it was in cubicle so that there will be no

interactions between panelists.

Floating particles

Liquid phase

solid phase


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Figure 4.16 Condition of sensory evaluation

There were four parameters that were examined, which were appearance, color, smell,

and taste. The panelists were also told to give their comment of the samples. The

graph below presented the result of hedonic test. Although there were triplicates,

average of each was determined.

Figure 4.17 Result of Hedonic Test

Based on the chart above, it can be seen that the score for appearance and color for

both product was not too different. Statistical analysis showed the same result that

there were no significant difference between two samples in appearance and color. In

smell parameter, the score showed that there was significant difference. From the


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graph, the difference was not really extreme but statistic showed that the difference

was significant. The smell of products from crude papain was more likeable than the

products from pure papain. The same goes for the taste. The score that was gained for

the pure papain and crude papain was really different. The graph showed that the one

hydrolyzed with crude papain had an average score of 4.69 (almost 5) whereas the

average score for hydrolysate from pure papain was 2.49. Most panelists said that the

samples that were hydrolyzed by pure papain gave out a bitter taste, which made it

less likeable. The score 2 meant that the panelist did not like the product. However,

the samples of crude papain had an average score 5 (like slightly) for its taste.

The comments from the panelists said that the hydrolysate from pure papain was

bitter, the color was more turbid, and the smell was less likeable. The hydrolysate

from crude papain on the other hand was salty, it was less turbid and the smell was

like smell of steamed shrimp. Some panelists said that the hydrolysate from crude

papain was too salty and suggested that sugar needs to be added as well to reduce

saltiness and improve the taste.

Bitterness from pure papain products was a result of hydrolysis. Hydrolysis cut down

long chain of protein to small peptides and amino acids. Some small peptides and

amino acids have bitter taste (Belitz, 2009), which cause the product to develop bitter

taste. In crude papain products these bitterness was masked by the additives of crude

papain, which are salt and sugar.


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CHAPTER 5 - CONCLUSIONS AND RECOMMENDATIONS

5.1. Conclusion

Both pure papain and crude papain can hydrolyze protein from shrimp head.

However, in terms of higher protein content, pure papain can hydrolyze better.

Concentration of enzyme did not affect the protein content, because the

protein content of 10, 20, and 30% enzyme concentration did not give any

significant difference. Then, it was true that the enzyme concentration at 10%

can hydrolyze as much protein as enzyme concentration 20% and 30%.

The temperature, on the other side, affected the protein content significantly.

There was significant difference between protein content of products

hydrolyzed at 45oC to 50oC, 55oC, and 60oC. However, there were no

significant differences between those three temperatures to the protein content.

The initial hypothesis that the optimum temperature was between 45-60oC

should be rejected because the optimum temperature was between 50-60oC.

In terms of sensory acceptance, there were four defining parameters. The level

of acceptance of appearance and color for both products were the same.

However, it was different in smell and taste parameters. It seemed that the

product hydrolyzed with crude papain was more likeable than the pure papain,

especially its taste. Therefore, it was true that crude papain produced product

with higher level of sensory acceptance.

5.2. Recommendation

The lowest enzyme concentration used in this enzyme was 10% and the result

did not give significant difference. Next time, the lower enzyme concentration

may be examined to see significant trend and to reduce cost of production.

Since protein concentrate in liquid form is unusual, drying process by freeze

or spray drying can be added to improve the palatability, diversity, and shelf

life of products.


Judith Salim

LIST OF REFERENCES

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Abun, Aisjah, T., & Saefulhadjar, D. (2006). Pemanfaatan Limbah Cair Ekstraksi Kitin dari Kulit Udang Produk Proses Kimiawi dan Biologis Sebagai Imbuhan Pakan dan Implikasinya Terhadap Pertumbuhan Ayam Broiler. Jatinangor: Universitas Padjadjaran.

Adrizal, Elihsridas, & Mirzah. (1999). Efek Pengolahan Terhadap Kualitas Protein pada Tepung Limbah Udang. Padang: Universitas Andalas.

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Binsan, W. (2007). Antioxidative Activity of Mungoong, An Extract Paste, from White Shrimp (Litopenaeus vannamei) Cephalothorax. Thailand.

Bondad-Reantaso, M., McGladdery, S., East, I., & Subasinghe, R. (2001). Asia Diagnostic Guid to Aquatic Animal Disease. Rome: Food and Agriculture Organization.

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Hosomi, R., Fukao, M., Fukunaga, K., Okuno, M., Yagita, R., & Kanda, S. (2010). Effect of Fish Protein and Peptides on Lipid Absorption in Rats. Trace Nutrients Research, 27, 21-27.

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Mizani, A., & Aminlari, B. (2007). A New Process for Deproteinization of Chitin from Shrimp Head Waste. Proceedings of European Congress of Chemical Engineering (ECCE-6). Copenhagen.

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APPENDICES

Appendix 5 Standard curve of Lowry

Appendix 6 Standard Curve for Enzyme Activity


Judith Salim

Appendix 7 Statistical analysis of yield YIELD Three Way Analysis of Variance Variable analyzed: yield Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 19.033 19.033 21.468 0.000 0.016 Among Columns 2 85.178 42.589 48.037 0.000 0.073 Among Slices 3 493.557 164.519 185.562 0.000 0.428 A x B Inter. 2 70.242 35.121 39.613 0.000 0.060 A x C Inter. 3 205.270 68.423 77.175 0.000 0.177 B x C Inter. 6 119.224 19.871 22.412 0.000 0.099 AxBxC Inter. 6 131.396 21.899 24.700 0.000 0.110 Within Groups 24 21.278 0.887 Total 47 1145.179 24.366 Omega squared for combined effects = 0.963 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 72.297 0.040 0.199 Cell 1 1 2 2 73.540 0.067 0.259 Cell 1 1 3 2 74.270 0.000 0.015 Cell 1 1 4 2 68.683 2.204 1.485 Cell 1 2 1 2 72.451 0.031 0.177 Cell 1 2 2 2 70.271 16.892 4.110 Cell 1 2 3 2 83.108 0.169 0.411 Cell 1 2 4 2 70.290 0.003 0.058 Cell 1 3 1 2 71.994 0.071 0.267 Cell 1 3 2 2 73.212 0.001 0.038 Cell 1 3 3 2 84.105 0.158 0.398 Cell 1 3 4 2 83.754 0.643 0.802 Cell 2 1 1 2 77.165 0.000 0.001 Cell 2 1 2 2 71.069 0.039 0.198 Cell 2 1 3 2 77.580 0.104 0.322 Cell 2 1 4 2 67.816 0.227 0.476


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Cell 2 2 1 2 77.288 0.073 0.269 Cell 2 2 2 2 70.999 0.361 0.601 Cell 2 2 3 2 78.094 0.046 0.215 Cell 2 2 4 2 67.967 0.045 0.212 Cell 2 3 1 2 77.357 0.063 0.252 Cell 2 3 2 2 71.928 0.000 0.021 Cell 2 3 3 2 78.066 0.039 0.197 Cell 2 3 4 2 67.534 0.000 0.019 Row 1 24 74.831 30.281 5.503 Row 2 24 73.572 18.682 4.322 Col 1 16 72.803 12.131 3.483 Col 2 16 73.809 25.936 5.093 Col 3 16 75.994 32.600 5.710 Slice 1 12 74.759 6.928 2.632 Slice 2 12 71.837 3.131 1.770 Slice 3 12 79.204 12.550 3.543 Slice 4 12 71.007 36.628 6.052 TOTAL 48 74.202 24.366 4.936 TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 13532871.42 with deg.s freem: 6 and 1. Cochran C statistic = 0.79 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 141.58 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.006 q = 4.274 0.0157 YES 1 - 3 -3.191 q = 13.557 0.0000 YES --------------------------------------------------------------- 2 - 3 -2.185 q = 9.283 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 2.922 q = 10.751 0.0000 YES 1 - 3 -4.445 q = 16.354 0.0000 YES 1 - 4 3.751 q = 13.801 0.0000 YES --------------------------------------------------------------- 2 - 3 -7.367 q = 27.105 0.0000 YES 2 - 4 0.829 q = 3.050 0.1644 NO --------------------------------------------------------------- 3 - 4 8.196 q = 30.155 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS ---------------------------------------------------------------


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Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.607 q = 2.413 0.2234 NO 1 - 3 -15.071 q = 22.636 0.0000 YES --------------------------------------------------------------- 2 - 3 -13.465 q = 20.223 0.0000 YES --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.151 q = 0.226 0.9861 NO 1 - 3 0.282 q = 0.424 0.9517 NO --------------------------------------------------------------- 2 - 3 0.433 q = 0.651 0.8904 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.867 q = 1.302 0.3666 NO --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 2.323 q = 3.489 0.0212 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 16.221 q = 24.362 0.0001 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.123 q = 0.185 0.9907 NO


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1 - 3 -0.192 q = 0.288 0.9775 NO --------------------------------------------------------------- 2 - 3 -0.069 q = 0.104 0.9971 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.071 q = 0.106 0.9970 NO 1 - 3 -0.858 q = 1.289 0.6385 NO --------------------------------------------------------------- 2 - 3 -0.929 q = 1.396 0.5921 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.515 q = 0.773 0.8492 NO 1 - 3 -0.487 q = 0.731 0.8638 NO --------------------------------------------------------------- 2 - 3 0.028 q = 0.042 0.9996 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.151 q = 0.226 0.9861 NO 1 - 3 0.282 q = 0.424 0.9517 NO --------------------------------------------------------------- 2 - 3 0.433 q = 0.651 0.8904 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.123 q = 0.185 0.8972 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.071 q = 0.106 0.9406 NO ---------------------------------------------------------------


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SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.515 q = 0.773 0.5895 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.151 q = 0.226 0.8742 NO --------------------------------------------------------------- Appendix 8 Statistical analysis of water content WATER CONTENT ANALYSIS Three Way Analysis of Variance Variable analyzed: wc Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: concentration (Fixed Levels) Factor C (slices) variable: temperature (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 616.940 616.940 5954.305 0.000 0.753 Among Columns 2 99.313 49.656 479.252 0.000 0.121 Among Slices 3 6.596 2.199 21.219 0.000 0.008 A x B Inter. 2 84.015 42.008 405.431 0.000 0.102 A x C Inter. 3 4.560 1.520 14.669 0.000 0.005 B x C Inter. 6 1.680 0.280 2.702 0.038 0.001 AxBxC Inter. 6 3.366 0.561 5.415 0.001 0.003 Within Groups 24 2.487 0.104 Total 47 818.957 17.425 Omega squared for combined effects = 0.994 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 87.283 0.140 0.375


Judith Salim

Cell 1 1 2 2 88.283 0.140 0.375 Cell 1 1 3 2 87.311 0.007 0.082 Cell 1 1 4 2 88.712 0.008 0.090 Cell 1 2 1 2 84.212 0.052 0.229 Cell 1 2 2 2 84.219 0.511 0.715 Cell 1 2 3 2 83.941 0.156 0.394 Cell 1 2 4 2 83.634 0.733 0.856 Cell 1 3 1 2 81.902 0.087 0.294 Cell 1 3 2 2 81.465 0.043 0.208 Cell 1 3 3 2 80.719 0.149 0.386 Cell 1 3 4 2 80.569 0.259 0.509 Cell 2 1 1 2 92.829 0.005 0.073 Cell 2 1 2 2 91.525 0.016 0.127 Cell 2 1 3 2 91.324 0.025 0.158 Cell 2 1 4 2 90.832 0.009 0.093 Cell 2 2 1 2 92.557 0.001 0.035 Cell 2 2 2 2 91.315 0.137 0.370 Cell 2 2 3 2 91.203 0.002 0.049 Cell 2 2 4 2 91.390 0.001 0.030 Cell 2 3 1 2 92.457 0.003 0.051 Cell 2 3 2 2 90.900 0.001 0.035 Cell 2 3 3 2 91.171 0.000 0.009 Cell 2 3 4 2 90.790 0.001 0.030 Row 1 24 84.354 8.307 2.882 Row 2 24 91.524 0.476 0.690 Col 1 16 89.763 4.229 2.056 Col 2 16 87.809 15.760 3.970 Col 3 16 86.246 27.987 5.290 Slice 1 12 88.540 20.799 4.561 Slice 2 12 87.951 16.239 4.030 Slice 3 12 87.611 18.290 4.277 Slice 4 12 87.654 18.524 4.304 TOTAL 48 87.939 17.425 4.174 TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 10019.01 with deg.s freem: 6 and 1. Cochran C statistic = 0.29 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 73.47 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 1.954 q = 24.278 0.0000 YES 1 - 3 3.516 q = 43.694 0.0000 YES --------------------------------------------------------------- 2 - 3 1.562 q = 19.416 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant?


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--------------------------------------------------------------- 1 - 2 0.589 q = 6.336 0.0008 YES 1 - 3 0.929 q = 9.993 0.0000 YES 1 - 4 0.886 q = 9.530 0.0000 YES --------------------------------------------------------------- 2 - 3 0.340 q = 3.657 0.0718 NO 2 - 4 0.297 q = 3.194 0.1364 NO --------------------------------------------------------------- 3 - 4 -0.043 q = 0.462 0.9877 NO --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 5.079 q = 22.313 0.0000 YES 1 - 3 8.143 q = 35.777 0.0000 YES --------------------------------------------------------------- 2 - 3 3.065 q = 13.465 0.0000 YES --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.558 q = 2.453 0.2131 NO 1 - 3 0.042 q = 0.186 0.9906 NO --------------------------------------------------------------- 2 - 3 0.601 q = 2.639 0.1702 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -2.120 q = 9.313 0.0001 YES --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -7.757 q = 34.079 0.0001 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means


Judith Salim

alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -10.221 q = 44.905 0.0001 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.272 q = 1.195 0.6795 NO 1 - 3 0.372 q = 1.635 0.4901 NO --------------------------------------------------------------- 2 - 3 0.100 q = 0.441 0.9481 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.210 q = 0.922 0.7931 NO 1 - 3 0.626 q = 2.749 0.1482 NO --------------------------------------------------------------- 2 - 3 0.416 q = 1.827 0.4133 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.122 q = 0.534 0.9247 NO 1 - 3 0.154 q = 0.675 0.8827 NO --------------------------------------------------------------- 2 - 3 0.032 q = 0.141 0.9946 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.558 q = 2.453 0.2131 NO 1 - 3 0.042 q = 0.186 0.9906 NO --------------------------------------------------------------- 2 - 3 0.601 q = 2.639 0.1702 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH SLICE SLICE 1 COMPARISONS ---------------------------------------------------------------


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Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.272 q = 1.195 0.4065 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.210 q = 0.922 0.5205 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.122 q = 0.534 0.7089 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.558 q = 2.453 0.0957 NO --------------------------------------------------------------- Appendix 5 Statistical analysis of ash content ASH CONTENT Three Way Analysis of Variance Variable analyzed: ash Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 426.976 426.976 11459.905 0.000 0.778 Among Columns 2 60.163 30.081 807.372 0.000 0.110 Among Slices 3 0.240 0.080 2.143 0.121 0.000 A x B Inter. 2 59.875 29.937 803.512 0.000 0.109 A x C Inter. 3 0.083 0.028 0.741 0.538 0.000


Judith Salim

B x C Inter. 6 0.133 0.022 0.593 0.733 0.000 AxBxC Inter. 6 0.137 0.023 0.614 0.717 0.000 Within Groups 24 0.894 0.037 Total 47 548.500 11.670 Omega squared for combined effects = 0.997 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 3.713 0.001 0.032 Cell 1 1 2 2 3.795 0.003 0.057 Cell 1 1 3 2 3.749 0.000 0.022 Cell 1 1 4 2 3.602 0.003 0.054 Cell 1 2 1 2 6.223 0.408 0.639 Cell 1 2 2 2 6.466 0.182 0.426 Cell 1 2 3 2 6.738 0.005 0.070 Cell 1 2 4 2 6.570 0.042 0.204 Cell 1 3 1 2 8.921 0.036 0.190 Cell 1 3 2 2 9.254 0.161 0.401 Cell 1 3 3 2 9.209 0.046 0.214 Cell 1 3 4 2 9.386 0.006 0.079 Cell 2 1 1 2 0.457 0.000 0.014 Cell 2 1 2 2 0.459 0.000 0.003 Cell 2 1 3 2 0.544 0.000 0.002 Cell 2 1 4 2 0.545 0.000 0.016 Cell 2 2 1 2 0.460 0.000 0.003 Cell 2 2 2 2 0.465 0.000 0.011 Cell 2 2 3 2 0.541 0.000 0.001 Cell 2 2 4 2 0.545 0.000 0.003 Cell 2 3 1 2 0.459 0.000 0.008 Cell 2 3 2 2 0.473 0.000 0.005 Cell 2 3 3 2 0.551 0.000 0.001 Cell 2 3 4 2 0.547 0.000 0.005 Row 1 24 6.469 5.282 2.298 Row 2 24 0.504 0.002 0.043 Col 1 16 2.108 2.758 1.661 Col 2 16 3.501 9.651 3.107 Col 3 16 4.850 20.146 4.488 Slice 1 12 3.372 11.768 3.430 Slice 2 12 3.485 12.687 3.562 Slice 3 12 3.555 12.607 3.551 Slice 4 12 3.533 12.780 3.575 TOTAL 48 3.486 11.670 3.416 TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 411246.45 with deg.s freem: 6 and 1. Cochran C statistic = 0.46 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 176.65 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS


Judith Salim

--------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.393 q = 28.874 0.0000 YES 1 - 3 -2.742 q = 56.826 0.0000 YES --------------------------------------------------------------- 2 - 3 -1.349 q = 27.953 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -2.968 q = 21.745 0.0000 YES 1 - 3 -5.785 q = 42.381 0.0000 YES --------------------------------------------------------------- 2 - 3 -2.817 q = 20.636 0.0000 YES --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.000 q = 0.002 1.0000 NO 1 - 3 -0.002 q = 0.017 0.9999 NO --------------------------------------------------------------- 2 - 3 -0.002 q = 0.015 0.9999 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 3.057 q = 22.399 0.0001 YES --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 6.025 q = 44.142 0.0001 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means


Judith Salim

alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 8.839 q = 64.763 0.0001 YES --------------------------------------------------------------- Appendix 6 Statistical analysis of protein content PROTEIN CONTENT Three Way Analysis of Variance Variable analyzed: protein Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 38.401 38.401 174.560 0.000 0.618 Among Columns 2 0.354 0.177 0.806 0.458 0.000 Among Slices 3 15.075 5.025 22.842 0.000 0.233 A x B Inter. 2 0.199 0.100 0.453 0.641 0.000 A x C Inter. 3 0.236 0.079 0.358 0.784 0.000 B x C Inter. 6 0.936 0.156 0.709 0.645 0.000 AxBxC Inter. 6 1.095 0.182 0.830 0.559 0.000 Within Groups 24 5.280 0.220 Total 47 61.576 1.310 Omega squared for combined effects = 0.829 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 5.068 0.897 0.947 Cell 1 1 2 2 5.535 0.027 0.166 Cell 1 1 3 2 6.238 0.002 0.041 Cell 1 1 4 2 6.282 0.227 0.476 Cell 1 2 1 2 5.023 0.004 0.062 Cell 1 2 2 2 5.872 0.000 0.021 Cell 1 2 3 2 6.707 0.988 0.994 Cell 1 2 4 2 6.483 0.782 0.884 Cell 1 3 1 2 5.096 0.027 0.166 Cell 1 3 2 2 6.590 0.556 0.746 Cell 1 3 3 2 5.887 0.007 0.083 Cell 1 3 4 2 6.692 1.297 1.139 Cell 2 1 1 2 6.623 0.000 0.021 Cell 2 1 2 2 7.912 0.000 0.021


Judith Salim

Cell 2 1 3 2 7.947 0.006 0.075 Cell 2 1 4 2 8.420 0.155 0.393 Cell 2 2 1 2 6.736 0.002 0.041 Cell 2 2 2 2 8.098 0.072 0.269 Cell 2 2 3 2 7.816 0.026 0.162 Cell 2 2 4 2 7.951 0.011 0.104 Cell 2 3 1 2 6.941 0.043 0.207 Cell 2 3 2 2 7.981 0.124 0.352 Cell 2 3 3 2 7.946 0.015 0.122 Cell 2 3 4 2 8.566 0.011 0.104 Row 1 24 5.956 0.606 0.778 Row 2 24 7.745 0.402 0.634 Col 1 16 6.753 1.471 1.213 Col 2 16 6.836 1.215 1.102 Col 3 16 6.962 1.396 1.182 Slice 1 12 5.915 0.891 0.944 Slice 2 12 6.998 1.268 1.126 Slice 3 12 7.090 0.880 0.938 Slice 4 12 7.399 1.189 1.090 TOTAL 48 6.850 1.310 1.145 TESTS FOR HOMOGENEITY OF VARIANCE -------------------------------------------------------------------- Hartley Fmax test statistic = 3025.00 with deg.s freem: 6 and 1. Cochran C statistic = 0.25 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 88.50 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.083 q = 8.002 0.0000 YES 1 - 3 -1.176 q = 8.683 0.0000 YES 1 - 4 -1.485 q = 10.965 0.0000 YES --------------------------------------------------------------- 2 - 3 -0.092 q = 0.681 0.9625 NO 2 - 4 -0.401 q = 2.963 0.1832 NO --------------------------------------------------------------- 3 - 4 -0.309 q = 2.282 0.3903 NO --------------------------------------------------------------- Appendix 7 Statistical analysis of recovered protein Recovered Protein Three Way Analysis of Variance Variable analyzed: pro rec Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared


Judith Salim

Among Rows 1 4150.060 4150.060 149.061 0.000 0.279 Among Columns 2 240.376 120.188 4.317 0.025 0.013 Among Slices 3 4093.520 1364.507 49.010 0.000 0.272 A x B Inter. 2 247.672 123.836 4.448 0.023 0.013 A x C Inter. 3 4772.234 1590.745 57.136 0.000 0.318 B x C Inter. 6 357.881 59.647 2.142 0.085 0.013 AxBxC Inter. 6 206.034 34.339 1.233 0.324 0.003 Within Groups 24 668.193 27.841 Total 47 14735.970 313.531 Omega squared for combined effects = 0.909 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 52.103 1.077 1.038 Cell 1 1 2 2 114.302 11.704 3.421 Cell 1 1 3 2 75.231 0.249 0.499 Cell 1 1 4 2 73.314 30.899 5.559 Cell 1 2 1 2 63.492 0.617 0.785 Cell 1 2 2 2 104.787 0.137 0.370 Cell 1 2 3 2 77.666 132.510 11.511 Cell 1 2 4 2 73.685 101.066 10.053 Cell 1 3 1 2 55.831 3.295 1.815 Cell 1 3 2 2 108.781 151.467 12.307 Cell 1 3 3 2 66.371 0.872 0.934 Cell 1 3 4 2 75.506 165.153 12.851 Cell 2 1 1 2 87.217 0.074 0.273 Cell 2 1 2 2 89.020 0.300 0.547 Cell 2 1 3 2 90.706 7.324 2.706 Cell 2 1 4 2 97.043 0.065 0.254 Cell 2 2 1 2 96.749 10.346 3.216 Cell 2 2 2 2 96.502 18.122 4.257 Cell 2 2 3 2 102.899 0.937 0.968 Cell 2 2 4 2 106.882 4.915 2.217 Cell 2 3 1 2 104.492 2.584 1.608 Cell 2 3 2 2 99.318 21.541 4.641 Cell 2 3 3 2 93.365 1.478 1.216 Cell 2 3 4 2 100.036 1.462 1.209 Row 1 24 78.422 421.106 20.521 Row 2 24 97.019 39.151 6.257 Col 1 16 84.867 319.633 17.878 Col 2 16 90.333 268.303 16.380 Col 3 16 87.962 378.437 19.453 Slice 1 12 76.647 456.171 21.358 Slice 2 12 102.119 93.189 9.653 Slice 3 12 84.373 179.722 13.406 Slice 4 12 87.744 238.413 15.441 TOTAL 48 87.721 313.531 17.707


Judith Salim

TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 2559.89 with deg.s freem: 6 and 1. Cochran C statistic = 0.25 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 86.19 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -5.466 q = 4.143 0.0194 YES 1 - 3 -3.095 q = 2.346 0.2412 NO --------------------------------------------------------------- 2 - 3 2.370 q = 1.797 0.4250 NO --------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -25.471 q = 16.722 0.0000 YES 1 - 3 -7.726 q = 5.072 0.0076 YES 1 - 4 -11.097 q = 7.285 0.0001 YES --------------------------------------------------------------- 2 - 3 17.746 q = 11.650 0.0000 YES 2 - 4 14.374 q = 9.437 0.0000 YES --------------------------------------------------------------- 3 - 4 -3.371 q = 2.213 0.4166 NO --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.371 q = 0.099 0.9973 NO 1 - 3 -2.193 q = 0.588 0.9096 NO --------------------------------------------------------------- 2 - 3 -1.822 q = 0.488 0.9366 NO --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -9.838 q = 2.637 0.1707 NO 1 - 3 -2.993 q = 0.802 0.8387 NO


Judith Salim

--------------------------------------------------------------- 2 - 3 6.845 q = 1.835 0.4103 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -23.730 q = 6.360 0.0002 YES --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -33.197 q = 8.898 0.0001 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -24.530 q = 6.575 0.0002 YES --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -9.532 q = 2.555 0.0835 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -7.482 q = 2.005 0.1692 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -12.192 q = 3.268 0.0298 YES


Judith Salim

--------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -9.838 q = 2.637 0.0746 NO --------------------------------------------------------------- Appendix 8 Two-way ANOVA of appearance in hedonic test Anova: Two-Factor Without Replication

SUMMARY Count Sum Average Variance1 6 34 5.666667 0.2666672 6 24 4 03 6 32 5.333333 0.2666674 6 27 4.5 0.35 6 30 5 06 6 24 4 07 6 33 5.5 0.38 6 27 4.5 1.59 6 27 4.5 0.3

10 6 30 5 0.811 6 27 4.5 0.312 6 29 4.833333 0.56666713 6 33 5.5 0.3

PK3 13 63 4.846154 0.641026PM1 13 66 5.076923 0.910256PK1 13 62 4.769231 0.692308PM2 13 63 4.846154 0.641026PK2 13 63 4.846154 0.474359PM3 13 60 4.615385 0.423077

ANOVA Source of Variation SS df MS F P-value F crit

Rows 22.33333 12 1.861111 4.844271 1.66E-05 1.917396Columns 1.448718 5 0.289744 0.754171 0.586356 2.36827Error 23.05128 60 0.384188

Total 46.83333 77


Judith Salim

Appendix 9 Two-way ANOVA of Color in hedonic test Anova: Two-Factor Without Replication

SUMMARY Count Sum Average Variance 1 6 36 6 0.82 6 27 4.5 0.73 6 32 5.333333 0.2666674 6 27 4.5 0.35 6 22 3.666667 0.6666676 6 24 4 07 6 33 5.5 0.38 6 27 4.5 1.59 6 24 4 0

10 6 29 4.833333 0.56666711 6 27 4.5 0.312 6 23 3.833333 1.36666713 6 31 5.166667 0.166667

PK3 13 62 4.769231 0.692308PM1 13 64 4.923077 1.74359PK1 13 59 4.538462 0.602564PM2 13 60 4.615385 1.423077PK2 13 61 4.692308 0.397436PM3 13 56 4.307692 0.730769

ANOVA

Source of Variation SS df MS F P-value F crit


Total 69.9487 77 Appendix 10 Two-way ANOVA of smell in hedonic test Anova: Two-Factor Without Replication

SUMMARY Count Sum Average Variance1 6 34 5.666667 0.2666672 6 26 4.333333 1.0666673 6 24 4 0.8


Judith Salim

4 6 27 4.5 0.35 6 30 5 06 6 33 5.5 0.37 6 36 6 08 6 27 4.5 0.39 6 24 4 1.6

10 6 26 4.333333 1.46666711 6 28 4.666667 0.66666712 6 28 4.666667 1.06666713 6 32 5.333333 0.266667

PK3 13 68 5.230769 0.525641PM1 13 67 5.153846 0.641026PK1 13 64 4.923077 0.74359PM2 13 61 4.692308 0.897436PK2 13 62 4.769231 0.858974PM3 13 53 4.076923 1.24359



Total 70.11538 77 Appendix 11 t-test between samples for smell t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means

PK3 PK1 PM1 PK1

Mean 5.230769 4.923077 Mean 5.153846 4.923077

Variance 0.525641 0.74359 Variance 0.641026 0.74359

Observations 13 13 Observations 13 13

Pearson Correlation 0.164053Pearson Correlation 0.139272

Hypothesized Mean Difference 0


df 12 df 12

t Stat 1.075466 t Stat 0.762001

P(T<=t) one‐tail 0.151657 P(T<=t) one‐tail 0.230387

t Critical one‐tail 1.782288 t Critical one‐tail 1.782288

P(T<=t) two‐tail 0.303314 P(T<=t) two‐tail 0.460775


Judith Salim

t Critical two‐tail 2.178813 t Critical two‐tail 2.178813

t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means

PM1 PK3 PM2 PK3

Mean 5.153846 5.230769 Mean 4.692308 5.230769






df 12 df 12

t Stat ‐0.2907 t Stat ‐2.00684






PM3 PM1 PM3 PM2

Mean 4.076923 5.153846 Mean 4.076923 4.692308






df 12 df 12

t Stat ‐4.06981 t Stat ‐2.55117





t‐Test: Paired Two Sample for Means


Judith Salim


PM2 PK1 PK2 PK1

Mean 4.692308 4.923077 Mean 4.769231 4.923077



Pearson Correlation 0.172635 Pearson Correlation 0.705832 Hypothesized Mean Difference 0


df 12 df 12

t Stat ‐0.71375 t Stat ‐0.80539






PK2 PK3 PM2 PM1

Mean 4.769231 5.230769 Mean 4.692308 5.153846





df 12 df 12

t Stat ‐1.4771 t Stat ‐2.14377






PK2 PM2 PK2 PM3

Mean 4.769231 4.692308 Mean 4.769231 4.076923





df 12 df 12

t Stat 0.267261 t Stat 2.419798




Judith Salim




PM3 PK3 PM3 PK1

Mean 4.076923 5.230769 Mean 4.076923 4.923077





df 12 df 12

t Stat ‐4.21464 t Stat ‐3.09073






PK2 PM1

Mean 4.769231 5.153846

Variance 0.858974 0.641026

Observations 13 13

Pearson Correlation 0.276438Hypothesized Mean Difference 0

df 12

t Stat ‐1.32842

P(T<=t) one‐tail 0.104375

t Critical one‐tail 1.782288

P(T<=t) two‐tail 0.208749

t Critical two‐tail 2.178813


Judith Salim

Appendix 12 Two-way ANOVA of taste in hedonic test Anova: Two-Factor Without Replication

SUMMARY Count Sum Average Variance1 6 23 3.833333 1.7666672 6 23 3.833333 0.5666673 6 22 3.666667 0.6666674 6 24 4 1.25 6 15 2.5 1.16 6 19 3.166667 4.1666677 6 27 4.5 7.58 6 15 2.5 2.79 6 25 4.166667 0.966667

10 6 24 4 3.611 6 20 3.333333 1.86666712 6 21 3.5 2.713 6 22 3.666667 1.066667

PK3 13 67 5.153846 0.641026PM1 13 35 2.692308 0.897436PK1 13 56 4.307692 1.397436PM2 13 32 2.461538 0.935897PK2 13 60 4.615385 1.589744PM3 13 30 2.307692 0.730769


Rows 25.53846 12 2.128205 2.618297 0.007086 1.917396Columns 100.5641 5 20.11282 24.74448 1.95E-13 2.36827Error 48.76923 60 0.812821

Total 174.8718 77


Judith Salim

Appendix 13 t-test between samples for taste t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means

PK3 PM3 PM1 PM3

Mean 5.153846 2.307692 Mean 2.692308 2.307692





df 12 df 12

t Stat 8.973818 t Stat 1.443376

P(T<=t) one‐tail 5.69E‐07 P(T<=t) one‐tail 0.087254


P(T<=t) two‐tail 1.14E‐06 P(T<=t) two‐tail 0.174509



PK2 PM3 PK3 PK2

Mean 4.615385 2.307692 Mean 5.153846 4.615385





df 12 df 12

t Stat 5.57086 t Stat 2.213594

P(T<=t) one‐tail 6.08E‐05 P(T<=t) one‐tail 0.023488





PM2 PK2 PK3 PM2

Mean 2.461538 4.615385 Mean 5.153846 2.461538



Pearson Correlation ‐0.0473 Pearson Correlation 0.223454 Hypothesized Mean Difference 0


df 12 df 12

t Stat ‐4.77859 t Stat 8.75


Judith Salim

P(T<=t) one‐tail 0.000225 P(T<=t) one‐tail 7.43E‐07


P(T<=t) two‐tail 0.00045 P(T<=t) two‐tail 1.49E‐06



PK3 PK1 PM1 PK1

Mean 5.153846 4.307692 Mean 2.692308 4.307692



Pearson Correlation 0.562147 Pearson Correlation ‐0.20607 Hypothesized Mean Difference 0


df 12 df 12

t Stat 3.090733 t Stat ‐3.50813






PK1 PM3 PM2 PM3

Mean 4.307692 2.307692 Mean 2.461538 2.307692



Pearson Correlation ‐0.01903 Pearson Correlation 0.720865Hypothesized Mean Difference 0


df 12 df 12

t Stat 4.898979 t Stat 0.805387






PM1 PK2 PK1 PK2

Mean 2.692308 4.615385 Mean 4.307692 4.615385



Pearson Correlation ‐0.3864 Pearson Correlation 0.756935


Judith Salim



df 12 df 12

t Stat ‐3.7547 t Stat ‐1.29777






PM1 PM2 PK1 PM2

Mean 2.692308 2.461538 Mean 4.307692 2.461538



Pearson Correlation 0.622515 Pearson Correlation ‐0.13453Hypothesized Mean Difference 0


df 12 df 12

t Stat 1 t Stat 4.095937






PM1 PK3

Mean 2.692308 5.153846

Variance 0.897436 0.641026

Observations 13 13

Pearson Correlation 0.067612Hypothesized Mean Difference 0

df 12

t Stat ‐7.40656

P(T<=t) one‐tail 4.1E‐06

t Critical one‐tail 1.782288

P(T<=t) two‐tail 8.2E‐06

t Critical two‐tail 2.178813


CURRICULUM VITAE

Name : Judith Salim

Place of Birth : Jakarta

Date of Birth : 26 April 1989

Address : Jl. Karang Asri V C3/13

Lebak Bulus, Jakarta 12440

Education : 2007 – present Swiss German University, Serpong

(Majoring Food Technology)

2004 – 2007 SMA Labschool Kebayoran, Jakarta

2001 – 2004 SMP Labschool Kebayoran, Jakarta

Courses : English Course at EF, Jakarta

Piano Course at Yamaha

German Course at Goethe Institute, Jakarta

Work Experience : March 2010 – August 2010, Internship Program in

Kattendorfer Hof, Germany.

December 2008 – January 2009, Internship Program at

PT. Frisian Flag Indonesia, Jakarta.

September 2008 – November 2008, Internship Program

PT. Multi Bintang Indonesia, Tbk., Jakarta.

Seminars and Workshop : 2007, Robotics and Neuroprothesis in Theurapeutic

Science – Innovative Biomedical Engineering

Solutions to Improve Human Functioning, SGU.


Judith Salim

2008, Cross Transfer Effects on Muscular Training,

SGU.

2008, Bio – reaction Modelling, SGU.

2009, Management of Obesity, SGU.

2010, Plant Biotechnology, SGU.

Skills/Interests : Computer Ms. Office,

Language Indonesian (native speaker)

English (Intermediate)

German (Basics)

Hobbies Travelling, Cooking, Singing, Playing

piano, Swimming, and Jogging

production of protein concentrate by enzymatic hydrolysis of shrimp (l. vannamei) head

Documents

enzymatic hydrolysis

palatability of shrimp

vannamei head page

shrimp processing

major advisor shrimp

soluble protein content

highest protein content

crude papain