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

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

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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)

_____________________________ ________________________________________ ___________ Ir. Murniyati (Co-Advisor)

_____________________________ ______________________________________ _________ Chairman of the Examination Steering Committee

__________________ __________________ Date

__________________ __________________ Date

_________________ _________________ Date

Judith Salim


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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, How ever, the head itself takes up 29% of the shrimp s hrimp 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 o

o

o

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

o

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


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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


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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


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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


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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 Judith Salim


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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

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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, 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 i n 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 Judith Salim


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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 o

high temperature (50-60 C). 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 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 enzy me for hydrolysis.



To find the optimum condition for papain enzy me.



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%. o

3. The optimum condition for papain enzyme to hydrolyze is at 45 – 60 C Judith Salim


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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.

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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. A dult 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 ty pes 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 Judith Salim


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using sodium hydroxide (NaOH). Enzymatically, hydrolysis can be done by wide range of protease enzyme. For demineralization process, H 2SO4 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 nonessential 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. Judith Salim


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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 Judith Salim


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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 Judith Salim


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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 o

o

to be active at 50-57 C (Grzonka et al., 2007). Heating at 75 C 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 o

and active if stored at 4 C.

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2.4.

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Protein hydrolysis +

Hydrolysis is a reaction when H 2O 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 che mical 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 Judith Salim

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

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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 hu man 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 producing protein

hydrolysate.

2.5.1. Shrimp protein hydrolysate Shrimp is known to contain high content of protein. Whole s hrimp 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, s hrimp, 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 Judith Salim


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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 F or 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 Judith Salim


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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% Judith Salim


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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.

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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 Judith Salim


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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 o

freshness. The shrimps were then stored in freezer at -20 C. 3.2.2. Chemicals Crude papain 



Pure papain



Ethyl ether



N-hexane



H2SO4



HCl



Casein 1%



Buffer pH 7 and pH 8 (KH 2PO4 and K 2HPO4)



TCA (Trichloro acetic)



Folin Ciocalteau



Biuret reagent (Cu 2SO4, Na-K Tartarate, NaOH, Na 2SO3)



Na2CO3



Na2SO4



BSA (Bovine Serum Albumin)

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

3.3.

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Tyrosine

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) o

Crucibles were already prepared in oven overnight at 100 C 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 o

the oven (100 C) 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 = Judith Salim


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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 o

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

white. After that, the temperature is lowered to 40 C 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 (Na 2SO4) 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 o

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

evaporated. Round bottom flask with fat was put in oven at 105 C 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 flas k + 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 K 2SO4. Using 250 ml flask, H 2SO4 used was 10o

15 ml. Destruction was done at 410 C 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 250 μl enzyme of interest in an Eppendorf o

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

TCA was added to stop reaction. The tube was centrifuged 8000 rpm at 4 C 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 50 oC 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 o

followed by inactivation of enzyme at 90 C 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 o

again. Solution was centrifuged at 9000 rpm for 15 minutes at 4 C 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. Judith Salim


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1. Fat content o

Round bottom flask was put into oven at 105 C 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 nhexane 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 o

distilled using distillation set at 60 C. 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 + B j +Ck + ABij + ACik + BC jk + ABCijk + Eijk Yij

: Observation value

μ

: Mean value

Ai

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

B j

: 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 Judith Salim


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: 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 o

o

o

o

used as hydrolysis temperature, which were 45 C, 50 C, 55 C, and 60 C. A. Type of enzyme variation A1 = crude papain A2 = pure papain B. Concentration variation B1 = 10% B2 = 20% B3 = 30% C. Temperature Variation o

C1 = 45 C o

C2 = 50 C o

C3 = 55 C o

C4 = 60 C 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

Water (%) Ash (%) Fat (%)

L.vannamei

79.138 + 1.008 4.416 + 0.547 2.123 + 0.173

a

P. monodon

78.5 5 3.1 13.6

Protein (%) 11.599 + 0.518 from Teerasuntonwat and Raksakulthai (1995)

a

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.

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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.

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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 o

( C). CP stood for crude papain and PP stood for pure papain. The data displayed was average of each treatment plus minus its standard deviation.

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Table 4.2 Data summary of yield and proximate analyses WATER CONTENT (%) C

T 45

10

50 55 60 45

20

50 55 60 45

30

50 55 60

CP 87.28 0.375 88.28 0.375 87.31 0.082 88.71 0.090 84.21 0.229 84.22 0.715 83.94 0.394 83.63 0.856 81.90 0.294 81.47 0.208 80.72 0.386 80.57 0.509

PP ± 92.83 0.073 ± 91.53 0.127 ± 91.32 0.158 ± 90.83 0.093 ± 92.56 0.035 ± 91.32 0.370 ± 91.20 0.049 ± 91.39 0.030 ± 92.46 0.051 ± 90.90 0.035 ± 91.17 0.009 ± 90.79 0.030

ASH CONTENT (%) CP ± 3.71 0.032 ± 3.79 0.057 ± 3.75 0.022 ± 3.60 0.054 ± 6.22 0.639 ± 6.47 0.426 ± 6.74 0.070 ± 6.57 0.204 ± 8.92 0.190 ± 9.25 0.401 ± 9.21 0.214 ± 9.39 0.079

± ± ± ± ± ± ± ± ± ± ± ±

PP 0.46 0.014 0.46 0.003 0.54 0.002 0.54± 0.016 0.46 0.003 0.47 0.011 0.54 0.001 0.55 0.003 0.46 0.008 0.47 0.005 0.55 0.001 0.55 0.005

PROTEIN (%) CP ± 5.07 0.947 ± 5.54 0.166 ± 6.24 0.041 6.28 0.476 ± 5.02 0.062 ± 5.87 0.021 ± 6.71 0.994 ± 6.48 0.884 ± 5.10 0.166 ± 6.59 0.746 ± 5.89 0.083 ± 6.69 1.139

CONTENT

PP ± 6.62 0.021 ± 7.91 0.021 ± 7.95 0.075 ± 8.42 0.393 ± 6.74 0.041 ± 8.10 0.269 ± 7.82 0.162 ± 7.95 0.104 ± 6.94 0.207 ± 7.98 0.352 ± 7.95 0.122 ± 8.57 0.104

YIELD (%)

CP ± 72.30 0.199 ± 73.54 0.259 ± 74.27 0.015 ± 68.68 1.485 ± 72.45 0.177 ± 70.27 4.110 ± 83.11 0.411 ± 70.29 0.058 ± 71.99 0.267 ± 73.21 0.038 ± 84.10 0.397 ± 83.75 0.802

PP ± 77.17 0.001 ± 71.07 0.198 ± 77.58 0.322 ± 67.82 0.476 ± 77.29 0.269 ± 71.00 0.601 ± 78.09 0.215 ± 67.97 0.212 ± 77.36 0.252 ± 71.93 0.021 ± 78.07 0.197 ± 67.53 0.019

± ± ± ± ± ± ± ± ± ± ± ±


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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 o

o

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

o

at 45 C and 55 C. 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 45 oC were o

o

o

difference to those incubated at 50 C, 55 C, and 60 C. 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 s ignificantly 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 Judith Salim


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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 o Ash content was calculated based on the residue of excessive heating at 550 C. 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 s tatistical 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. Crude papain hydrolysate

Pure papain hydrolysate

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).

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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 o

at 50, 55, and 60 C. However, the protein result between the incubation temperature of 50, 55, and 60 oC 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.

o

Result showed that when temperature was 45 C, 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 60 oC. It was consistent to the literature that said that papain is o

o

optimum between temperature 50-60 C. Therefore, it was also predicted that at 45 C, soluble protein will be less than at 50-60 oC. With consideration of denaturation and energy use, it could be concluded that the effective incubation temperature for o

hydrolysis of shrimp head waste by both papain was 50 C.

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 o (%) ( C) Crude Papain

10

20

30

Pure Papain

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

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

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 Judith Salim


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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 o

highest recovered protein seemed to come from 50 C incubation temperature. o

Therefore, it supported the analysis of protein content conclusion that 50 C 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 o

temperature that was chosen was 50 C because based on the statistical analysis; it o

showed that from 50 C 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.

Liquid phase

Solid phase Figure 4.14 Result of centrifugation of hydrolysate from pure papain

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Floating particles

Liquid phase

solid phase 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/cm

3

while water’s density is 1 g/cm

3

(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.

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Figure 4.16 Condition of sensory evaluation There were four parameters that were examined, which w ere 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 o

o

o

o

hydrolyzed at 45 C to 50 C, 55 C, and 60 C. However, there were no significant differences between those three temperatures to the protein content. o

The initial hypothesis that the optimum temperature was between 45-60 C o

should be rejected because the optimum temperature was between 50-60 C.

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.

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LIST OF REFERENCES

“Chemistry Review: Physical Change http://www.inquiryinaction.org/chemistryreview/solids/, 2011.

in accessed

Solids.” July 2,

“Mineral Solubility” http://www.chemistry.wustl.edu/~courses/genchem/Tutorials/Vitamins/minera l.html, accessed June 30, 2011 “Penaeus Genome Database” http://sysbio.iis.sinica.edu.tw/page/, accessed June 20, 2011 “The Case for Zero Waste” http://www.zerowaste.org/case.htm, accessed July 4, 2011 Abun. (2009). Pengolahan Limbah Udang Windu Secara Kimiawi dengan NaOH dan H 2SO4 Terhadap Protein dan Mineral Terlarut. Jatinangor : Universitas Padjadjaran. 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. Anonymous. (2006). A Zero Waste Local Economy Myth, Mystery, or Magic? ICLEI Conference. Cape Town: Institute for Zero Waste in Africa. Anonymous. (2009). Indonesian Fisheries Statistic Index 2009. Jakarta: Ministry of Marine Affairs and Fisheries. Anonymous. (2010). Code of Federal Regulations. FDA. Ariyani, F., Saleh, M., Tazwir, & Hak, N. (2003). Optimasi Proses Produksi Hidrolisat Protein Ikan dari Ikan Mujair (Oreochromis mossambicus). Jurnal Penelitian Perikanan Indonesia, 9(5). Belitz, H.-D., Grosch, W., & Schieberle, P. (2009). Food Chemistry 4th Revised and Extended Edition. Heidelberg: Springer-Verlag Berlin. Benjakul , S., & Morrissey, M. (1997). Protein Hydrolysates from Pacific Whiting Solid Wastes. J. Agric. Food Chem., 45(9), 3423-3430.

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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. Briggs, M., Funge-Smith, S., Subasinghe, R., & Phillips, M. (2004). Introductions and Movement of Penaeus vannamei and Penaeus stylirostris in Asian and the Pacific. Bangkok: FAO RAP. Bueno-Solano, C., Lopez-Cervantes, J., Campas-Baypoli, O., Lauterio-Garcia, R., Adan-Bante, N., & Sanchez-Machado, D. (2009). Chemical and Biological Characteristics of Protein Hydrolysates from Fermented Shrimp By-Products. Food Chemistry, 112(3), 671-675. Bugg, T. (2004). Introduction to Enzyme and Coenzyme Chemistry 2nd Edition. Oxford: Blackwell . Chang. (2000). “Salt Water vs. Fresh Water – Ghyben-Herzberg Lens” http://geography.about.com/library/misc/ucghyben.htm, accessed July 2, 2011 Dong, Y., Sheng, G., Fu, J., & Wen, K. (2005). Chemical Characterization and AntiAnemia Activity of FIsh Protein Hydrolysate from Saurida elongata. Journal of the Science of Food and Agriculture, 85, 2033-2039. FDA, “CFR – Code of Federal Regulations Title 21” http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=1 72.340, accessed May 5, 2011 Gbogouri, G., Linder, M., Fanni, J., & Parmentier, M. (2004). Influence of Hydrolysis Degree on Functional Properties of Sal mon Byproducts Hydrolysate. Journal of Food Science, 69(8). Grzonka, Z., Jankowska, E., Kasprzykowski, F., Kasprzykowska, R., Łankiewicz, L., Wiczk, W., Wieczerzak, E., Ciarkowski, J. Drabik, P., Janowski, R., Kozak, M., Jaskolski, M. and Grubb, A. (2001) Structural studies of cysteine proteases and their inhibitors. Acta. Bioch. Pol. 48 , 1–20. Hastuti, H. (1996). Pemanfaatan Air Rebusan Kepala Udang Sebagai Atraktan Pakan Udang. Bogor: Institut Pertanian Bogor. Hidayat, T. (2005). Pembuatan Hidrolisat Protein dari Ikan Selar Kuning (Caranx leptolepis) dengan Menggunakan Enzim Papain. Bogor: Institut Pertanian Bogor.

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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. Januri. (2004). Pengaruh Waktu Penirisan dan Penyimpanan Udang Headless B eku Terhadap Perubahan Berat dalam Kaitannya Dengan HACCP. Bogor: Institut Pertanian Bogor. Junianto, Mangunwidjadja, D., Suprihatin, Mulyorini, & Wahyuntari, B. (2009). Pengaruh Tingkat Aerasi dan Kecapatan Agitasi terhadap Tingkat Hidrolisis Protein Kulit Udang pada Tahapan Ekstraksi Kitin Secara Biologis. Bionatura, 11(2), 107-116. Khoerunnisa, Suryahadi, H., & Trisyulianti, E. (2002). Pengaruh Penggunaan Papain dalam Meningkatkan Kecernaan Protein Kedelai Secara In Vitro. Bogor: Institut Pertanian Bogor. Kristinsson H.G., Rasco, B.A. (2000). Fish Protein Hydrolysates: Production, Biochemical, and functional Properties. Critical of reviews in Food Science. Vol 40 (1). 43-81. Lehninger, A., Nelson, D., & Cox, M. (2005). Lehninger Principles of Biochemistry. W.H. Freeman. Liceaga-Gesualdo, A., & Li-Chan, E. (1999). Functional Properties of Fish Protein Hydrolysate from Herring (Clupea harengus). Journal of Food Science, 10001004. Limam, Z., Sadok, S., & El Abed, A. (2008). Enzymatic Hydrolysis of Shrimp Head Waste: Functional and Biochemical Properties. Food Biotechnology, 22, 352362. Liu, B., & Chiang, P. (2008). Production of Hydrolysate with Antioxidative Activity and Functional Properties by Enzymatic Hydrolysis of Defatted Sesame (Sesamum indicum L.). International Journal of Applied Science and Engineering, 6 (2), 73-83. Lowry, O., Rosebrough, N., Farr, A., & Randall, R. (1951). Protein Measurement with the Folin Phenol Reagent. Washington University School of Medicine , 265-275. Mahata, M., Dharma, A., Ryanto, H., & Rizal, Y. (2008). Effect of Substituting Shrimp Waste Hydrolysate of Penaeus merguensis for Fish Meal in Broiler Performance. Pakistan Journal of Nutrition, 7 (6), 806-810.

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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. Nielsen, S. (2010). Food Analysis 4th Edition. New York: Springer. Nilsang, S., Lertsiri, S., Suphantharika, M., & Assavanig, A. (2005). Optimization of Enzymatic Hydrolysis of Fish Soluble Concentrate by Commercial Proteases. Journal of Food Engineering, 70(4), 571-578. Ovissipour, M., Benjakul, S., Safari, R., & Motamedzadegan, A. (2010). Fish Protein Hydrolysate Production from Yellowfin Tuna Thunnus albacares H ead Using Alcalase and Protamex. International Aquatic Research, 2, 87-95. Polaina, J., & MacCabe, A. (2007). Industrial Enzymes Structure, Function and Applications. Valencia: Springer. Rahman. (2003). Produksi Pepton dari Limbah Industri Bir dengan Enzim Papain untuk Medium Pertumbuhan Bakteri. Bogor: Institut Pertanian Bogor. Ruttanapornvareesakul, Y., Hara, K., Osatomi, K., & Osako, K. (2005). Concentration Dependent Effect of Shrimp Head Protein Hy drolysate on Freeze-Induced Denaturation of Lizardfish Myofibrillar Protein during Frozen S torage. Food Science Technology Research, 11(3), 261-268. Simpson, B., Nayeri, G., Yaylayan, V., & Ashie, I. (1998). Enzymatic Hydrolysis of Shrimp Meat. Food Chemistry, 61, 131-138. Sulastri, S. (2009). Karakterisik dan Bentuk Olahan Udang Vannamei (L. vannamei). Bogor: Institut Pertanian Bogor. Synowiecki, J., & Al-Khateeb, N. (2000). The Recovery of Protein Hydrolysate during Enzymatic Isolation of Chitin from Shrimp Crangon crangon Processing Discards. Food Chemistry, 68 , 147-152. Teeransuntonwat, P., & Raksakulthai, N. (1995). Production of F lavouring Agent from Shrimp Heads. ASEAN Food Journal, 10(4), 131-138. Tsopmo, A., Cooper, A., & Jodayree, S. (2010). Enzymatic Hydrolysis of Oat Flour Protein Isolates to Enhance Antioxidative Properties. Advance Journal of Food Science and Technology, 2(4), 206-212. Valdez Pena, A., Espinoza-Perez, J., Sandoval-Fabian, G., Balagurusamy, N., Hernandez-Rivera, A., Garza, I., et al. (2010). Screening of Industrial Enzymes for Deproteinization of Shrimp Head for Chitin Recovery. Food Science and Biotechnology, 19(2), 553-557.

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Wickins, J.F., Lee, D.O’C. (2002). Crustacean Farming Ranching and Culture . Oxford: Blackwell science Ltd.

WHO. (2007). Protein and Amino Acid Requirements in Human Nutrition. Geneva: World Health Organization. Wijayanti, A. (2009). Kajian Penyaringan dan Lama Penyimpanan dalam Pembuatan Fish Peptone dari Ikan Selar Kuning (Caranx leptolepis). Bogor: Institut Pertanian Bogor. Windsor, M. (2001). Fish Protein Concentrate. Food and Agriculture Organization. http://www.fao.org/wairdocs/tan/x5917E/x5917e01.htm, accessed April 7, 2011 Yeung, D.L., Laquatra, I. (2003). Heinz Handbook of Nutrition. H.J. Heinz Company Yap, W., Funge-Smith, S., & Ponia, B. (2006). Regional Review on Aquaculture Development 3. Asia and The Pacific-2005. Rome: FAO Fisheries. You, L., Zhao, M., Cui, C., Zhao, H., & Yang, B. (2009). Effect of Degree of Hydrolysis on the Antioxidant Activity of Loach (Misgurnus anguillicaudatus) Protein Hydrolysate. Innovative Food Science and Emerging Technologies, 235-240. Yulistianti, L. (2009). Produksi Bubuk Flavour Enhancer Udang dari Limbah Kepala Udang Windu (Penaeus monodon). Jakarta: UIN.

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APPENDICES Appendix 5 Standard curve of Lowry

Appendix 6 Standard Curve for Enzyme Activity

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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 Omega Squared

D.F.

SS

MS

Among Rows 0.016 Among Columns 0.073 Among Slices 0.428 A x B Inter. 0.060 A x C Inter. 0.177 B x C Inter. 0.099 AxBxC Inter. 0.110 Within Groups Total

1

19.033

19.033

21.468

0.000

2

85.178

42.589

48.037

0.000

3

493.557

164.519

185.562

0.000

2

70.242

35.121

39.613

0.000

3

205.270

68.423

77.175

0.000

6

119.224

19.871

22.412

0.000

6

131.396

21.899

24.700

0.000

24 47

21.278 1145.179

0.887 24.366

Omega squared for combined effects =

F

PROB.> F

0.963

Note: MSErr denominator for all F ratios.

Descriptive Statistics GROUP Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell

1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2

1 1 1 1 2 2 2 2 3 3 3 3 1 1 1 1

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

MEAN 72.297 73.540 74.270 68.683 72.451 70.271 83.108 70.290 71.994 73.212 84.105 83.754 77.165 71.069 77.580 67.816

VARIANCE 0.040 0.067 0.000 2.204 0.031 16.892 0.169 0.003 0.071 0.001 0.158 0.643 0.000 0.039 0.104 0.227

STD.DEV. 0.199 0.259 0.015 1.485 0.177 4.110 0.411 0.058 0.267 0.038 0.398 0.802 0.001 0.198 0.322 0.476

Judith Salim

Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp ( L.vannamei) Head Cell Cell Cell Cell Cell Cell Cell Cell Row Row Col Col Col Slice Slice Slice Slice TOTAL

2 2 2 2 2 2 2 2 1 2 1 2 3 1 2 3 4

2 2 2 2 3 3 3 3

1 2 3 4 1 2 3 4

2 2 2 2 2 2 2 2 24 24 16 16 16 12 12 12 12 48

77.288 70.999 78.094 67.967 77.357 71.928 78.066 67.534 74.831 73.572 72.803 73.809 75.994 74.759 71.837 79.204 71.007 74.202

0.073 0.361 0.046 0.045 0.063 0.000 0.039 0.000 30.281 18.682 12.131 25.936 32.600 6.928 3.131 12.550 36.628 24.366

Page 74 of 100 0.269 0.601 0.215 0.212 0.252 0.021 0.197 0.019 5.503 4.322 3.483 5.093 5.710 2.632 1.770 3.543 6.052 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 Omega Squared

D.F.

SS

MS


1

616.940

616.940

5954.305

0.000

2

99.313

49.656

479.252

0.000

3

6.596

2.199

21.219

0.000

2

84.015

42.008

405.431

0.000

3

4.560

1.520

14.669

0.000

6

1.680

0.280

2.702

0.038

6

3.366

0.561

5.415

0.001

24 47

2.487 818.957

0.104 17.425


F

PROB.> F

0.994


Descriptive Statistics GROUP Cell

1

1

1

N 2

MEAN 87.283

VARIANCE 0.140

STD.DEV. 0.375

Judith Salim

Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp ( L.vannamei) Head Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Row Row Col Col Col Slice Slice Slice Slice TOTAL

1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 3 1 2 3 4

1 1 1 2 2 2 2 3 3 3 3 1 1 1 1 2 2 2 2 3 3 3 3

2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 24 24 16 16 16 12 12 12 12 48

88.283 87.311 88.712 84.212 84.219 83.941 83.634 81.902 81.465 80.719 80.569 92.829 91.525 91.324 90.832 92.557 91.315 91.203 91.390 92.457 90.900 91.171 90.790 84.354 91.524 89.763 87.809 86.246 88.540 87.951 87.611 87.654 87.939

0.140 0.007 0.008 0.052 0.511 0.156 0.733 0.087 0.043 0.149 0.259 0.005 0.016 0.025 0.009 0.001 0.137 0.002 0.001 0.003 0.001 0.000 0.001 8.307 0.476 4.229 15.760 27.987 20.799 16.239 18.290 18.524 17.425

Page 78 of 100 0.375 0.082 0.090 0.229 0.715 0.394 0.856 0.294 0.208 0.386 0.509 0.073 0.127 0.158 0.093 0.035 0.370 0.049 0.030 0.051 0.035 0.009 0.030 2.882 0.690 2.056 3.970 5.290 4.561 4.030 4.277 4.304 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

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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 Omega Squared Among 0.778 Among 0.110 Among 0.000 A x B 0.109 A x C 0.000

D.F.

SS

MS

F

PROB.> F

Rows

1

426.976

426.976

11459.905

0.000

Columns

2

60.163

30.081

807.372

0.000

Slices

3

0.240

0.080

2.143

0.121

Inter.

2

59.875

29.937

803.512

0.000

Inter.

3

0.083

0.028

0.741

0.538

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Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp ( L.vannamei) Head B x C Inter. 0.000 AxBxC Inter. 0.000 Within Groups Total

Page 82 of 100

6

0.133

0.022

0.593

0.733

6

0.137

0.023

0.614

0.717

24 47

0.894 548.500

0.037 11.670


0.997


Descriptive Statistics GROUP Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Row Row Col Col Col Slice Slice Slice Slice TOTAL

1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 3 1 2 3 4

1 1 1 1 2 2 2 2 3 3 3 3 1 1 1 1 2 2 2 2 3 3 3 3

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 24 24 16 16 16 12 12 12 12 48

MEAN VARIANCE STD.DEV. 3.713 0.001 0.032 3.795 0.003 0.057 3.749 0.000 0.022 3.602 0.003 0.054 6.223 0.408 0.639 6.466 0.182 0.426 6.738 0.005 0.070 6.570 0.042 0.204 8.921 0.036 0.190 9.254 0.161 0.401 9.209 0.046 0.214 9.386 0.006 0.079 0.457 0.000 0.014 0.459 0.000 0.003 0.544 0.000 0.002 0.545 0.000 0.016 0.460 0.000 0.003 0.465 0.000 0.011 0.541 0.000 0.001 0.545 0.000 0.003 0.459 0.000 0.008 0.473 0.000 0.005 0.551 0.000 0.001 0.547 0.000 0.005 6.469 5.282 2.298 0.504 0.002 0.043 2.108 2.758 1.661 3.501 9.651 3.107 4.850 20.146 4.488 3.372 11.768 3.430 3.485 12.687 3.562 3.555 12.607 3.551 3.533 12.780 3.575 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


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--------------------------------------------------------------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


Page 84 of 100

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 Omega Squared

D.F.

SS

MS


1

38.401

38.401

174.560

0.000

2

0.354

0.177

0.806

0.458

3

15.075

5.025

22.842

0.000

2

0.199

0.100

0.453

0.641

3

0.236

0.079

0.358

0.784

6

0.936

0.156

0.709

0.645

6

1.095

0.182

0.830

0.559

24 47

5.280 61.576

0.220 1.310


F

PROB.> F

0.829


Descriptive Statistics GROUP Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell

1 1 1 1 1 1 1 1 1 1 1 1 2 2

1 1 1 1 2 2 2 2 3 3 3 3 1 1

1 2 3 4 1 2 3 4 1 2 3 4 1 2

N 2 2 2 2 2 2 2 2 2 2 2 2 2 2

MEAN 5.068 5.535 6.238 6.282 5.023 5.872 6.707 6.483 5.096 6.590 5.887 6.692 6.623 7.912

VARIANCE 0.897 0.027 0.002 0.227 0.004 0.000 0.988 0.782 0.027 0.556 0.007 1.297 0.000 0.000

STD.DEV. 0.947 0.166 0.041 0.476 0.062 0.021 0.994 0.884 0.166 0.746 0.083 1.139 0.021 0.021

Judith Salim

Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp ( L.vannamei) Head Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Row Row Col Col Col Slice Slice Slice Slice TOTAL

2 2 2 2 2 2 2 2 2 2 1 2 1 2 3 1 2 3 4

1 1 2 2 2 2 3 3 3 3

3 4 1 2 3 4 1 2 3 4

2 2 2 2 2 2 2 2 2 2 24 24 16 16 16 12 12 12 12 48

7.947 8.420 6.736 8.098 7.816 7.951 6.941 7.981 7.946 8.566 5.956 7.745 6.753 6.836 6.962 5.915 6.998 7.090 7.399 6.850

0.006 0.155 0.002 0.072 0.026 0.011 0.043 0.124 0.015 0.011 0.606 0.402 1.471 1.215 1.396 0.891 1.268 0.880 1.189 1.310

Page 85 of 100 0.075 0.393 0.041 0.269 0.162 0.104 0.207 0.352 0.122 0.104 0.778 0.634 1.213 1.102 1.182 0.944 1.126 0.938 1.090 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 Omega Squared

D.F.

SS

MS

F

PROB.> F

Judith Salim



Page 86 of 100

1

4150.060

4150.060

149.061

0.000

2

240.376

120.188

4.317

0.025

3

4093.520

1364.507

49.010

0.000

2

247.672

123.836

4.448

0.023

3

4772.234

1590.745

57.136

0.000

6

357.881

59.647

2.142

0.085

6

206.034

34.339

1.233

0.324

24 47

668.193 14735.970

27.841 313.531


0.909


Descriptive Statistics GROUP Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Row Row Col Col Col Slice Slice Slice Slice TOTAL

1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 3 1 2 3 4

1 1 1 1 2 2 2 2 3 3 3 3 1 1 1 1 2 2 2 2 3 3 3 3

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 24 24 16 16 16 12 12 12 12 48

MEAN VARIANCE STD.DEV. 52.103 1.077 1.038 114.302 11.704 3.421 75.231 0.249 0.499 73.314 30.899 5.559 63.492 0.617 0.785 104.787 0.137 0.370 77.666 132.510 11.511 73.685 101.066 10.053 55.831 3.295 1.815 108.781 151.467 12.307 66.371 0.872 0.934 75.506 165.153 12.851 87.217 0.074 0.273 89.020 0.300 0.547 90.706 7.324 2.706 97.043 0.065 0.254 96.749 10.346 3.216 96.502 18.122 4.257 102.899 0.937 0.968 106.882 4.915 2.217 104.492 2.584 1.608 99.318 21.541 4.641 93.365 1.478 1.216 100.036 1.462 1.209 78.422 421.106 20.521 97.019 39.151 6.257 84.867 319.633 17.878 90.333 268.303 16.380 87.962 378.437 19.453 76.647 456.171 21.358 102.119 93.189 9.653 84.373 179.722 13.406 87.744 238.413 15.441 87.721 313.531 17.707

Judith Salim


Page 87 of 100

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


Page 88 of 100

--------------------------------------------------------------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


Page 89 of 100

--------------------------------------------------------------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 1 2 3 4 5 6 7 8 9 10 11 12 13

PK3 PM1 PK1 PM2 PK2 PM3

Count

6 6 6 6 6 6 6 6 6 6 6 6 6 13 13 13 13 13 13

ANOVA Source of Variation Rows Columns Error

SS 22.33333 1.448718 23.05128

Total

46.83333

Sum Average Variance 34 5.666667 0.266667 24 4 0 32 5.333333 0.266667 27 4.5 0.3 30 5 0 24 4 0 33 5.5 0.3 27 4.5 1.5 27 4.5 0.3 30 5 0.8 27 4.5 0.3 29 4.833333 0.566667 33 5.5 0.3

63 66 62 63 63 60

4.846154 5.076923 4.769231 4.846154 4.846154 4.615385

0.641026 0.910256 0.692308 0.641026 0.474359 0.423077

df MS F P-value 12 1.861111 4.844271 1.66E-05 5 0.289744 0.754171 0.586356 60 0.384188

F crit 1.917396 2.36827

77

Judith Salim


Page 90 of 100

Appendix 9 Two-way ANOVA of Color in hedonic test Anova: Two-Factor Without Replication SUMMARY 1 2 3 4 5 6 7 8 9 10 11 12 13

Count Sum 6 36 6 27 6 32 6 27 6 22 6 24 6 33 6 27 6 24 6 29 6 27 6 23 6 31

PK3 PM1 PK1 PM2 PK2 PM3

Average

6 4.5 5.333333 4.5 3.666667 4 5.5 4.5 4 4.833333 4.5 3.833333 5.166667

Variance 0.8 0.7 0.266667 0.3 0.666667 0 0.3 1.5 0 0.566667 0.3 1.366667 0.166667

13 13 13 13 13 13

62 64 59 60 61 56

4.769231 4.923077 4.538462 4.615385 4.692308 4.307692

0.692308 1.74359 0.602564 1.423077 0.397436 0.730769


SS 35.2820 2.87179 31.7948

df 12 5 60

MS 2.940171 0.574359 0.529915

F 5.54838 1.083871

Total

69.9487

77

P-value F crit 2.85E-06 1.917396 0.378548 2.36827

Appendix 10 Two-way ANOVA of smell in hedonic test

Anova: Two-Factor Without Replication SUMMARY

Count

1 2 3

Sum Average Variance 6 34 5.666667 0.266667 6 26 4.333333 1.066667 6 24 4 0.8

Judith Salim


4 5 6 7 8 9 10 11 12 13 PK3 PM1 PK1 PM2 PK2 PM3

Page 91 of 100

6 6 6 6 6 6 6 6 6 6

27 30 33 36 27 24 26 28 28 32

4.5 5 5.5 6 4.5 4 4.333333 4.666667 4.666667 5.333333

0.3 0 0.3 0 0.3 1.6 1.466667 0.666667 1.066667 0.266667

13 13 13 13 13 13

68 67 64 61 62 53

5.230769 5.153846 4.923077 4.692308 4.769231 4.076923

0.525641 0.641026 0.74359 0.897436 0.858974 1.24359


SS 29.61538 11.19231 29.30769

df MS F P-value F crit 12 2.467949 5.052493 9.79E-06 1.917396 5 2.238462 4.582677 0.001319 2.36827 60 0.488462

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

Mean

5.230769

4.923077

Variance

0.525641

0.74359

13

13

Observations Pearson Correlation Hypothesized Mean Difference df

0.164053 0 12

PM1

PK1

Mean

5.153846

4.923077

Variance

0.641026

0.74359

13

13


0.139272 0

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


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‐Test: Paired Two Sample for Means PK3

Mean

5.153846

5.230769

Variance

0.641026

0.525641

13

13

Pearson Correlation Hypothesized Mean Difference df t Stat

2.178813


PM1

Observations

Page 92 of 100

0.220863 0 12

PM2

PK3

Mean

4.692308

5.230769

Variance

0.897436

0.525641

13

13


‐0.2907

0.35466 0

12

t Stat

‐2.00684

P(T<=t) one‐tail

0.388122

P(T<=t) one‐tail

0.033918


1.782288


1.782288

P(T<=t) two‐tail

0.776243

P(T<=t) two‐tail

0.067836


2.178813


2.178813


Mean Variance Observations Pearson Correlation Hypothesized Mean Difference df


PM3

PM1

4.076923

5.153846

Mean

1.24359

0.641026

Variance

13

13

0.545648 0 12


PM3

PM2

4.076923

4.692308

1.24359

0.897436

13

13

0.655328 0

12

t Stat

‐4.06981

t Stat

‐2.55117

P(T<=t) one‐tail

0.000777

P(T<=t) one‐tail

0.012705


1.782288


1.782288

P(T<=t) two‐tail

0.001554

P(T<=t) two‐tail

0.025411


2.178813


2.178813

t‐Test: Paired Two Sample for Means Judith Salim


Page 93 of 100

t‐Test: Paired Two Sample for Means PM2

PK1

Mean

4.692308

4.923077

Variance

0.897436

0.74359

13

13

Observations Pearson Correlation Hypothesized Mean Difference

0.172635

PK1

Mean

4.769231

4.923077

Variance

0.858974

0.74359

13

13


0

df

PK2

12

df

0.705832 0

12

t Stat

‐0.71375

t Stat

‐0.80539

P(T<=t) one‐tail

0.244517

P(T<=t) one‐tail

0.218132


1.782288


1.782288

P(T<=t) two‐tail

0.489034

P(T<=t) two‐tail

0.436265


2.178813


2.178813



PK2

PK3

Mean

4.769231

5.230769

Variance

0.858974

0.525641

13

13


0.085858

t Stat

PM1

Mean

4.692308

5.153846

Variance

0.897436

0.641026

13

13


0

df

PM2

12

df

‐1.4771

0.616963 0

12

t Stat

‐2.14377

P(T<=t) one‐tail

0.082703

P(T<=t) one‐tail

0.026616


1.782288


1.782288

P(T<=t) two‐tail

0.165407

P(T<=t) two‐tail

0.053232


2.178813


2.178813



PK2

PM2

Mean

4.769231

4.692308

Variance

0.858974

0.897436

13

13


0.386954 0 12

PK2

PM3

Mean

4.769231

4.076923

Variance

0.858974

1.24359

13

13


0.502379 0

12

t Stat

0.267261

t Stat

2.419798

P(T<=t) one‐tail

0.396903

P(T<=t) one‐tail

0.016165


1.782288


1.782288

Judith Salim


Page 94 of 100

P(T<=t) two‐tail

0.793806

P(T<=t) two‐tail

0.032329


2.178813


2.178813


Mean Variance

PM3

PK3

4.076923

5.230769

Mean

1.24359

0.525641

Variance

13

13


0.491568

P(T<=t) one‐tail t Critical one‐tail P(T<=t) two‐tail t Critical two‐tail


0

df t Stat


12

df

‐4.21464 0.0006 1.782288 0.0012 2.178813

PM3

PK1

4.076923

4.923077

1.24359

0.74359

13

13

0.52662 0

12

t Stat

‐3.09073

P(T<=t) one‐tail

0.004675


1.782288

P(T<=t) two‐tail

0.00935


2.178813

t‐Test: Paired Two Sample for Means PK2

PM1

Mean

4.769231

5.153846

Variance

0.858974

0.641026

13

13


0.276438 0 12

t Stat

‐1.32842

P(T<=t) one‐tail

0.104375


1.782288

P(T<=t) two‐tail

0.208749


2.178813

Judith Salim


Page 95 of 100

Appendix 12 Two-way ANOVA of taste in hedonic test

Anova: Two-Factor Without Replication SUMMARY

1 2 3 4 5 6 7 8 9 10 11 12 13 PK3 PM1 PK1 PM2 PK2 PM3

Count

6 6 6 6 6 6 6 6 6 6 6 6 6

Sum 23 23 22 24 15 19 27 15 25 24 20 21 22

Average 3.833333 3.833333 3.666667 4 2.5 3.166667 4.5 2.5 4.166667 4 3.333333 3.5 3.666667

Variance 1.766667 0.566667 0.666667 1.2 1.1 4.166667 7.5 2.7 0.966667 3.6 1.866667 2.7 1.066667

13 13 13 13 13 13

67 35 56 32 60 30

5.153846 2.692308 4.307692 2.461538 4.615385 2.307692

0.641026 0.897436 1.397436 0.935897 1.589744 0.730769


SS 25.53846 100.5641 48.76923

df MS F P-value F crit 12 2.128205 2.618297 0.007086 1.917396 5 20.11282 24.74448 1.95E-13 2.36827 60 0.812821

Total

174.8718

77

Judith Salim


Page 96 of 100

Appendix 13 t-test between samples for taste t‐Test: Paired Two Sample for Means


PK3

PM3

Mean

5.153846

2.307692

Variance

0.641026

0.730769

13

13


0.046829

PM3

Mean

2.692308

2.307692

Variance

0.897436

0.730769

13

13


0

df

PM1

12

df

0.435358 0

12

t Stat

8.973818

t Stat

1.443376

P(T<=t) one‐tail

5.69E‐07

P(T<=t) one‐tail

0.087254


1.782288


1.782288

P(T<=t) two‐tail

1.14E‐06

P(T<=t) two‐tail

0.174509


2.178813


2.178813



PK2

PM3

Mean

4.615385

2.307692

Variance

1.589744

0.730769

13

13

Observations Pearson Correlation Hypothesized Mean Difference df t Stat

0.041631

PK3

PK2

Mean

5.153846

4.615385

Variance

0.641026

1.589744

13

13


0 12

df

5.57086

0.723901 0

12

t Stat

2.213594

P(T<=t) one‐tail

6.08E‐05

P(T<=t) one‐tail

0.023488


1.782288


1.782288

P(T<=t) two‐tail

0.000122

P(T<=t) two‐tail

0.046976


2.178813


2.178813



PM2

PK2

Mean

2.461538

4.615385

Variance

0.935897

1.589744

13

13


‐0.0473 0 12

‐4.77859

PK3

PM2

Mean

5.153846

2.461538

Variance

0.641026

0.935897

13

13


0.223454 0

12

t Stat

8.75

Judith Salim


Page 97 of 100

P(T<=t) one‐tail

0.000225

P(T<=t) one‐tail

7.43E‐07


1.782288


1.782288

P(T<=t) two‐tail

0.00045

P(T<=t) two‐tail

1.49E‐06


2.178813


2.178813



PK3

PK1

Mean

5.153846

4.307692

Variance

0.641026

1.397436

13

13


0.562147

PK1

Mean

2.692308

4.307692

Variance

0.897436

1.397436

13

13


0

df

PM1

12

df

‐0.20607 0

12

t Stat

3.090733

t Stat

‐3.50813

P(T<=t) one‐tail

0.004675

P(T<=t) one‐tail

0.002158


1.782288


1.782288

P(T<=t) two‐tail

0.00935

P(T<=t) two‐tail

0.004317


2.178813


2.178813



PK1

PM3

Mean

4.307692

2.307692

Variance

1.397436

0.730769

13

13


‐0.01903

PM2

PM3

Mean

2.461538

2.307692

Variance

0.935897

0.730769

13

13


0 12

df

0.720865 0

12

t Stat

4.898979

t Stat

0.805387

P(T<=t) one‐tail

0.000183

P(T<=t) one‐tail

0.218132


1.782288


1.782288

P(T<=t) two‐tail

0.000367

P(T<=t) two‐tail

0.436265


2.178813


2.178813



PM1

PK2

Mean

2.692308

4.615385

Variance

0.897436

1.589744

13

13

Observations Pearson Correlation

‐0.3864

PK1

PK2

Mean

4.307692

4.615385

Variance

1.397436

1.589744

13

13

Observations Pearson Correlation

0.756935 Judith Salim


Hypothesized Mean Difference df t Stat

Page 98 of 100

Hypothesized Mean Difference

0 12

df

‐3.7547

0

12

t Stat

‐1.29777

P(T<=t) one‐tail

0.001374

P(T<=t) one‐tail

0.109379


1.782288


1.782288

P(T<=t) two‐tail

0.002747

P(T<=t) two‐tail

0.218758


2.178813


2.178813



PM1

PM2

Mean

2.692308

2.461538

Variance

0.897436

0.935897

13

13


0.622515

PM2

Mean

4.307692

2.461538

Variance

1.397436

0.935897

13

13


0 12

t Stat

PK1

df

1

‐0.13453 0

12

t Stat

4.095937

P(T<=t) one‐tail

0.168525

P(T<=t) one‐tail

0.000742


1.782288


1.782288

P(T<=t) two‐tail

0.337049

P(T<=t) two‐tail

0.001483


2.178813


2.178813

t‐Test: Paired Two Sample for Means PM1

PK3

Mean

2.692308

5.153846

Variance

0.897436

0.641026

13

13


0.067612 0 12

P(T<=t) one‐tail

‐7.40656 4.1E‐06


1.782288

P(T<=t) two‐tail t Critical two‐tail

8.2E‐06 2.178813

Judith Salim


Page 99 of 100

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)

Courses

2004 – 2007

SMA Labschool Kebayoran, Jakarta

2001 – 2004

SMP Labschool Kebayoran, Jakarta

: 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.

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

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