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PRODUCTION OF PROTEIN CONCENTRATE BY ENZYMATIC HYDROLYSIS OF SHRIMP (L. vannamei) HEAD
By
JUDITH SALIM
A Bachelor’s Thesis
Submitted to the Faculty of
LIFE SCIENCE
Department of FOOD TECHNOLOGY
in partial fulfillment of the
requirements for
BACHELOR’S DEGREE
IN
FOOD TECHNOLOGY
Swiss German University
EduTown BSDCity Tangerang 15339
INDONESIA
July 2011
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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) Date
________________________________________ __________________
Ir. Murniyati (Co-Advisor) Date
______________________________________ _________________
Chairman of the Examination Steering Committee Date
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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, the head itself takes up 29% of the shrimp and contains many
nutrients. One of the most abundant nutrients is protein. To improve the digestibility
and palatability of shrimp head, enzymatic hydrolysis was done. There were two types
of enzyme that were used, which were pure and crude papain. The treatments were
enzyme concentration (10%, 20%, and 30%) and temperature (45oC, 50oC, 55oC,
60oC). The highest protein content was produced by pure papain at 50oC incubation
temperature. The concentration did not seem to have a significant effect on soluble
protein content. However, the sensory level of acceptance of products hydrolyzed
with pure enzyme was lower than those hydrolyzed by crude papain. On the other
hand, hydrolysis by crude papain produced products with lower water content and
higher ash content of minerals compared to hydrolysis products of pure papain.
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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.
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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.
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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
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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
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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, which was in 2001. There
is a significant increase in the production from 5000 tons in 2002 (Briggs et
al., 2004) to approximately 198 kilo tons in 2009 (Ministry of Marine Affairs
and Fisheries, 2009).
In L. vannamei processing (IQF shrimp or block frozen shrimp), the shell and
head are usually thrown away, producing a terrible odor. The head itself took
about 36-49% of total weight of the shrimp (Purwaningsih, 2000 in Sulastri,
2009). By turning the shrimp head to protein concentrate, it may increase the
value of the waste and producing an alternative nutraceutical for human. The
protein concentrate that exists in Indonesia comes from various sources, such
as milk, soy bean, sesame, and also fish. However, these sources are able to be
sold in their unprocessed condition, whereas the waste cannot.
To obtain such protein concentrate from the shrimp head, enzymatic
hydrolysis of the head will be needed. Shrimp head contains chitin, which bind
to the protein and makes it not digestible by human digestion system. Both of
them can be separated by hydrolyzing the shrimp head by either enzymes or
chemicals. This study will use enzyme instead of chemical because enzyme is
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specific and not toxic, consume less energy, and produce less byproducts. It is
more environmental friendly than chemicals because some chemicals can
produce toxic waste which can harm the environment.
1.2. Research Problem
Conducting hydrolysis using enzyme must be done correctly. The first
consideration is the type of enzyme. Enzyme is divided to 6 classes but the
one that is used to hydrolyze substance is hydrolase. Hydrolase can hydrolyze
large molecules to smaller ones. In this case, protein is the molecules that will
be degraded to small peptides and amino acids. Therefore, the type of
hydrolase enzyme that will be used is protease.
The consideration of enzyme selection depends on price, availability,
concentration needed, and condition of hydrolysis. Past researches about
shrimp hydrolysate indicates that serine protease, such as trypsin (Limam et
al., 2008), chymotrypsin (Simpson et al., 1997), and Alcalase © (Mizani et al.,
2007) were used to hydrolyze the shrimp head. However, these enzymes are
not available widely in Indonesia and it might cause a problem during the
research. Instead papain enzyme, which is a cysteine protease, is used as
hydrolase enzyme for protein hydrolysate. Papain enzyme has been used to
hydrolyze both shrimp (Valdez-Pena et al., 2010) and fish (Hosomi et al.,
2010). However, the hydrolysis of shrimp did by Valdez-Pena only a
comparison of hydrolytic activity between crude enzymes and not find a way
to optimize amount of the protein hydrolysate produced.
There are two types of papain enzyme that will be used in this experiment.
The first one is crude papain, which is sold widely in supermarket as meat
tenderizer. This enzyme also contains other additives like salt and sugar.
Crude papain doesn’t undergo purification process. Hence, it has lower
enzyme activity than pure papain. The second one is pure papain. This is a
product of purification. Pure papain does not contain any additives. It also has
higher enzyme activity. However, the crude papain is cheaper and it exists
widely. Therefore, the ability of pure papain and crude papain to hydrolyze
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should be monitored whether both enzymes, which concentration has been
equalized, produce insignificantly different protein concentrate.
Papain works optimally at neutral pH but it catalyzes reaction at relatively
high temperature (50-60oC). If the temperature is too high, it will result in
denaturation of protein and causing the protein to lose its beneficial contents.
Temperature should be examined carefully in order to avoid denaturation of
protein. Concentration of enzyme added will also affect the catalytic activity.
The higher the enzyme activity, the higher the rate of reaction will be.
However, it also has a maximum rate of reaction, so another addition after that
will not affect the rate anymore. Therefore, it is important to know how much
papain should be added to the concentration.
Another common problem in seafood hydrolysates is bitterness. This
bitterness is caused by some amino acids and small peptides (Belitz et al.,
2009). The protein that is produced for human consumption should not have
bitter taste because it is somehow unlikable. Descriptive sensory evaluation
must be conducted in order to examine the taste and the odor of the protein
concentrate.
1.3. Research Objectives
The objectives of this research are
To determine which papain enzyme produce protein concentrate with
better protein content.
To find the effective concentration of papain enzyme for hydrolysis.
To find the optimum condition for papain enzyme.
To determine which protein concentrate has more acceptable sensory
score.
1.4. Hypothesis
1. Crude papain can hydrolyze shrimp head as good as pure papain, in
terms of protein content.
2. The effective concentration of papain enzyme for hydrolysis is 10%.
3. The optimum condition for papain enzyme to hydrolyze is at 45 – 60oC
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4. Protein concentrate from hydrolysis of crude papain will have a better
sensory score than the pure papain.
1.5. Research Scope
This study was targeted to hydrolyze the waste from shrimp production
(shrimp head) to produce nutritive protein concentrate. Protein concentrate
was aimed to be consumable product for human. Since the separation of
quality in shrimp protein concentrate has not existed, the quality reference
would be from fish protein concentrate that was regulated from FAO.
Hydrolysis using enzyme was done so that the product was environmentally
friendly and safe for human consumption. Enzyme that was used for this
hydrolysis was papain because it was available widely in Indonesia, has a
good hydrolysis capability, and was available at low price. The optimum
condition and concentration for this hydrolysis was experimented, so that
hydrolysate contained high yield of protein.
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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. Adult shrimp is
a part of marine animal, but during their early stages of development
(juvenile), they move to the estuaries and after reaching adult phase they move
back to the ocean (Wickins and Lee, 2002). The taxonomy of L. vannamei can
be seen below (Boone, 1931 in Sulastri, 1999).
Kingdom : Animalia
Subkingdom : Metozoa
Phylum : Arthropoda
Subphylum : Crustacea
Class : Malacostraca
Subclass : Eumalacosteraca
Superordo : Euracida
Ordo : Decapoda
Subordo : Denderobrachiata
Family : Penaeidae
Genus : Litopeneus
Species : Litopenaus vannamei
Figure 2.2 Litopenaeus vannamei Source: http://sysbio.iis.sinica.edu.tw/page/ (2011)
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L. vannamei’s body is made of two branches (Biromous), which are exopodite
and endopodite. Vannamei’s anatomy can be divided to two parts:
Head (thorax)
Its head contains antenula, antenna, mandibular, and two pair maxillae. It also
has three pairs of maxilliped (organ which is used to take up food), 5 pairs of
pereipodes (this is used to walk). Pereiopodes are segmented. It is divided to
type, with clamp or without. The fourth and fifth pereipodes don’t have
clamps but the first until third pereipodes have clamps.
Stomach (abdomen)
The abdomen is made of six segments. At this part, there are five pairs of
pleopods (this organ is used to swim) and a pair of uropodes (this is similar to
tail) which is fan-like shaped.
Figure 2.3 Anatomy of L. vannamei Source: Bondad-Reantoso et al. (2001)
Due to the several advantages, Indonesia started to culture this shrimp in 2000.
The originally cultured shrimp species in Indonesia is Penaeus monodon and
Penaeus merguiensis. However, there was an outbreak of WSSV and YHV in
Penaeus monodon (Bondad-Reantoso et al., 2001). This was the main reason
for importing the broodstock for L. vannamei. The shrimp that is produced in
Indonesia is imported from Hawaii (Yap et al. 2005). The production of the
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shrimp itself is still increasing. For example, the shrimp production in 2002
was only 5000 tons but in 2003, it increased to 20000 tons (Briggs et al.,
2004). Within the big production of this shrimp, there will be great amount of
waste too. The waste of shrimp (shell, head, or tail) from the industrial shrimp
factory is usually thrown away after production. They bury the waste with soil
to remove its smell and through time the waste will become compost.
2.1.2. Utilization of shrimp waste In industrial frozen shrimp processing there are many types of shape, like head
on or headless, peeled shrimp is also popular. Peeled shrimp is usually
headless and contains no shell. The yield that lies on the meat itself is 57%,
the yield from head is 33%, and 10% from the shell (Sulastri, 2009). It can be
concluded that the yield from waste is almost half of the total mass.
There are some alternatives to process this usually discarded waste. It can be
used as protein feed for fish meal (Nwanna et al., 2004) or animal feed
because of the high content of amino acids. Aside from utilization as feed,
shrimp waste can also be used as flavoring agent (Teerasuntonwat and
Raksakulthai, 1995). In Indonesia, processed shrimp waste known as terasi is
very popular (Abun, 2009). It is actually fermented shrimp waste or small
shrimp, and it is usually used quite widely as flavoring in Indonesia despite the
fact that it produces a terrible smell because of the ammonia produced.
Surprisingly, not only Indonesia uses this fermented shrimp as flavoring, other
neighboring countries such as Kamboja, The Philippines, and Japan.
Other use of shrimp waste is as source of pigment. Shrimp waste contains
carotenoids and it can be extracted by using organic solvents and solvent
mixtures (Sachindra et al., 2006). Aside from pigment, the commonly
utilization of shrimp waste these days is to produce chitin. The shell and
carapace of shrimp contain high content of chitin and it is therefore extracted.
There are two main processes in producing chitin; the first one is
deproteinization and followed by demineralization. Deproteinization can be
done enzymatically and chemically. Chemically, the shell can be hydrolyzed
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using sodium hydroxide (NaOH). Enzymatically, hydrolysis can be done by
wide range of protease enzyme. For demineralization process, H2SO4 can be
used so that the mineral will be extracted from the shell (Abun, 2000). The
extraction of chitin can also be optimized by combining it with production of
protein hydrolysate (Synowiecki et al., 2000). The firstly demineralized shell
was hydrolysed using Alcalase and NaOH. It was inactivated using HCl and
precipitated using centrifugation. From this point forward, the processing of
chitin and protein hydrolysate was separated. The chitin was stored in the shell
and it was dried. Therefore, crude chitin was produced. The protein was
solubilized from the hydrolysis, so the protein will exist in the solution. The
solution will be lyophilized and the product will be in the form of protein
powder.
2.2. Protein
Protein is one of macromolecules and it is also the most abundant. It is also a
very important constituent of our body and a great source of nutrient and
energy. Protein is constructed from its monomer which is amino acid. These
amino acids are linked covalently using peptide bond.
The functions of protein are for growth, maintenance, and repair of cells by
their action as:
enzymes that catalyze metabolic reactions
structural proteins that maintain the shape of cell
hormones that regulates cell activities
antibodies that provide defense mechanism
contractile proteins, transport proteins, toxins, and components of
intracellular structures
energy source (4kcal)
(Yeung and Laquatra, 2003)
Amino acid is what makes up the protein. Amino acid consists of R groups,
carboxyl, and amino group that bond to carbon atom. R groups are the
determining molecules that differentiate each amino acid. In general, R groups
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are divided to 5 groups. The hydrophobic ones are the nonpolar aliphatic
(glycine, alanine, proline, valine, leucine, isoleucine, methionine) and
aromatic groups (phenylalanine, tyrosine, and tryptophan). Hydrophobic
means that it doesn’t like water, the opposite is hydrophilic which means
water loving. The hydrophilic R groups are the polar uncharged (serine,
threonine, cysteine, asparagine, and glutamine), positively charged (lysine,
histidine, and arginine) and negatively charged (aspartic acid and glutamic
acid). (Lehninger et al., 2005)
The 20 common amino acids in our body are divided to 2 main groups based
on its ability to be synthesized in the body: essential amino acids and non-
essential amino acids. Essential amino acids are those amino acids that human
requires, whereas non-essential amino acid can be produced in the body. There
are 9 essential amino acids: histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine. Alanine, arginine,
asparagine, aspartic acid, cysteine, glycine, cysteine, glutamic acid, proline,
tyrosine, serine, glutamine are the non-essential amino acids that can be
synthesized in human body (Yeung and Laquatra, 2003).
Protein is varied from the simple form to the complex form. The simplest form
is primary structure which is sequence of amino acids in peptide chain.
Secondary structure is determined by hydrogen bond between amino acids
within the polypeptide chain (alpha-helix of beta-conformation).The tertiary
structure is more complex because it is the 3D conformation of the protein.
Based on the shape, tertiary structure is divided to fibrous and globular
protein. The most complex form of protein are quaternary structure which is a
joined of two or more polypeptide subunits.
In order to be digested in body, large protein molecules must be degraded by
proteolytic enzyme in gastrointestinal tract. The product will be peptides
(small chain of protein) and amino acids that are easier to be absorbed. The
enzymes of gastrointestinal tract are pepsin and trypsin. The amino acid
requirements for human can be seen below.
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Table 2.1 Indispensable (essential) amino acid requirement
Source: WHO (2007)
2.3. Enzyme
Enzyme is a component that can catalyze a reaction. Most of enzymes are
protein in their tertiary structure. Enzyme can increase the rate of reaction by
lowering its activation energy (Ea). Some enzymes do not need any chemical
groups for activity other than the amino acid residues. However, there are also
some enzymes that need additional components. These components are called
cofactor. It can either be inorganic ions or complex, which is usually called
coenzyme (Bugg, 2004).
To catalyze a reaction, enzyme needs substrate. Substrate is molecule that
bound to the active site of enzyme. When substrate is bound to enzyme,
substrate will alter its structure and becoming the product of the reaction.
Afterwards, the enzyme will release itself from the product and it can be used
again for other substrates. The simple reaction of enzyme and substrates can
be described below.
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Where E is enzyme, S is substrate, P is product, ES is enzyme and substrate
complex, and EP is enzyme and product complex. Most of enzyme reactions
are reversible. It means that the product can be turned back to its original state
by the same enzyme. However, some reactions are irreversible or it will need
other enzyme to change back (Lehninger et al., 2005).
Enzyme works specifically. It means that only the right substrates can bind to
the enzyme and start the reaction. The mechanism of enzyme and substrate
binding has been explained in many ways. Two of the famous mechanisms are
lock and key mechanism and induced fit mechanism. Lock and key
mechanism stated that enzyme and substrate is comparable to lock and key. If
a key doesn’t fit to its lock, the lock will not be opened. Same with the enzyme
and substrate, if it doesn’t fit, it will not react. However, there was also
another opinion. In induced fit mechanism, it is said that the enzyme can alter
themselves a little to fit the substrate. It means that the shape of enzyme can
change.
To work at their optimum condition, enzyme is affected by several factors.
The factors that affect the enzymes are temperature, pH, salts, and organic
solvents, and concentration. Enzymes usually work within range. Outside of
their optimum range, this enzyme will not work effectively. Temperature is
one of the crucial factors of enzyme’s work. Some enzymes will work
optimally at low temperature and room temperature, but there are some that
work optimally at high temperature. However, since enzyme is protein, it can
denature. If the temperature is too high, the enzyme will lose its catalytic
activity due to the denaturation.
The acidity also affects the work of enzyme. There are three classification of
enzymes based on its optimum pH: acid, neutral, and base (Rahman, 2003). It
is important to know which pH is suitable for the enzyme. Salts can either be a
cofactor or an inhibitor. Cofactor, as explained earlier, can activate the works
of enzyme. However, the inhibitor can inhibit the reaction. There are two main
types of inhibitor: reversible and irreversible. Reversible inhibitor is further
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classified to three types, which are competitive inhibitor, uncompetitive
inhibitor, and mixed inhibitor. Salt is most probably categorized as
uncompetitive inhibitor which binds on the non-active site of enzyme.
Based on reaction types, enzyme is classified to six main classes:
oxidoreductase, transferase, lyase, hydrolase, isomerase, and ligase. The most
important enzyme for hydrolysis reaction is hydrolase. Hydrolysis is a process
of hydration to the molecules. Hydrolase usually degrades large molecules to
smaller molecules. There are many types of hydrolase, for example protease,
amylase, and lipase.
2.3.1. Protease Protease is classified as hydrolase. It is an enzyme that hydrolyzed peptide
bonds. Protease can also be called peptidase. Peptidase is really important for
survival and it makes up about 2 % of genes in all organisms (Polaina and
MacCabe, 2007). Protease is used in many industrial process, for example in
cheese processing, rennet or ficin is used as clotting agent, it can also be used
as clarifying agent for beer, tenderize meat, and hair removal in leather
processing.
Protease can be classified based on its catalytic type. The catalytic type is
related to the group responsible for catalysis of peptide bond hydrolysis. There
are six specific catalytic types: serine protease, threonine protease, cysteine
protease, aspartic protease, glutamic protease, and metallo protease (Polaina
and MacCabe, 2007).
Since protease exists in all organisms, protease can also be extracted from
some organisms like animals, plants, and microorganisms. Animal proteases
are trypsin, pepsin, and rennin. Proteases from plant source are ficin,
bromelain, and papain. Nowadays, many industrial enzymes are extracted
from microorganism. Especially those commercially produced ones, like
Alcalase, Neutrase, and Protamex.
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2.3.2. Papain Papain (EC 3.4.22.2) is part of cysteine protease. Cysteine protease is divided
to 2 main classes: exopeptidase and endopeptidase. Papain is included in
endopeptidase. It means that papain will cleave bonds that are distant from N
and C termini. Papain is drived from Carica papaya. The crude dried latex
from papaya usually contains 4 cysteine proteases (papain, chymopapain,
caricain, and glycyl endopeptidase) and other enzymes.
Papaya with high quality usually produces the highest quality and activity
comes from tropical areas. This is very suitable to be applied to Indonesia.
There are some methods that can be used to purified crude papain. Water
extraction with reducing and chelating agents, salt precipitation, and solvent
extraction are usually done. To produce pure papain, some additional process
may be added. It is usually done by affinity chromatography methods. There
are some significant difference between crude papain and pure papain.
Table 2.2 Comparison of crude papain and pure papain Characteristics Crude Papain Pure Papain
Color Brown to white White
Smell Not preferred More preferred
Non dissolve material Up to 30% Maximum 0.05%
Water content Up to 18% Maximum 6%
Ash content Up to 14% Maximum 5%
Proteolytic activity (U/g) 70-500 70-1000
Source: Muchtadi et al. (1992) in Rahman (2003)
If dissolved in water, the crude papain will leave brownish particles around
them, but not with pure papain. Pure papain is pure white and it will be sticky
if it comes in contact with water. Since crude papain contains additives,
sometimes the materials cannot be dissolved completely. Therefore there will
be some loss of activity.
Papain is the model enzyme of cysteine protease. Its mechanism has been well
studied. Papain’s enzymatic activity is utilized by catalytic dyad formed by
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cysteine and histidine residues, which in pH between 3.5 and 8.0 forms an ion-
pair. Asparagine is used for orientation of imidazolium ring or histidine in
catalytic cleft. To be able to catalyze, thiol group of enzyme should be
reduced. Hence, the protease require reducing and acidic environment to be
active. Formation of S-acyl enzyme is a basic step in hydrolysis. Afterwards,
water molecule reacts with intermediate, N-terminal fragment is released, and
free cysteine protease molecule can begin a new cycle. (Polaina and MacCabe,
2007)
Figure 2.4 Hydrolytic mechanism of papain Source: Polaina and MacCabe (2007)
Papain has a broad specificity and is able to split many peptide bonds. is said
to be active at 50-57oC (Grzonka et al., 2007). Heating at 75oC for 30 minutes
will decrease the enzyme activity up to 20%. The optimum pH for papain is
between 5.0 and 7.0 and its isoelectric point is at 8.75. Papain is quite stable
and active if stored at 4oC.
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2.4. Protein hydrolysis
Hydrolysis is a reaction when H2O becomes H+ (hydrogen cation) and OH-
(hydroxide anion) (Wijayanti, 2009). Protein hydrolysis will degrade large
protein to small peptides. There are many purposes that can be achieved
through hydrolysis. It can increase the nutritive value, prevent damage,
provide texture, increase and decrease solubility, and increase foaming and
coagulations, add emulsion capacity, and eliminate toxin (Rahman, 2003).
Protein can be hydrolyzed by using chemical and enzyme.
2.4.1. Chemical hydrolysis Chemical hydrolysis can be done in acid condition or base condition. It is easy
to be done and relatively inexpensive. However, it is usually hard to control
and the products can produce different chemical composition and functional
properties. Chemical hydrolysis is usually done at extreme temperatures and
pH. The downsides are the product will have reduced nutritional qualities and
poor functionality.
Acid hydrolysis is more common than alkaline hydrolysis. The process is
harsh and hard to control. Vegetables protein is preferred for this kind of
process. Acid hydrolysis has some disadvantage, tryptophan, asparagine,
glutamine, and some other amino acids are destroyed. The product of acid
hydrolysis is not suitable for human consumption and usually is used as
fertilizer.
Opposite to acid hydrolysis, alkaline hydrolysis use alkali reactants like
sodium hydroxide to hydrolyze protein. However its product usually has a
poor functionality and can affect nutritive value of hydrolysate. Alkaline
hydrolysis is almost exclusively used for determination of tryptophan. Because
of formations of lysinoalanine, ornithinoalanine, and lanthionine, toxic
substances can be produced (Binsan, 2007).
2.4.2. Enzymatic hydrolysis Enzymatic protein hydrolysis can be done by using proteolytic enzyme. This
method is more preferable because it can improve the functionality and avoid
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the destruction of product. Hydrolysis using non-specific protease like papain
can increase the solubility of hydrophobic protein (Rahman, 2003). However,
this hydrolysis might produce bitter-tasting peptides which can affect its
sensory properties. The bitterness is due to hydrophobicity as well as
molecular configuration. Bitter amino acids are generally within L-series. The
bitterest amino acids are L-tryptophan and L-tyrosine but D-tryptophan named
as the sweetest one (Belitz et al., 2009).
Nowadays, enzymatic hydrolysis is more preferable for the making of protein
concentrate. It is also safer for human and animal consumption because
enzyme is naturally exists in body. There are some protease enzymes that are
usually used for producing protein concentrate, like papain, bromelain,
trypsin, chymotrypsin, Alcalase, Neutrase, Protamex. In order for enzymatic
hydrolysis to succeed, the optimum condition for each enzyme should be taken
into account.
2.5. Protein hydrolysate
In order to be healthy and grow well, human needs to consume protein in
adequate amount. However, consuming food is sometimes not enough. There
are some nutrients that are harder to find than others. Nowadays, human can
also consume supplements and nutraceuticals. This compound is designed to
fulfill human needs of nutrients. Protein hydrolysate can be consumed in the
form of powder and liquid.
The same goes for protein. Although almost all foods contain protein, not all
food contains the required essential amino acids in adequate amount. In human
diet, they do not usually consider or count the required amount of nutrients
they need. By consuming protein hydrolysate, this problem might be solved.
Protein hydrolysate is the product of protein extraction from its raw material.
The raw material can vary from plants, animals, and even microbes. The
example of protein hydrolysate from plant is soy protein isolate. Nowadays,
the most popular one is protein concentrate from milk. However, milk is
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relatively expensive in some countries, so researchers tried to find alternatives.
One of the alternatives is by harnessing the waste of seafood production.
Fish and shrimp production usually produce waste. The waste can be from
scales, shells, head, tail, or even the small fishes that are not acceptable
according to standards. These wastes can be used for producing protein
hydrolysate.
2.5.1. Shrimp protein hydrolysate Shrimp is known to contain high content of protein. Whole shrimp is predicted
to contain protein from 18% to 21%. Therefore, the use of shrimp as protein
hydrolysate has been researched frequently. The hydrolysate itself can be
produced from several parts of the shrimp, from meat (Simpson et al., 1998),
shell (Synowiecki et al., 2000), or head (Limam et al., 2008). However, the
meat of shrimp already has high value. Therefore many researches are focused
on the utilization of waste (shell and head).
There are many ways to produce shrimp protein hydrolysate, for example by
adding proteolytic enzymes (Ruttanapornvareesakul et al., 2005) or chemicals
like sodium hydroxide (Abun, 2006), biological process by fermentation
(Junianto et al., 2009) and also physical treatment (Adrizal et al., 1999) to the
sample can also extract the protein.
The focus of today’s researches is in the enzymatic hydrolysis because it
produces less undesirable by-products and also increases its functional and
nutritional value (Kristinsson and Rasco, 2000). There are some key processes
in making shrimp protein hydrolysate by enzyme.
1. Mincing
Mincing is meant to downsize the size of sample. The size needs to be
smaller because enzyme will work more effective if the smaller is given in
the smaller size and in larger amount rather than larger size but small
amount. Mincing can be done manually or automatically. Since mincing
manually spends more energy and time, machines are usually used to
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mince. The machines that are usually used are blender, food processor, and
meat grinder. Blender and food processor are used if the sample is not
produced in large scale. For large scale production, it is better to use meat
grinder for industrial process.
2. Incubation
Incubation is the most crucial part of the hydrolysis process. The factors
that need to be considered are time, temperature, pH, and salt addition
because enzyme is used. It is important to set the condition precisely for
each enzyme to get the best result because some enzymes work in acidic
condition, others in base condition.
Temperature should also be considered as crucial, because some enzymes
can only work in low temperature, and the effect of denaturation if the
temperature is too high should also be considered because denaturation of
enzyme can affect its functionality. Time will not affect the hydrolysis
process but it will affect the end result. If the time is too short, the process
will not be effective and if it is too long it will not be efficient.
Researchers stated that the optimum time for incubation was between 1 to
5 hours. Although there are some enzymes that don’t need salt, there are
also those that need it. Salt can act as the cofactor of the enzyme, so that it
will activate the work of enzyme. Salt addition to improve the activity of
enzyme had been done by Mizani and Aminlari (2007)
3. Inactivation of enzyme
The purpose of inactivation is to stop the enzyme catalyzed reaction. If the
reaction is not stopped, enzyme will continue to degrade the product which
can lead to microbial contamination. The other purpose is to pasteurize the
product. Note that pasteurization is only for inactivation that uses
temperature. Pasteurization is done to kill the contaminant in product and
also increase the shelf life of product.
Enzyme can be inactivated by several ways; the easiest is to raise the
temperature. Raising temperature to certain level can cause the enzyme to
denature and lose its functionality. Some enzymes can withstand high
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temperature, so the temperature should be considered carefully. In
contrary, heating the product can also cause the protein of product to
denature, so it is better to inactivate the enzyme in short time. The other
way is by adding chemicals that induce denaturation. Acid is usually added
to put the sample in extreme pH (Synowiecki et al., 2000), so that
denaturation occurs.
4. Filtration
Filtration is an optional process. Some researches stated that after enzyme
inactivation, they went straight to centrifugation. Others eliminated the
centrifugation process and did filtration instead (Valdez-Pena et al., 2010).
The purpose of filtration is to separate the large pieces of the shrimp head
and the small molecules, soluble solid, and liquid. Filtration may be done
using sieve, muslin or cheese cloth, and vacuum filter.
5. Centrifugation
Centrifugation is a process of separation based on the density of product.
In hydrolysis of shrimp head, the protein will be soluble in the distilled
water. To remove the impurities of hydrolysate, centrifugation is one of
the most efficient ways. The impurities can come from the shrimp head
that are not soluble, sand and minerals from the shrimp head, and also the
fat. According to Synowiecki et al. (2000), centrifugation will remove
chitin residue from hydrolysate. Centrifugation process may reduce the
yield of dried product but increase the quality and purity of the
hydrolysate.
2.5.2 Quality Standards for Fish Protein Hydrolysate Since shrimp protein concentrate hasn’t had any quality standards, the quality
standards of shrimp protein hydrolysate will be compared to those regulations
from fish protein concentrate. FAO has divided quality of fish protein
concentrate to three types (Windsor, 2001).
Type A: This product is virtually odorless and tasteless powder. The
maximum total fat content is 0.75%
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Type B: This product has no specific limit of odor and flavor, but it still has
fishy flavor. The maximum fat content of this product is 3%
Type C: this product will not be consumed by human. It is usually used as fish
meal and is produced under satisfactorily hygienic conditions.
The reason why fat is one of the requirement of quality is because of the
rancid taste that fat will produce during oxidation. In dehydrated fish protein,
the protein content of the product can reach up to 65% and up to 80% in type
A fish protein concentrate.
The standards of fish protein concentrate (fish protein isolates as referred by
FDA) also exist in FDA (Food and Drug Administration). It is written in
section 172.340 noted as fish protein isolate. If fish protein isolate is about to
be used as food additive as food supplement, it should follow several
prescribed conditions and specifications of product (FDA, 2010):
1. Additive shall consist dried fish protein prepared from edible portions of
fish after removal of head, fins, tails, bones, scales, viscera, and intestinal
content.
2. Additive shall be derived from bony fish that are recognized by qualified
scientists as safe for human consumption and can be processed as
prescribed to meet the required specifications.
3. Only wholesome fresh fish suitable for human consumption may be used.
It shall be handled expeditiously under sanitary conditions (GMP).
4. Additive shall be prepared by extraction with hexane and food-grade
ethanol to remove fat and moisture. Solvent residues shall be reduced by
drying.
5. Protein content (Kjedahl method), shall not be less than 90% by weight of
final product.
6. Moisture content shall not be more than 10% by weight of final product.
7. Fat content shall not exceed 0.5% by weight of final product.
8. Solvent residues in final product shall not be more than 5 ppm (parts per
million) of hexane and 3.5 percent ethanol by weight.
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2.6. Zero Waste Concept
Waste can cause great loss of value and resources. By identifying and
harnessing waste, mankind can save money, save the world by making
sustainable resources. Zero waste is a concept, in which all part of resources
used in processing will not produce any waste. Waste is recalled as
inefficiency in zero waste concepts.
Instead of throwing the waste away, waste can be considered as potential
resource to be harnessed. Waste is not a burden anymore but it is an
opportunity. It will reduce costs for discarding waste and increase profits at
the same time. On the positive side, the application of zero waste concepts can
solve the environmental issue and also provide sustainability of resources.
This concept is applicable to various kind of organization from community,
business, and school, industry wide and even at home.
Figure 2.4 below showed the material flows in today’s industry. The raw
materials are processed and used and discarded after its life ended. There are
recovery and disposal. The recovered materials will be used as compost and
the rest of the waste will be disposed in land fill, which cause terrible smell
and also exploit space, or incinerated causing pollution to the air.
Figure 2.5 Material flows today Source: http://www.zerowaste.org/case.htm (2011)
This concept of material flows is what the zero waste concept tries to avoid. A
zero waste society will not disposed and incinerated the waste. Instead, it will
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be used as reusable and recycled material. The rest of the waste will be used as
compost.
Figure 2.6 Improved material flows Source: http://www.zerowaste.org/case.htm (2011)
The example of zero waste concept can be taken from frozen shrimp
processing. In frozen shrimp processing, it is usually only the meat that was
used while head and shell are usually discarded or used as compost. This head
and shell can be reused completely to produce another product. The head and
shell can be hydrolyzed and producing shrimp protein concentrate. This
process will produce waste as undissolved materials. However this materials
can be used to produce chitin. Both protein concentrate and chitin are valuable
and can even have higher price than the shrimp itself. If all industry can apply
this concept to their production process, they can gain a lot more profits and
also protect the environment.
However, it is hard to apply this concept to all industries. Considering that to
do research about how to harness the waste is time and money consuming.
Other things that will be disadvantages to this concept is that processing of
waste itself will need other materials that are not related to the industry which
will produce other costs. The industry is usually already feel fine about
discarding their waste and do not have any intention to protect the
environment. This awareness of environmental problem should come from the
company itself and maybe by support of environmentalists.
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CHAPTER 3 – METHODOLOGY
3.1. Time and Venue
Research was conducted within 4 months from March 2011 until June 2011.
This was done in Research Center for Marine and Fisheries Product
Processing and Biotechnology (Jakarta, Indonesia) and Swiss Germany
University (BSD, Tangerang). The research used more than one laboratory,
which are Chemistry Laboratory, Biotechnology Laboratory, Sensory
Laboratory, and Product Processing Laboratory.
3.2. Materials
3.2.1. Raw Materials Raw material for this research was shrimp head of Litopenaeus vannamei. L.
vannamei for this research was taken from a shrimp processing company in
Ancol, Jakarta. The shrimps were taken right after the production to retain its
freshness. The shrimps were then stored in freezer at -20oC.
3.2.2. Chemicals Crude papain
Pure papain
Ethyl ether
N-hexane
H2SO4
HCl
Casein 1%
Buffer pH 7 and pH 8 (KH2PO4 and K2HPO4)
TCA (Trichloro acetic)
Folin Ciocalteau
Biuret reagent (Cu2SO4, Na-K Tartarate, NaOH, Na2SO3)
Na2CO3
Na2SO4
BSA (Bovine Serum Albumin)
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Tyrosine
3.3. Equipments
1. Analytical Mass Balance
2. Oven
3. Furnace
4. Soxhlet
5. Kjeltec 2300 FOSS
6. Water Bath Shaker
7. Hot Plate
8. Spectrophotometer
9. Distillation set
10. Micropipette
11. Centrifuge
12. Crucibles
13. Dessicator
14. Round bottom flasks
15. Erlenemeyer flasks
16. Beaker glasses
3.4. Procedures
3.4.1. Examination of raw material Raw material was examined using proximate analysis. Proximate analysis
included moisture content, ash content, fat content, and protein content. Before
the proximate analysis, the sample was ground first using blender. The method
used in this analysis followed SNI (Standar Nasional Indonesia).
1. Water content ( SNI 01-2354.2-2006)
Crucibles were already prepared in oven overnight at 100oC and were put
into dessicator for 15 minutes. The crucible was weighed afterward and 2
g of sample was put into the crucible. Crucible with sample was put into
the oven (100oC) and left overnight. It was moved to dessicator for 15
minutes and was weighed once more. The moisture content was measured
by this equation
Water content =
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This experiment was replicated 2 times.
2. Ash content (SNI 01-2354.1-2006)
The dried crucible and sample from previous experiment was put into
furnace (550oC) and was left there for 8 hours or until the color turned to
white. After that, the temperature is lowered to 40oC and the crucible was
put in the dessicator for 30 minutes. If the sample was not white, it should
be put in the furnace again, but first it was given distilled water and dried
on hot plate, then was dried again. The experiment was done until ash was
white and weight was constant. To calculate the ash content, equation
below was used.
% Total ash =
This experiment was replicated 2 times.
3. Lipid content (SNI 01-2354.3-2006)
Sample was put into an extraction thimble. Extraction thimble is made
from filter paper. If sample was wet, sodium sulfate (Na2SO4) should be
added twice the weight of sample to dry it. Sample was later put into the
extraction soxhlet. Round flask for extraction was weighed and boiling
stone should be added inside. Soxhlet was assembled and 150 ml ether
was added. The extraction was done at 60oC for 8 hours. Fat and ether was
evaporated. Round bottom flask with fat was put in oven at 105oC for 2
hours to eliminate ether and vapor. Flask was weighed again. This analysis
was done twice. The equation for lipid content is displayed below.
% fat =
A= weight of empty round bottom flask (g), B= weight of sample (sodium
hydroxide was excluded) (g), C= weight of round bottom flask + fat (g)
4. Protein content (SNI 01-2354.4-2006)
1 g of sample was weighed and added with 0.2 N HCl. Sample destruction
used H2SO4 with catalyst K2SO4. Using 250 ml flask, H2SO4 used was 10-
15 ml. Destruction was done at 410oC for 2 hours until the solution was
clear. This is indication of perfect destruction. Afterward, the sample was
distilled and titrated using Kjeltec 2300 (FOSS).
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3.4.2. Assay of enzyme papain (food grade and pure) Determining enzyme activity was separated to two main parts. The first was
determining enzyme activity. This was done by combining 250 μl casein 1%
as substrate, 250 μl Buffer pH 7, and 250μl enzyme of interest in an Eppendorf
tube. The incubation time was 20 minutes at 50oC. After incubation 750 μl
TCA was added to stop reaction. The tube was centrifuged 8000 rpm at 4oC
for 10 minutes. 300 μl supernatant from tube was taken and mixed with
1000μl Na2CO3 and 200μl Folin Ciocalteau. Sample was put into
spectrophotometer to measure absorbance at 578 nm. Standard curve was
generated from tyrosine sample to determine protein activity. Volume activity
was determined after this experiment.
3.4.3. Hydrolysis of shrimp head (Limam, 2008)
Initially, research was done to find the optimum time for incubation (3, 4, and
5 hours), pH that was suitable for research (pH 7, pH 8, and distilled water).
These experiments were combined with the effect of centrifugation and
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filtration to protein content and the effect of filtration after hydrolysate was
recovered from centrifugation. The enzyme used in this experiment was only
the crude papain at 10% concentration. The incubation was done at 50oC in
water bath shaker with occasional stirring.
The protein content on early experiments was analyzed and conclusion was
made. The optimum time for incubation was 4 hours and distilled water was
used instead of buffer. Both filtration and centrifugation were done but the
hydrolysate was not filtered once again.
The method of making shrimp head protein concentrate followed Limam et al.
(2008) with slight modification. Shrimp head was ground using blender. 100 g
of the shrimp head was taken for each hydrolysis. Then, it was added with
distilled water at ratio 1:1 (w/v) that was already added with enzyme (the
enzyme used in this experiment was two types of papain: pure papain and
crude papain). This solution is homogenized using glass rod. Incubation period
was 4 hours and during this period, it was occasionally stirred. Incubation was
followed by inactivation of enzyme at 90oC for 5 minutes. After the solution
had cooled down, the solution was filtered using muslin cloth. The waste from
the unfiltered material was thrown and the liquid part was processed once
again. Solution was centrifuged at 9000 rpm for 15 minutes at 4oC and the
supernatant were taken as the protein hydrolysate. The treatments for this
experiment were type of enzyme (crude and pure papain), concentration of
enzyme (10, 20, and 30%), and temperature of incubation (45, 50, 55, and
60oC).
3.4.4. Analysis of proximate composition and yield of hydrolysate Proximate analyses that will be done are water, ash, protein, and fat. Fat
analysis was done to two samples from each enzyme that had the highest
protein concentration. The proximate analysis was done with the same method
as analysis of raw material except the protein and fat content. Soluble protein
was determined using Lowry assay and fat content was analyzed using batch
solvent extraction method.
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1. Fat content
Round bottom flask was put into oven at 105oC for one night. After that, it
was put into dessicator for 15 minutes and weighed. The round bottom
flask was put into the oven again for an hour and afterwards it was put into
dessicator for 15 minutes and was weighed. The weight of the first and the
second measurement should not exceed 0.004 g.
15 g of liquid sample was taken and put into separator funnel. 15 ml of n-
hexane was added to the separator funnel. The solution was homogenized
by shaking the separator funnel manually. The solution was left to settle
and produced two separate parts. The n-hexane were put into the round
bottom flask. Sample was added with n-hexane again and the process was
repeated three times. The round bottom flask containing n-hexane was
distilled using distillation set at 60oC. After all n-hexane evaporated, the
round bottom flask containing fat was put into oven for two hours.
Afterwards, it was put into dessicator for 15 minutes and was weighed.
The fat content was calculated by the equation below,
where A was weight of empty round bottom flask, B was weight of round
bottom flask plus fat, and C was the initial weight of sample.
2. Lowry Assay (Lowry, 1951)
In Lowry assay, 0.6 ml protein sample will be mixed with 3 ml Biuret
solution. This solution was vortexed. After ten minutes, this solution was
added with 0.15ml Folin-Ciocalteau (1:2 v/v). The solution was left for 30
minutes and afterwards the absorbance of sample was measured at 650 nm.
To know the concentration of protein in sample, standard curve should be
made using BSA (bovine serum albumin) solution with concentration 0.1,
0.2, 0.4, 0.6, 0.8, 1.0 mg/ml. The procedure of absorbance was the same as
the protein sample.
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Other than the protein content, recovered protein from the process was also
calculated. Recovered protein is percentage of soluble protein in
hydrolysate from total protein in raw material.
Yield was calculated based on the initial mass of the mixture and the mass
after centrifugation. It means that the weight of water and enzyme were taken
into account. Yield was calculated by the equation below.
3.4.5. Sensory evaluation The samples that will be examined were the two samples from each enzyme
that has the highest protein content. The type of sensory test that will be used
is hedonic test to know the preferred product between the shrimp head
hydrolyzed with pure and crude papain. The result was obtained from three
replications. The parameters that were be used are taste, appearance, color,
and smell. The scoring were from level 1 (very dislike) to 7 (very like). The
hedonic test used 7 trained panelists and 6 semi-trained panelists.
3.5. Experimental Design
Experimental Design that was used on this research was Completely
Randomized Factorial Design with three factorials as the treatments.
Replication was done twice. The model of the experimental design was as
following.
Yij = μ + Ai + Bj +Ck+ ABij + ACik + BCjk + ABCijk + Eijk
Yij : Observation value
μ : Mean value
Ai : Influence of Treatment A, where i=1 and 2
Bj : Influence of Treatment B, where j=1, 2, 3
Ck : Influence of Treatment C, where k= 1, 2, 3, and 4
ABij : Influence of interaction between Treatment A and Treatment B
ACik : Influence of interaction between Treatment A and Treatment C
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BCjk : Influence of interaction between Treatment B and Treatment C
ABCijk: Influence of interaction between Treatment A, B, and C
Eijk : Influence of error
There were 3 factors used as treatments on this research. They were called as
Treatment A, Treatment B and Treatment C. Treatment A covered the type of
enzyme used for hydrolysis, which were crude papain and pure papain.
Treatment B covered various concentration of enzyme papain used in
hydrolysis, which were 10%, 20%, 30%. Treatment C covered the temperature
used as hydrolysis temperature, which were 45oC, 50oC, 55oC, and 60oC.
A. Type of enzyme variation
A1 = crude papain
A2 = pure papain
B. Concentration variation
B1 = 10%
B2 = 20%
B3 = 30%
C. Temperature Variation
C1 = 45oC
C2 = 50oC
C3 = 55oC
C4 = 60oC
3.6. Data Analysis
Data analysis for proximate composition and yield of products were done
using OpenStat with three-way ANOVA (Analysis of Variance) with 95% of
confidence level. Post hoc analysis was done using Tukey HSD (Honestly
Significant Difference). Sensory test was analyzed using two-way ANOVA in
Microsoft Excel Data Analysis with 95% of confidence level, and t-test
between samples for mean was done to the parameters that showed significant
difference.
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CHAPTER 4 – RESULT & DISCUSSION
4.1. Proximate composition of shrimp head (L.vannamei)
The raw material was analyzed for its water, ash, protein, and fat content. The
table below showed the result of the analysis.
Table 4.1 Proximate composition of L. vannamei head and P. monodon head
Parameter L.vannamei P. monodona
Water (%) 79.138 + 1.008 78.5
Ash (%) 4.416 + 0.547 5
Fat (%) 2.123 + 0.173 3.1
Protein (%) 11.599 + 0.518 13.6 afrom Teerasuntonwat and Raksakulthai (1995)
The objective of finding the proximate composition of raw material was to
compare it to the proximate composition of products. More specifically, the
protein content of raw material was used to determine the recovered protein
from the result. Water content of whole shrimp is 75.86 %. It has 1.2 % ash
content and about 20.31% protein. The fat content is 1.73%. Compared to the
literature, the water, ash and fat content were higher.
Protein content in shrimp head is lower than the protein of reference. It ought
to be noted that the composition in the literature was for whole shrimp. If
compared to other research on shrimp head with different species, this
proximate composition was not really different. It can be concluded that
shrimp head contain less protein than whole shrimp but more fat, ash, and
water.
4.2. Determination of enzyme activity
The enzyme activities of both pure and crude papain were determined. To fit
the standard curve, the enzyme should be diluted first. The pure papain was
diluted 1000 times and the crude papain was diluted 5 times. The enzyme
activity of pure papain was 2375.78 U/g and for crude papain was 21.2 U/g.
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This determination of enzyme activity was very important because both
enzymes were used to hydrolyze the same thing.
The enzyme activity of crude papain was much lower than the pure papain.
Thus, it was not possible to compare the hydrolytic capability of both enzymes
by its concentration. Therefore, the approximate enzyme activity was the one
that was being equalized. Since the treatment of the experiment also required
enzyme concentration, concentration of crude papain was made as the
reference. The enzyme concentration 10, 20, and 30% was derived to mass
unit as 10 g, 20 g, and 30 g. By equalizing the activity of both enzymes, the
pure papain added as 10% was 0.89g, 20% was 0.178g, and 30% was 0.267g.
4.3. Effect of incubation time and filtration after centrifugation to protein concentration
From the experiment, statistical analysis showed that there were no significant
difference between incubation time 3, 4, and 5 hours. However, from the
graph, there was an increase of protein concentration at 4 hours incubation
time. So, it was decided that the appropriate time for incubation was 4 hours.
The decrease of protein content after 4 hours was the result of ununiformed
stirring, quality of raw material was not the same, or capability of enzyme to
hydrolyze was not the same (Wijayanti, 2009).
There were three phases after centrifugation of hydrolysate: the solid part at
the bottom, which contained insoluble material and solid waste, the middle
part which contained the hydrolysate, and the third phase which stuck to the
wall of the centrifuge tube and floated around the hydrolysate. To get rid of
this flocculent, another process of filtration using filter paper was done. To
compare this process, the hydrolysate which were not filtrated, were also
prepared. It seems that there was a significant difference in protein content
between the filtration and non- filtration. The products which were not filtered
contained more protein than the one that was filtered. It means that the
flocculent were also a part of the protein, which means it was important.
Therefore, the filtration process after centrifugation process was eliminated.
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4.4. Effect of pH to protein concentration
When working with enzyme, pH is one of the factors to be considered in order
to obtain the best result. Each enzyme has its own optimum pH. Papain is
considered to be active in broad range of pH. However, it is optimum between
the pH 6-8. Therefore, the experiment was to determine whether or not, there
was a significant influence of using pH buffer to maintain the pH or not.
In the experiment, the crude papain was dissolved in three different solvent,
which are buffer pH 7, buffer pH 8, and distilled water. The pH of the shrimp
head with distilled water was controlled using pH paper every hour during
incubation. The pH was 8 and it was stable during the incubation. After the
process, the protein content was analyzed. Apparently from the result it can be
concluded that there was no significant difference between the use of buffer
and distilled water. Hence, the main research used the distilled water as
solvent.
4.5. Effect of centrifugation and filtration using muslin cloth to protein content
In this experiment, two key processes were examined. Some of the products of
incubation were filtered using muslin cloth and others were centrifuged. In the
result, it showed that there was no significant difference in protein content
between filtration and centrifugation.
However, both products had a significant difference in appearance. The
product that was only filtered was filled with many flocculent and it was
darker, hazier and there were some precipitations. In comparison, the
centrifuged product was lighter, had less flocculent, was also clearer and smell
less fishy than another. In the end, the combination of filtration and
centrifugation were done in the main research.
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4.6. Analyses of yield and proximate compositions to treatments type of papain, concentration of papain, and temperature of incubation
There were a total of 24 treatments for the shrimp protein hydrolysate. Each
treatment was analyzed for its yield, water content, ash content, and protein
content. The data summary can be viewed on Table 4.1. C indicated the
concentraton of enzyme (%) and T indicated the temperature of incubation
(oC). CP stood for crude papain and PP stood for pure papain. The data
displayed was average of each treatment plus minus its standard deviation.
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WATER CONTENT (%)
ASH CONTENT (%)
PROTEIN CONTENT (%)
YIELD (%)
C T CP PP CP PP CP PP CP PP
10
45 87.28 ± 0.375
92.83 ± 0.073
3.71 ± 0.032
0.46 ± 0.014
5.07 ± 0.947
6.62 ± 0.021
72.30 ± 0.199
77.17 ± 0.001
50 88.28 ± 0.375
91.53 ± 0.127
3.79 ± 0.057
0.46 ± 0.003
5.54 ± 0.166
7.91 ± 0.021
73.54 ± 0.259
71.07 ± 0.198
55 87.31 ± 0.082
91.32 ± 0.158
3.75 ± 0.022
0.54 ± 0.002
6.24 ± 0.041
7.95 ± 0.075
74.27 ± 0.015
77.58 ± 0.322
60 88.71 ± 0.090
90.83 ± 0.093
3.60 ± 0.054
0.54± 0.016
6.28 ± 0.476
8.42 ± 0.393
68.68 ± 1.485
67.82 ± 0.476
20
45 84.21 ± 0.229
92.56 ± 0.035
6.22 ± 0.639
0.46 ± 0.003
5.02 ± 0.062
6.74 ± 0.041
72.45 ± 0.177
77.29 ± 0.269
50 84.22 ± 0.715
91.32 ± 0.370
6.47 ± 0.426
0.47 ± 0.011
5.87 ± 0.021
8.10 ± 0.269
70.27 ± 4.110
71.00 ± 0.601
55 83.94 ± 0.394
91.20 ± 0.049
6.74 ± 0.070
0.54 ± 0.001
6.71 ± 0.994
7.82 ± 0.162
83.11 ± 0.411
78.09 ± 0.215
60 83.63 ± 0.856
91.39 ± 0.030
6.57 ± 0.204
0.55 ± 0.003
6.48 ± 0.884
7.95 ± 0.104
70.29 ± 0.058
67.97 ± 0.212
30
45 81.90 ± 0.294
92.46 ± 0.051
8.92 ± 0.190
0.46 ± 0.008
5.10 ± 0.166
6.94 ± 0.207
71.99 ± 0.267
77.36 ± 0.252
50 81.47 ± 0.208
90.90 ± 0.035
9.25 ± 0.401
0.47 ± 0.005
6.59 ± 0.746
7.98 ± 0.352
73.21 ± 0.038
71.93 ± 0.021
55 80.72 ± 0.386
91.17 ± 0.009
9.21 ± 0.214
0.55 ± 0.001
5.89 ± 0.083
7.95 ± 0.122
84.10 ± 0.397
78.07 ± 0.197
60 80.57 ± 0.509
90.79 ± 0.030
9.39 ± 0.079
0.55 ± 0.005
6.69 ± 1.139
8.57 ± 0.104
83.75 ± 0.802
67.53 ± 0.019
Table 4.2 Data summary of yield and proximate analyses
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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
can be seen that the yield for pure papain at 50oC and 60oC were lower than the yield
at 45oC and 55oC. The same went for the crude papain. Although statistical analysis
showed that temperature had effect on yield, result of graph temperature versus yield
was irrelevant compared to any literature. It did not show any significant increment or
decrement. The factors that may affect this result was the filtration using muslin cloth.
The filtration using muslin cloth was done manually. Therefore, the work done in the
products may not be uniform. This may cause the fluctuation of the data. According
to Yulistianti (2009), liquid flavor (liquid concentrate in this case) is volatile and
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chemically unstable against air, light, humidity, and temperature on storage, this may
be the factor why the yield was unstable as well.
4.6.2. Water Content The water content of the product was analyzed. Since the protein concentrate was in
its liquid phase, the water content was definitely higher than 80%. The data that was
obtained from the analysis of water content can be seen below.
Based on statistical analysis using three way analysis of variance (ANOVA), it was
known that there was significant difference between the water content of product
hydrolyzed by crude papain and pure papain (Appendix 4). There were also
significant differences in water content among concentration of enzyme and
incubation temperature. There were significance interactions between type of enzyme
and its concentration, type of enzyme and incubation temperature, concentration of
enzyme and incubation temperature, and type of enzyme with its concentration and
incubation temperature.
Figure 4.3 Graph of concentration versus water content
Since the early result showed that there was significant difference in water content
between the concentrations, statistical analysis showed that water content hydrolyzed
with 10% enzyme concentration was different to 20% and 30%, and 20% enzyme
concentration was also different from the 30%. This difference was easier to see in the
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graph (Fig 4.3). The water content of products decreased as the concentration of
enzyme increase.
Figure 4.4 Graph of temperature versus water content
Besides the effect of concentration, temperature also gave a significant effect to water
content. Therefore, the difference needed to be pointed out. From statistical analysis
(Appendix 4), it was known that water content with products incubated at 45oC were
difference to those incubated at 50oC, 55oC, and 60oC. However, the difference of
water content between 50oC, 55oC, and 60oC was insignificant.
From the result, it showed that the water content of products hydrolyzed with crude
enzyme at several concentrations was significantly different. When crude enzyme was
applied at 10% concentration, the water content was different from 20% and 30%
enzyme concentration. The products with 20% and 30% enzyme concentration also
showed different water content. However, the products hydrolyzed with pure enzyme
didn’t show any significant difference among its concentration. To equalize the
enzyme activity of crude papain, the pure papain added to the solution was
approximately hundred times less than the crude papain. Therefore, more crude
papain dissolved to the solution which automatically decreased the water content. The
pure papain added to equalize the crude papain were 0.089 g, 0.178 g, and 0.267 g,
which didn’t affect the water content significantly. Within each incubation
temperature, the average water content from each concentration showed no significant
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difference. It means that temperature did not affect the enzyme concentration effect
on water content.
Crude papain contains additives like sugar and salt. Both materials are soluble in
water. Because of the solubility of these materials, the free bound water in the
products will also decrease (Anonymous, 2011). This phenomenon contributed to the
decrease of water content.
The increase of water soluble material can either be adventageous or disadventageous.
It is adventegous if the soluble material that are dissolved are the protein part. This
process is considered not successful if most of the soluble material is the salt and
sugar, not the protein. The difference between the water content of hydrolysate that
was hydrolyzed with pure papain and crude papain can be seen clearly. The water
contents of the hydrolysates from crude papain were under 90% while the water
contents of hydrolysates from pure papain were over 90%. The pure enzyme added to
the solution was hundred times less than crude enzyme added. Moreover, the pure
papain contains only enzyme and no additives, which means the only dissolved
material from the powder was the enzyme. If the process were to be continued to
drying, the products hydrolyzed with crude enzyme will probably contain high sugar
and salt concentration.
4.6.3. Ash Content Ash content was calculated based on the residue of excessive heating at 550oC. Ash
content gives a quick glance of mineral trace in the product. Mineral was not volatile
and it can withstand high temperature. Therefore, when it is burned at high
temperature, other organic materials will evaporate which leave the ash behind
Statistical analysis (Appendix 5) showed that there was significant difference between
the ash content of products hydrolyzed by crude papain and pure papain. The
difference of ash content among the concentration of enzymes was also significant.
However, temperature did not have significant impact to the ash content. The
significant interaction was only between the type of enzyme and concentration. The
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interaction between type of enzyme and temperature, concentration and temperature,
and the three of them were insignificant.
Figure 4.5 displayed the relationship between ash content and concentration of
enzyme for both types of enzyme. The ash content of products hydrolyzed by crude
papain increased as the concentration increased. However, it can be seen that the ash
content of pure papain products did not increase or decrease as the concentration
increase. It made an almost linear line. The difference between ash content produced
by the pure and crude papain was also significant, as it can be seen on the graph. The
range of ash content for crude papain was between 3 to 9 % and the range of ash
content for pure papain was only 0.4-0.5%.
Figure 4.5 Graph of Concentration versus ash content
To be specific of the difference, the statistical difference among the concentration was
divided per type of enzyme. Results showed that hydrolysis using crude papain at
several concentrations will produce significantly different ash content. As the
concentration of crude enzyme got higher, the ash content also increased. The ash
content of 10% concentration was significantly different to the 20% and 30%. The
products with 20% enzyme concentration were also different from the 30%. However,
the ash content of products from pure papain did not show significant difference
among the concentrations. At 10, 20, and 30% concentration, there were significant
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differences between the ash content of products from pure and crude papain
hydrolysis, it was the same conclusion as the interpretation of the graph.
Figure 4.6 Graph of temperature versus ash content
The difference between the ash from crude and pure papain hydrolysate was not only
in percent but also in appearance. Products from pure papain produced totally white
ash and it takes shorter time to ash it. In comparison, the products from crude papain
produce white ash with some trace of black particles. It also took longer time to
produce the ash. This result was most probably due to the content of crude papain.
Figure 4.7 Comparison of ash of hydrolysate
Crude papain contains not only papain but also salt and sugar. The proportion of salt
and sugar was not clearly described but both were soluble in distilled water. Most of
minerals that are water soluble are usually in the form of salts (Traverso, 2004).
Crude papain hydrolysate
Pure papain hydrolysate
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Hence, salt is also considered as mineral, so it will not evaporate when put into the
furnace. This explained the high ash content in products of crude papain hydrolysis.
4.6.4. Protein Content Protein content was the essential part of this product. The higher the yield of protein
was the better. However, it is also important not to waste sources and energy even if
the yield is high. Therefore, statistical analysis to protein data should be done.
Based on the statistical analysis (Appendix 6), it can be concluded that there was
difference between the types of enzyme used. The temperature also gave significantly
different protein result. However, there was no interaction between all the treatments.
Figure 4.8 Graph of concentration versus protein content
Figure 4.8 showed the relationship between papain concentration and protein
concentration. It was said that concentration apparently did not have any significant
effect to the protein content. The correlation can be seen by looking at the graph. The
protein content tend to be constant even though the concentration of enzyme
increased. It also means that 10% enzyme concentration is already sufficient to
produce a good protein hydrolysate. The protein content produced by both enzymes
was significantly different. This graph indicated that the average content of protein of
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products hydrolyzed by crude enzyme was 6%, while the others were approximatelly
8%.
Figure 4.9 Graph of temperature versus protein content
Temperature was shown to have significant effect on protein. Figure 4.9 represented
the effect of temperature to protein content. For both types of papain, there were slight
increases of protein content as the temperature got higher. Therefore, the point of
difference should be located. It was known that when incubation temperature was
45oC, it produced significantly different protein results from those that are incubated
at 50, 55, and 60oC. However, the protein result between the incubation temperature
of 50, 55, and 60oC showed no significant difference.
From the experiment, results showed that pure papain produced higher protein
content. This result was expected because even though the activity has been
equalized, crude papain still contained high amount of additives. These additives
might affect the work of the enzyme itself so that the hydrolysis will be disrupted. The
other problem with using crude papain was that it was not totally soluble in the water.
The amount of enzyme that was added was 10, 20, and 30% of the water weight.
During the experiment, there were some particles that could not dissolve to the
distilled water. This may be the cause of ineffective hydrolysis. The particles that
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could not dissolve were not able to hydrolyze and cause the protein unable to dissolve
in water.
Result showed that when temperature was 45oC, it could not produce protein with
higher result, which means that the rate of hydrolysis was not as good as those
incubated at 50, 55, and 60oC. It was consistent to the literature that said that papain is
optimum between temperature 50-60oC. Therefore, it was also predicted that at 45oC,
soluble protein will be less than at 50-60oC. With consideration of denaturation and
energy use, it could be concluded that the effective incubation temperature for
hydrolysis of shrimp head waste by both papain was 50oC.
Concentration of enzyme was said to have no significant difference in the protein
content. It can be concluded that 10% enzyme concentration was adequate to be used
for hydrolysis of L. vannamei hydrolysis. For crude papain, 10% was probably
adequate because when the concentration was higher than 10%, there were more
insoluble particles, which made it inefficient. When concentration of enzyme added
reaches a certain point, the increase of soluble protein in hydrolysate will not increase
significantly or even does not increase at all. This may be the reason why the
concentration did not provide higher protein result for the pure papain.
Shrimp head hydrolysis was a process to degrade the long protein chain in shrimp
head to small peptides and amino acids. It also takes out and degrades protein that is
stored in chitin. Chitin is a part of the shell and head. The digestibility of protein will
decrease if it is stored inside chitin (Adrizal et al., 1999). To know how effective the
process of protein hydrolysis went, recovered protein should be calculated. The data
below showed the recovered protein from the head.
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Table 4.3 Recovered protein of each treatment Concentration (%)
Temperature (oC) Crude Papain Pure Papain
10
45 52.10 ± 1.038 87.22 ± 0.273
50 114.30 ±3.421 89.02 ± 0.547
55 75.23 ± 0.499 90.71 ± 2.706
60 73.31 ± 5.559 97.04 ± 0.254
20
45 63.49 ± 0.785 96.75 ± 3.216
50 104.79 ± 0.370 96.50 ± 4.257
55 77.67 ± 11.511 102.90 ± 0.968
60 73.68 ± 10.053 106.88 ± 2.217
30
45 55.83 ± 1.815 104.49 ± 1.608
50 108.78 ± 12.307 99.32 ± 4.641
55 66.37 ± 0.934 93.36 ± 1.216
60 75.51 ± 12.851 100.04 ± 1.209
The protein recovered was the ratio of soluble protein in hydrolysate and protein in
raw material (shrimp head). Statistical analysis using three-way ANOVA showed that
there were significant differences among type of enzyme, concentration, and
incubation temperature to the protein recovered. However, the significant interactions
were only from type of enzyme and concentration of enzyme and type of enzyme and
incubation temperature.
Figure 4.10 Graph of concentration versus recovered protein
Figure 4.10 showed the relationship between papain concentration and recovered
protein. From the graph, recovered protein from products hydrolyzed with pure
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papain was higher than the crude papain. The difference of protein recovered between
the enzyme concentrations laid between 10% and 20% concentration. However,
protein recovered from 10% and 30% and 20% and 30% showed no significant
difference. Among incubation temperatures, protein recovered was different
significantly. The differences were between all of the temperature, except protein
recovered between 55oC and 60oC.
In graph temperature versus recovered protein, the relationships were not linear. The
highest recovered protein seemed to come from 50oC incubation temperature.
Therefore, it supported the analysis of protein content conclusion that 50oC was the
best temperature to incubate the head of L.vannamei.
Figure 4.11 Graph of temperature versus recovered protein
If the effect of enzyme concentration were to look at separately based on the type of
enzyme, they would have shown that there was no significant difference on protein
recovered between the enzyme concentration on both crude and pure enzyme. Protein
recovered gave rough view of whether the enzyme hydrolyzed properly. Most of the
protein recovered results were more than 50%. It was considered as effective because
it has hydrolyze the protein from chitin and obtained half of the protein content in the
shrimp head. There were also some data of protein recovered that exceeds 100%. This
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showed a very effective hydrolysis. It was possible because in determining the protein
content in raw material was not all uniform. Some shrimp heads might contain higher
protein content than the result.
Other than recovered protein, the other important determination of which enzymes
produced better result was done by comparing the dry basis of both products.
Comparing dry basis gave a rough indication of the percentage of protein in product
in solid part.
Figure 4.12 Graph of concentration versus protein content (dry basis)
This graph (Figure 4.12) showed that concentration of enzyme affected the dry basis
protein content. Although in wet basis calculation, there seemed to be no difference
between protein content hydrolyzed with different concentrations, the difference can
be seen in this graph. The protein content decreased as the papain concentration
increased. This may happen because of the proportion of high ash content. As said
earlier, the process of hydrolysis can be considered a success if the protein content
was higher than the ash content. However, in these products, the ash content seemed
to be higher.
In comparison, products hydrolyzed with pure papain did not really show any changes
in protein content. The difference between protein content of products hydrolyzed by
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crude and pure papain was different significantly. The protein content of crude papain
hydrolysis was about 30-50% but it is up to 90% in pure papain hydrolysate.
Protein content for each enzyme was insignifantly different based on the graph of
temperature versus protein (Figure 4.13). The line was almost linear and that means
that temperature has no effect in protein content. Statistical analysis also supported
this prediction. Once again, the protein content of the of pure and crude papain was
significantly different.
Figure 4.13 Graph of temperature versus protein content (dry basis)
4.6.5. Fat Content Fat content was determined for two products from each enzyme that produced the best
protein result according to statistical analysis. Since the statistical analysis done to
protein content showed that concentration gave no significant difference to protein
content, the enzyme concentration used for this fat analysis was 10%. The
temperature that was chosen was 50oC because based on the statistical analysis; it
showed that from 50oC onwards there were no significant changes in protein content.
The fat content from the sample that was hydrolyzed with pure papain was 0.068%
and the fat content from sample hydrolyzed with crude papain was 0.16%. Both
results showed that the hydrolysates had lower fat content than the raw material
because raw material contains about 2.1% fat. The result of fat in the hydrolysate was
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lower because this calculation was calculated from wet basis. The dry basis
calculation showed that the raw materials contained about 10.18% fat content. The
products however, contained 0.8% for pure papain hydrolysate and 1.4% for crude
papain hydrolysate. That means not all the fat that existed in the shrimp head entered
the hydrolysate. It may be because the fat was not filtered through the muslin cloth.
There was also one possibility why fat did not enter the hydrolysate. Earlier in this
chapter, it was said that centrifugation phase left out three phases: the solid part, the
liquid part, and the floating solid part, which usually remained on the wall of tube.
The floating part is most probably contained the fat. However, because in preliminary
research filtering the floating particles cause significance changes in protein content,
it was not filtered in the main research.
The fat content of product hydrolyzed with pure papain was lower rather than the one
hydrolyzed with crude papain. During centrifugation of product hydrolyzed by crude
papain, there were a lot of floating particles on the top but not in the product of pure
papain hydrolysis. Product of pure papain hydrolysis also had three phases, but the
floating particles of crude papain hydrolysis existed in the middle of the supernatant
and the solid waste.
Figure 4.14 Result of centrifugation of hydrolysate from pure papain
Liquid phase
Solid phase
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Figure 4.15 Result of centrifugation of hydrolysate from crude papain
The crude papain contains salt and when salt is dissolved in water, the water will
become salt water and will have higher density than water. The salt water density is
1.025 g/cm3 while water’s density is 1 g/cm3 (Chang, 2000). The principle of
centrifugation is separation based specific gravity. If the floating particles did not
float in the protein concentrate hydrolyzed by pure enzyme, it means that the floating
particles were supposed to be in the bottom but due to the higher density of salt water,
the particles identified as fat will be floating.
4.7. Sensory Evaluation
Hedonic test was done to two products which had the highest protein content. Since
sensory analysis requires the sample to be fresh, new batch of hydrolysates were
made. Although there were only two products, they were made into three replicates.
The reason in doing this was to compare the homogeneity of panelists score. The
samples were displayed in small transparent glass with lid. The lid was applied to
prevent the smell to evaporate. The room used for this test was a special room for
sensory purpose only. It was bright and it was in cubicle so that there will be no
interactions between panelists.
Floating particles
Liquid phase
solid phase
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Figure 4.16 Condition of sensory evaluation
There were four parameters that were examined, which were appearance, color, smell,
and taste. The panelists were also told to give their comment of the samples. The
graph below presented the result of hedonic test. Although there were triplicates,
average of each was determined.
Figure 4.17 Result of Hedonic Test
Based on the chart above, it can be seen that the score for appearance and color for
both product was not too different. Statistical analysis showed the same result that
there were no significant difference between two samples in appearance and color. In
smell parameter, the score showed that there was significant difference. From the
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graph, the difference was not really extreme but statistic showed that the difference
was significant. The smell of products from crude papain was more likeable than the
products from pure papain. The same goes for the taste. The score that was gained for
the pure papain and crude papain was really different. The graph showed that the one
hydrolyzed with crude papain had an average score of 4.69 (almost 5) whereas the
average score for hydrolysate from pure papain was 2.49. Most panelists said that the
samples that were hydrolyzed by pure papain gave out a bitter taste, which made it
less likeable. The score 2 meant that the panelist did not like the product. However,
the samples of crude papain had an average score 5 (like slightly) for its taste.
The comments from the panelists said that the hydrolysate from pure papain was
bitter, the color was more turbid, and the smell was less likeable. The hydrolysate
from crude papain on the other hand was salty, it was less turbid and the smell was
like smell of steamed shrimp. Some panelists said that the hydrolysate from crude
papain was too salty and suggested that sugar needs to be added as well to reduce
saltiness and improve the taste.
Bitterness from pure papain products was a result of hydrolysis. Hydrolysis cut down
long chain of protein to small peptides and amino acids. Some small peptides and
amino acids have bitter taste (Belitz, 2009), which cause the product to develop bitter
taste. In crude papain products these bitterness was masked by the additives of crude
papain, which are salt and sugar.
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CHAPTER 5 - CONCLUSIONS AND RECOMMENDATIONS
5.1. Conclusion
Both pure papain and crude papain can hydrolyze protein from shrimp head.
However, in terms of higher protein content, pure papain can hydrolyze better.
Concentration of enzyme did not affect the protein content, because the
protein content of 10, 20, and 30% enzyme concentration did not give any
significant difference. Then, it was true that the enzyme concentration at 10%
can hydrolyze as much protein as enzyme concentration 20% and 30%.
The temperature, on the other side, affected the protein content significantly.
There was significant difference between protein content of products
hydrolyzed at 45oC to 50oC, 55oC, and 60oC. However, there were no
significant differences between those three temperatures to the protein content.
The initial hypothesis that the optimum temperature was between 45-60oC
should be rejected because the optimum temperature was between 50-60oC.
In terms of sensory acceptance, there were four defining parameters. The level
of acceptance of appearance and color for both products were the same.
However, it was different in smell and taste parameters. It seemed that the
product hydrolyzed with crude papain was more likeable than the pure papain,
especially its taste. Therefore, it was true that crude papain produced product
with higher level of sensory acceptance.
5.2. Recommendation
The lowest enzyme concentration used in this enzyme was 10% and the result
did not give significant difference. Next time, the lower enzyme concentration
may be examined to see significant trend and to reduce cost of production.
Since protein concentrate in liquid form is unusual, drying process by freeze
or spray drying can be added to improve the palatability, diversity, and shelf
life of products.
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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 D.F. SS MS F PROB.> F Omega Squared Among Rows 1 19.033 19.033 21.468 0.000 0.016 Among Columns 2 85.178 42.589 48.037 0.000 0.073 Among Slices 3 493.557 164.519 185.562 0.000 0.428 A x B Inter. 2 70.242 35.121 39.613 0.000 0.060 A x C Inter. 3 205.270 68.423 77.175 0.000 0.177 B x C Inter. 6 119.224 19.871 22.412 0.000 0.099 AxBxC Inter. 6 131.396 21.899 24.700 0.000 0.110 Within Groups 24 21.278 0.887 Total 47 1145.179 24.366 Omega squared for combined effects = 0.963 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 72.297 0.040 0.199 Cell 1 1 2 2 73.540 0.067 0.259 Cell 1 1 3 2 74.270 0.000 0.015 Cell 1 1 4 2 68.683 2.204 1.485 Cell 1 2 1 2 72.451 0.031 0.177 Cell 1 2 2 2 70.271 16.892 4.110 Cell 1 2 3 2 83.108 0.169 0.411 Cell 1 2 4 2 70.290 0.003 0.058 Cell 1 3 1 2 71.994 0.071 0.267 Cell 1 3 2 2 73.212 0.001 0.038 Cell 1 3 3 2 84.105 0.158 0.398 Cell 1 3 4 2 83.754 0.643 0.802 Cell 2 1 1 2 77.165 0.000 0.001 Cell 2 1 2 2 71.069 0.039 0.198 Cell 2 1 3 2 77.580 0.104 0.322 Cell 2 1 4 2 67.816 0.227 0.476
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Cell 2 2 1 2 77.288 0.073 0.269 Cell 2 2 2 2 70.999 0.361 0.601 Cell 2 2 3 2 78.094 0.046 0.215 Cell 2 2 4 2 67.967 0.045 0.212 Cell 2 3 1 2 77.357 0.063 0.252 Cell 2 3 2 2 71.928 0.000 0.021 Cell 2 3 3 2 78.066 0.039 0.197 Cell 2 3 4 2 67.534 0.000 0.019 Row 1 24 74.831 30.281 5.503 Row 2 24 73.572 18.682 4.322 Col 1 16 72.803 12.131 3.483 Col 2 16 73.809 25.936 5.093 Col 3 16 75.994 32.600 5.710 Slice 1 12 74.759 6.928 2.632 Slice 2 12 71.837 3.131 1.770 Slice 3 12 79.204 12.550 3.543 Slice 4 12 71.007 36.628 6.052 TOTAL 48 74.202 24.366 4.936 TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 13532871.42 with deg.s freem: 6 and 1. Cochran C statistic = 0.79 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 141.58 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.006 q = 4.274 0.0157 YES 1 - 3 -3.191 q = 13.557 0.0000 YES --------------------------------------------------------------- 2 - 3 -2.185 q = 9.283 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 2.922 q = 10.751 0.0000 YES 1 - 3 -4.445 q = 16.354 0.0000 YES 1 - 4 3.751 q = 13.801 0.0000 YES --------------------------------------------------------------- 2 - 3 -7.367 q = 27.105 0.0000 YES 2 - 4 0.829 q = 3.050 0.1644 NO --------------------------------------------------------------- 3 - 4 8.196 q = 30.155 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS ---------------------------------------------------------------
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Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.607 q = 2.413 0.2234 NO 1 - 3 -15.071 q = 22.636 0.0000 YES --------------------------------------------------------------- 2 - 3 -13.465 q = 20.223 0.0000 YES --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.151 q = 0.226 0.9861 NO 1 - 3 0.282 q = 0.424 0.9517 NO --------------------------------------------------------------- 2 - 3 0.433 q = 0.651 0.8904 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.867 q = 1.302 0.3666 NO --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 2.323 q = 3.489 0.0212 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 16.221 q = 24.362 0.0001 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.123 q = 0.185 0.9907 NO
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1 - 3 -0.192 q = 0.288 0.9775 NO --------------------------------------------------------------- 2 - 3 -0.069 q = 0.104 0.9971 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.071 q = 0.106 0.9970 NO 1 - 3 -0.858 q = 1.289 0.6385 NO --------------------------------------------------------------- 2 - 3 -0.929 q = 1.396 0.5921 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.515 q = 0.773 0.8492 NO 1 - 3 -0.487 q = 0.731 0.8638 NO --------------------------------------------------------------- 2 - 3 0.028 q = 0.042 0.9996 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.151 q = 0.226 0.9861 NO 1 - 3 0.282 q = 0.424 0.9517 NO --------------------------------------------------------------- 2 - 3 0.433 q = 0.651 0.8904 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.123 q = 0.185 0.8972 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.071 q = 0.106 0.9406 NO ---------------------------------------------------------------
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SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.515 q = 0.773 0.5895 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.151 q = 0.226 0.8742 NO --------------------------------------------------------------- Appendix 8 Statistical analysis of water content WATER CONTENT ANALYSIS Three Way Analysis of Variance Variable analyzed: wc Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: concentration (Fixed Levels) Factor C (slices) variable: temperature (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 616.940 616.940 5954.305 0.000 0.753 Among Columns 2 99.313 49.656 479.252 0.000 0.121 Among Slices 3 6.596 2.199 21.219 0.000 0.008 A x B Inter. 2 84.015 42.008 405.431 0.000 0.102 A x C Inter. 3 4.560 1.520 14.669 0.000 0.005 B x C Inter. 6 1.680 0.280 2.702 0.038 0.001 AxBxC Inter. 6 3.366 0.561 5.415 0.001 0.003 Within Groups 24 2.487 0.104 Total 47 818.957 17.425 Omega squared for combined effects = 0.994 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 87.283 0.140 0.375
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Cell 1 1 2 2 88.283 0.140 0.375 Cell 1 1 3 2 87.311 0.007 0.082 Cell 1 1 4 2 88.712 0.008 0.090 Cell 1 2 1 2 84.212 0.052 0.229 Cell 1 2 2 2 84.219 0.511 0.715 Cell 1 2 3 2 83.941 0.156 0.394 Cell 1 2 4 2 83.634 0.733 0.856 Cell 1 3 1 2 81.902 0.087 0.294 Cell 1 3 2 2 81.465 0.043 0.208 Cell 1 3 3 2 80.719 0.149 0.386 Cell 1 3 4 2 80.569 0.259 0.509 Cell 2 1 1 2 92.829 0.005 0.073 Cell 2 1 2 2 91.525 0.016 0.127 Cell 2 1 3 2 91.324 0.025 0.158 Cell 2 1 4 2 90.832 0.009 0.093 Cell 2 2 1 2 92.557 0.001 0.035 Cell 2 2 2 2 91.315 0.137 0.370 Cell 2 2 3 2 91.203 0.002 0.049 Cell 2 2 4 2 91.390 0.001 0.030 Cell 2 3 1 2 92.457 0.003 0.051 Cell 2 3 2 2 90.900 0.001 0.035 Cell 2 3 3 2 91.171 0.000 0.009 Cell 2 3 4 2 90.790 0.001 0.030 Row 1 24 84.354 8.307 2.882 Row 2 24 91.524 0.476 0.690 Col 1 16 89.763 4.229 2.056 Col 2 16 87.809 15.760 3.970 Col 3 16 86.246 27.987 5.290 Slice 1 12 88.540 20.799 4.561 Slice 2 12 87.951 16.239 4.030 Slice 3 12 87.611 18.290 4.277 Slice 4 12 87.654 18.524 4.304 TOTAL 48 87.939 17.425 4.174 TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 10019.01 with deg.s freem: 6 and 1. Cochran C statistic = 0.29 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 73.47 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 1.954 q = 24.278 0.0000 YES 1 - 3 3.516 q = 43.694 0.0000 YES --------------------------------------------------------------- 2 - 3 1.562 q = 19.416 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant?
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 79 of 100
Judith Salim
--------------------------------------------------------------- 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
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 80 of 100
Judith Salim
alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -10.221 q = 44.905 0.0001 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.272 q = 1.195 0.6795 NO 1 - 3 0.372 q = 1.635 0.4901 NO --------------------------------------------------------------- 2 - 3 0.100 q = 0.441 0.9481 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.210 q = 0.922 0.7931 NO 1 - 3 0.626 q = 2.749 0.1482 NO --------------------------------------------------------------- 2 - 3 0.416 q = 1.827 0.4133 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.122 q = 0.534 0.9247 NO 1 - 3 0.154 q = 0.675 0.8827 NO --------------------------------------------------------------- 2 - 3 0.032 q = 0.141 0.9946 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.558 q = 2.453 0.2131 NO 1 - 3 0.042 q = 0.186 0.9906 NO --------------------------------------------------------------- 2 - 3 0.601 q = 2.639 0.1702 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH SLICE SLICE 1 COMPARISONS ---------------------------------------------------------------
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 81 of 100
Judith Salim
Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.272 q = 1.195 0.4065 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.210 q = 0.922 0.5205 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 0.122 q = 0.534 0.7089 NO --------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.558 q = 2.453 0.0957 NO --------------------------------------------------------------- Appendix 5 Statistical analysis of ash content ASH CONTENT Three Way Analysis of Variance Variable analyzed: ash Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 426.976 426.976 11459.905 0.000 0.778 Among Columns 2 60.163 30.081 807.372 0.000 0.110 Among Slices 3 0.240 0.080 2.143 0.121 0.000 A x B Inter. 2 59.875 29.937 803.512 0.000 0.109 A x C Inter. 3 0.083 0.028 0.741 0.538 0.000
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 82 of 100
Judith Salim
B x C Inter. 6 0.133 0.022 0.593 0.733 0.000 AxBxC Inter. 6 0.137 0.023 0.614 0.717 0.000 Within Groups 24 0.894 0.037 Total 47 548.500 11.670 Omega squared for combined effects = 0.997 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 3.713 0.001 0.032 Cell 1 1 2 2 3.795 0.003 0.057 Cell 1 1 3 2 3.749 0.000 0.022 Cell 1 1 4 2 3.602 0.003 0.054 Cell 1 2 1 2 6.223 0.408 0.639 Cell 1 2 2 2 6.466 0.182 0.426 Cell 1 2 3 2 6.738 0.005 0.070 Cell 1 2 4 2 6.570 0.042 0.204 Cell 1 3 1 2 8.921 0.036 0.190 Cell 1 3 2 2 9.254 0.161 0.401 Cell 1 3 3 2 9.209 0.046 0.214 Cell 1 3 4 2 9.386 0.006 0.079 Cell 2 1 1 2 0.457 0.000 0.014 Cell 2 1 2 2 0.459 0.000 0.003 Cell 2 1 3 2 0.544 0.000 0.002 Cell 2 1 4 2 0.545 0.000 0.016 Cell 2 2 1 2 0.460 0.000 0.003 Cell 2 2 2 2 0.465 0.000 0.011 Cell 2 2 3 2 0.541 0.000 0.001 Cell 2 2 4 2 0.545 0.000 0.003 Cell 2 3 1 2 0.459 0.000 0.008 Cell 2 3 2 2 0.473 0.000 0.005 Cell 2 3 3 2 0.551 0.000 0.001 Cell 2 3 4 2 0.547 0.000 0.005 Row 1 24 6.469 5.282 2.298 Row 2 24 0.504 0.002 0.043 Col 1 16 2.108 2.758 1.661 Col 2 16 3.501 9.651 3.107 Col 3 16 4.850 20.146 4.488 Slice 1 12 3.372 11.768 3.430 Slice 2 12 3.485 12.687 3.562 Slice 3 12 3.555 12.607 3.551 Slice 4 12 3.533 12.780 3.575 TOTAL 48 3.486 11.670 3.416 TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 411246.45 with deg.s freem: 6 and 1. Cochran C statistic = 0.46 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 176.65 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 83 of 100
Judith Salim
--------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.393 q = 28.874 0.0000 YES 1 - 3 -2.742 q = 56.826 0.0000 YES --------------------------------------------------------------- 2 - 3 -1.349 q = 27.953 0.0000 YES --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -2.968 q = 21.745 0.0000 YES 1 - 3 -5.785 q = 42.381 0.0000 YES --------------------------------------------------------------- 2 - 3 -2.817 q = 20.636 0.0000 YES --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.000 q = 0.002 1.0000 NO 1 - 3 -0.002 q = 0.017 0.9999 NO --------------------------------------------------------------- 2 - 3 -0.002 q = 0.015 0.9999 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 3.057 q = 22.399 0.0001 YES --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 6.025 q = 44.142 0.0001 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 84 of 100
Judith Salim
alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 8.839 q = 64.763 0.0001 YES --------------------------------------------------------------- Appendix 6 Statistical analysis of protein content PROTEIN CONTENT Three Way Analysis of Variance Variable analyzed: protein Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared Among Rows 1 38.401 38.401 174.560 0.000 0.618 Among Columns 2 0.354 0.177 0.806 0.458 0.000 Among Slices 3 15.075 5.025 22.842 0.000 0.233 A x B Inter. 2 0.199 0.100 0.453 0.641 0.000 A x C Inter. 3 0.236 0.079 0.358 0.784 0.000 B x C Inter. 6 0.936 0.156 0.709 0.645 0.000 AxBxC Inter. 6 1.095 0.182 0.830 0.559 0.000 Within Groups 24 5.280 0.220 Total 47 61.576 1.310 Omega squared for combined effects = 0.829 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 5.068 0.897 0.947 Cell 1 1 2 2 5.535 0.027 0.166 Cell 1 1 3 2 6.238 0.002 0.041 Cell 1 1 4 2 6.282 0.227 0.476 Cell 1 2 1 2 5.023 0.004 0.062 Cell 1 2 2 2 5.872 0.000 0.021 Cell 1 2 3 2 6.707 0.988 0.994 Cell 1 2 4 2 6.483 0.782 0.884 Cell 1 3 1 2 5.096 0.027 0.166 Cell 1 3 2 2 6.590 0.556 0.746 Cell 1 3 3 2 5.887 0.007 0.083 Cell 1 3 4 2 6.692 1.297 1.139 Cell 2 1 1 2 6.623 0.000 0.021 Cell 2 1 2 2 7.912 0.000 0.021
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 85 of 100
Judith Salim
Cell 2 1 3 2 7.947 0.006 0.075 Cell 2 1 4 2 8.420 0.155 0.393 Cell 2 2 1 2 6.736 0.002 0.041 Cell 2 2 2 2 8.098 0.072 0.269 Cell 2 2 3 2 7.816 0.026 0.162 Cell 2 2 4 2 7.951 0.011 0.104 Cell 2 3 1 2 6.941 0.043 0.207 Cell 2 3 2 2 7.981 0.124 0.352 Cell 2 3 3 2 7.946 0.015 0.122 Cell 2 3 4 2 8.566 0.011 0.104 Row 1 24 5.956 0.606 0.778 Row 2 24 7.745 0.402 0.634 Col 1 16 6.753 1.471 1.213 Col 2 16 6.836 1.215 1.102 Col 3 16 6.962 1.396 1.182 Slice 1 12 5.915 0.891 0.944 Slice 2 12 6.998 1.268 1.126 Slice 3 12 7.090 0.880 0.938 Slice 4 12 7.399 1.189 1.090 TOTAL 48 6.850 1.310 1.145 TESTS FOR HOMOGENEITY OF VARIANCE -------------------------------------------------------------------- Hartley Fmax test statistic = 3025.00 with deg.s freem: 6 and 1. Cochran C statistic = 0.25 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 88.50 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -1.083 q = 8.002 0.0000 YES 1 - 3 -1.176 q = 8.683 0.0000 YES 1 - 4 -1.485 q = 10.965 0.0000 YES --------------------------------------------------------------- 2 - 3 -0.092 q = 0.681 0.9625 NO 2 - 4 -0.401 q = 2.963 0.1832 NO --------------------------------------------------------------- 3 - 4 -0.309 q = 2.282 0.3903 NO --------------------------------------------------------------- Appendix 7 Statistical analysis of recovered protein Recovered Protein Three Way Analysis of Variance Variable analyzed: pro rec Factor A (rows) variable: type (Fixed Levels) Factor B (columns) variable: conc (Fixed Levels) Factor C (slices) variable: temp (Fixed Levels) SOURCE D.F. SS MS F PROB.> F Omega Squared
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 86 of 100
Judith Salim
Among Rows 1 4150.060 4150.060 149.061 0.000 0.279 Among Columns 2 240.376 120.188 4.317 0.025 0.013 Among Slices 3 4093.520 1364.507 49.010 0.000 0.272 A x B Inter. 2 247.672 123.836 4.448 0.023 0.013 A x C Inter. 3 4772.234 1590.745 57.136 0.000 0.318 B x C Inter. 6 357.881 59.647 2.142 0.085 0.013 AxBxC Inter. 6 206.034 34.339 1.233 0.324 0.003 Within Groups 24 668.193 27.841 Total 47 14735.970 313.531 Omega squared for combined effects = 0.909 Note: MSErr denominator for all F ratios. Descriptive Statistics GROUP N MEAN VARIANCE STD.DEV. Cell 1 1 1 2 52.103 1.077 1.038 Cell 1 1 2 2 114.302 11.704 3.421 Cell 1 1 3 2 75.231 0.249 0.499 Cell 1 1 4 2 73.314 30.899 5.559 Cell 1 2 1 2 63.492 0.617 0.785 Cell 1 2 2 2 104.787 0.137 0.370 Cell 1 2 3 2 77.666 132.510 11.511 Cell 1 2 4 2 73.685 101.066 10.053 Cell 1 3 1 2 55.831 3.295 1.815 Cell 1 3 2 2 108.781 151.467 12.307 Cell 1 3 3 2 66.371 0.872 0.934 Cell 1 3 4 2 75.506 165.153 12.851 Cell 2 1 1 2 87.217 0.074 0.273 Cell 2 1 2 2 89.020 0.300 0.547 Cell 2 1 3 2 90.706 7.324 2.706 Cell 2 1 4 2 97.043 0.065 0.254 Cell 2 2 1 2 96.749 10.346 3.216 Cell 2 2 2 2 96.502 18.122 4.257 Cell 2 2 3 2 102.899 0.937 0.968 Cell 2 2 4 2 106.882 4.915 2.217 Cell 2 3 1 2 104.492 2.584 1.608 Cell 2 3 2 2 99.318 21.541 4.641 Cell 2 3 3 2 93.365 1.478 1.216 Cell 2 3 4 2 100.036 1.462 1.209 Row 1 24 78.422 421.106 20.521 Row 2 24 97.019 39.151 6.257 Col 1 16 84.867 319.633 17.878 Col 2 16 90.333 268.303 16.380 Col 3 16 87.962 378.437 19.453 Slice 1 12 76.647 456.171 21.358 Slice 2 12 102.119 93.189 9.653 Slice 3 12 84.373 179.722 13.406 Slice 4 12 87.744 238.413 15.441 TOTAL 48 87.721 313.531 17.707
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 87 of 100
Judith Salim
TESTS FOR HOMOGENEITY OF VARIANCE --------------------------------------------------------------------- Hartley Fmax test statistic = 2559.89 with deg.s freem: 6 and 1. Cochran C statistic = 0.25 with deg.s freem: 6 and 1. Bartlett Chi-square statistic = 86.19 with 5 D.F. Prob. larger = 0.000 --------------------------------------------------------------------- COMPARISONS AMONG COLUMNS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -5.466 q = 4.143 0.0194 YES 1 - 3 -3.095 q = 2.346 0.2412 NO --------------------------------------------------------------- 2 - 3 2.370 q = 1.797 0.4250 NO --------------------------------------------------------------- COMPARISONS AMONG SLICES --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -25.471 q = 16.722 0.0000 YES 1 - 3 -7.726 q = 5.072 0.0076 YES 1 - 4 -11.097 q = 7.285 0.0001 YES --------------------------------------------------------------- 2 - 3 17.746 q = 11.650 0.0000 YES 2 - 4 14.374 q = 9.437 0.0000 YES --------------------------------------------------------------- 3 - 4 -3.371 q = 2.213 0.4166 NO --------------------------------------------------------------- COMPARISONS AMONG COLUMNS WITHIN EACH ROW ROW 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -0.371 q = 0.099 0.9973 NO 1 - 3 -2.193 q = 0.588 0.9096 NO --------------------------------------------------------------- 2 - 3 -1.822 q = 0.488 0.9366 NO --------------------------------------------------------------- ROW 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -9.838 q = 2.637 0.1707 NO 1 - 3 -2.993 q = 0.802 0.8387 NO
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 88 of 100
Judith Salim
--------------------------------------------------------------- 2 - 3 6.845 q = 1.835 0.4103 NO --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH COLUMN COLUMN 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -23.730 q = 6.360 0.0002 YES --------------------------------------------------------------- COLUMN 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -33.197 q = 8.898 0.0001 YES --------------------------------------------------------------- COLUMN 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -24.530 q = 6.575 0.0002 YES --------------------------------------------------------------- COMPARISONS AMONG ROWS WITHIN EACH SLICE SLICE 1 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -9.532 q = 2.555 0.0835 NO --------------------------------------------------------------- SLICE 2 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -7.482 q = 2.005 0.1692 NO --------------------------------------------------------------- SLICE 3 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -12.192 q = 3.268 0.0298 YES
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 89 of 100
Judith Salim
--------------------------------------------------------------- SLICE 4 COMPARISONS --------------------------------------------------------------- Tukey HSD Test for (Differences Between Means alpha selected = 0.05 Groups Difference Statistic Probability Significant? --------------------------------------------------------------- 1 - 2 -9.838 q = 2.637 0.0746 NO --------------------------------------------------------------- Appendix 8 Two-way ANOVA of appearance in hedonic test Anova: Two-Factor Without Replication
SUMMARY Count Sum Average Variance1 6 34 5.666667 0.2666672 6 24 4 03 6 32 5.333333 0.2666674 6 27 4.5 0.35 6 30 5 06 6 24 4 07 6 33 5.5 0.38 6 27 4.5 1.59 6 27 4.5 0.3
10 6 30 5 0.811 6 27 4.5 0.312 6 29 4.833333 0.56666713 6 33 5.5 0.3
PK3 13 63 4.846154 0.641026PM1 13 66 5.076923 0.910256PK1 13 62 4.769231 0.692308PM2 13 63 4.846154 0.641026PK2 13 63 4.846154 0.474359PM3 13 60 4.615385 0.423077
ANOVA Source of Variation SS df MS F P-value F crit
Rows 22.33333 12 1.861111 4.844271 1.66E-05 1.917396Columns 1.448718 5 0.289744 0.754171 0.586356 2.36827Error 23.05128 60 0.384188
Total 46.83333 77
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 90 of 100
Judith Salim
Appendix 9 Two-way ANOVA of Color in hedonic test Anova: Two-Factor Without Replication
SUMMARY Count Sum Average Variance 1 6 36 6 0.82 6 27 4.5 0.73 6 32 5.333333 0.2666674 6 27 4.5 0.35 6 22 3.666667 0.6666676 6 24 4 07 6 33 5.5 0.38 6 27 4.5 1.59 6 24 4 0
10 6 29 4.833333 0.56666711 6 27 4.5 0.312 6 23 3.833333 1.36666713 6 31 5.166667 0.166667
PK3 13 62 4.769231 0.692308PM1 13 64 4.923077 1.74359PK1 13 59 4.538462 0.602564PM2 13 60 4.615385 1.423077PK2 13 61 4.692308 0.397436PM3 13 56 4.307692 0.730769
ANOVA
Source of Variation SS df MS F P-value F crit
Rows 35.2820 12 2.940171 5.54838 2.85E-06 1.917396Columns 2.87179 5 0.574359 1.083871 0.378548 2.36827Error 31.7948 60 0.529915
Total 69.9487 77 Appendix 10 Two-way ANOVA of smell in hedonic test Anova: Two-Factor Without Replication
SUMMARY Count Sum Average Variance1 6 34 5.666667 0.2666672 6 26 4.333333 1.0666673 6 24 4 0.8
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Judith Salim
4 6 27 4.5 0.35 6 30 5 06 6 33 5.5 0.37 6 36 6 08 6 27 4.5 0.39 6 24 4 1.6
10 6 26 4.333333 1.46666711 6 28 4.666667 0.66666712 6 28 4.666667 1.06666713 6 32 5.333333 0.266667
PK3 13 68 5.230769 0.525641PM1 13 67 5.153846 0.641026PK1 13 64 4.923077 0.74359PM2 13 61 4.692308 0.897436PK2 13 62 4.769231 0.858974PM3 13 53 4.076923 1.24359
ANOVA Source of Variation SS df MS F P-value F crit
Rows 29.61538 12 2.467949 5.052493 9.79E-06 1.917396Columns 11.19231 5 2.238462 4.582677 0.001319 2.36827Error 29.30769 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 PM1 PK1
Mean 5.230769 4.923077 Mean 5.153846 4.923077
Variance 0.525641 0.74359 Variance 0.641026 0.74359
Observations 13 13 Observations 13 13
Pearson Correlation 0.164053Pearson Correlation 0.139272
Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 1.075466 t Stat 0.762001
P(T<=t) one‐tail 0.151657 P(T<=t) one‐tail 0.230387
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.303314 P(T<=t) two‐tail 0.460775
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 92 of 100
Judith Salim
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PM1 PK3 PM2 PK3
Mean 5.153846 5.230769 Mean 4.692308 5.230769
Variance 0.641026 0.525641 Variance 0.897436 0.525641
Observations 13 13 Observations 13 13
Pearson Correlation 0.220863Pearson Correlation 0.35466
Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐0.2907 t Stat ‐2.00684
P(T<=t) one‐tail 0.388122 P(T<=t) one‐tail 0.033918
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.776243 P(T<=t) two‐tail 0.067836
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PM3 PM1 PM3 PM2
Mean 4.076923 5.153846 Mean 4.076923 4.692308
Variance 1.24359 0.641026 Variance 1.24359 0.897436
Observations 13 13 Observations 13 13
Pearson Correlation 0.545648Pearson Correlation 0.655328
Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐4.06981 t Stat ‐2.55117
P(T<=t) one‐tail 0.000777 P(T<=t) one‐tail 0.012705
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.001554 P(T<=t) two‐tail 0.025411
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means
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Judith Salim
t‐Test: Paired Two Sample for Means
PM2 PK1 PK2 PK1
Mean 4.692308 4.923077 Mean 4.769231 4.923077
Variance 0.897436 0.74359 Variance 0.858974 0.74359
Observations 13 13 Observations 13 13
Pearson Correlation 0.172635 Pearson Correlation 0.705832 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐0.71375 t Stat ‐0.80539
P(T<=t) one‐tail 0.244517 P(T<=t) one‐tail 0.218132
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.489034 P(T<=t) two‐tail 0.436265
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PK2 PK3 PM2 PM1
Mean 4.769231 5.230769 Mean 4.692308 5.153846
Variance 0.858974 0.525641 Variance 0.897436 0.641026
Observations 13 13 Observations 13 13
Pearson Correlation 0.085858 Pearson Correlation 0.616963 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐1.4771 t Stat ‐2.14377
P(T<=t) one‐tail 0.082703 P(T<=t) one‐tail 0.026616
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.165407 P(T<=t) two‐tail 0.053232
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PK2 PM2 PK2 PM3
Mean 4.769231 4.692308 Mean 4.769231 4.076923
Variance 0.858974 0.897436 Variance 0.858974 1.24359
Observations 13 13 Observations 13 13
Pearson Correlation 0.386954 Pearson Correlation 0.502379 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 0.267261 t Stat 2.419798
P(T<=t) one‐tail 0.396903 P(T<=t) one‐tail 0.016165
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
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Judith Salim
P(T<=t) two‐tail 0.793806 P(T<=t) two‐tail 0.032329
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PM3 PK3 PM3 PK1
Mean 4.076923 5.230769 Mean 4.076923 4.923077
Variance 1.24359 0.525641 Variance 1.24359 0.74359
Observations 13 13 Observations 13 13
Pearson Correlation 0.491568 Pearson Correlation 0.52662 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐4.21464 t Stat ‐3.09073
P(T<=t) one‐tail 0.0006 P(T<=t) one‐tail 0.004675
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.0012 P(T<=t) two‐tail 0.00935
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means
PK2 PM1
Mean 4.769231 5.153846
Variance 0.858974 0.641026
Observations 13 13
Pearson Correlation 0.276438Hypothesized Mean Difference 0
df 12
t Stat ‐1.32842
P(T<=t) one‐tail 0.104375
t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.208749
t Critical two‐tail 2.178813
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Appendix 12 Two-way ANOVA of taste in hedonic test Anova: Two-Factor Without Replication
SUMMARY Count Sum Average Variance1 6 23 3.833333 1.7666672 6 23 3.833333 0.5666673 6 22 3.666667 0.6666674 6 24 4 1.25 6 15 2.5 1.16 6 19 3.166667 4.1666677 6 27 4.5 7.58 6 15 2.5 2.79 6 25 4.166667 0.966667
10 6 24 4 3.611 6 20 3.333333 1.86666712 6 21 3.5 2.713 6 22 3.666667 1.066667
PK3 13 67 5.153846 0.641026PM1 13 35 2.692308 0.897436PK1 13 56 4.307692 1.397436PM2 13 32 2.461538 0.935897PK2 13 60 4.615385 1.589744PM3 13 30 2.307692 0.730769
ANOVA Source of Variation SS df MS F P-value F crit
Rows 25.53846 12 2.128205 2.618297 0.007086 1.917396Columns 100.5641 5 20.11282 24.74448 1.95E-13 2.36827Error 48.76923 60 0.812821
Total 174.8718 77
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Appendix 13 t-test between samples for taste t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PK3 PM3 PM1 PM3
Mean 5.153846 2.307692 Mean 2.692308 2.307692
Variance 0.641026 0.730769 Variance 0.897436 0.730769
Observations 13 13 Observations 13 13
Pearson Correlation 0.046829 Pearson Correlation 0.435358 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 8.973818 t Stat 1.443376
P(T<=t) one‐tail 5.69E‐07 P(T<=t) one‐tail 0.087254
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 1.14E‐06 P(T<=t) two‐tail 0.174509
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PK2 PM3 PK3 PK2
Mean 4.615385 2.307692 Mean 5.153846 4.615385
Variance 1.589744 0.730769 Variance 0.641026 1.589744
Observations 13 13 Observations 13 13
Pearson Correlation 0.041631 Pearson Correlation 0.723901 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 5.57086 t Stat 2.213594
P(T<=t) one‐tail 6.08E‐05 P(T<=t) one‐tail 0.023488
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.000122 P(T<=t) two‐tail 0.046976
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PM2 PK2 PK3 PM2
Mean 2.461538 4.615385 Mean 5.153846 2.461538
Variance 0.935897 1.589744 Variance 0.641026 0.935897
Observations 13 13 Observations 13 13
Pearson Correlation ‐0.0473 Pearson Correlation 0.223454 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐4.77859 t Stat 8.75
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Judith Salim
P(T<=t) one‐tail 0.000225 P(T<=t) one‐tail 7.43E‐07
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.00045 P(T<=t) two‐tail 1.49E‐06
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PK3 PK1 PM1 PK1
Mean 5.153846 4.307692 Mean 2.692308 4.307692
Variance 0.641026 1.397436 Variance 0.897436 1.397436
Observations 13 13 Observations 13 13
Pearson Correlation 0.562147 Pearson Correlation ‐0.20607 Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 3.090733 t Stat ‐3.50813
P(T<=t) one‐tail 0.004675 P(T<=t) one‐tail 0.002158
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.00935 P(T<=t) two‐tail 0.004317
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PK1 PM3 PM2 PM3
Mean 4.307692 2.307692 Mean 2.461538 2.307692
Variance 1.397436 0.730769 Variance 0.935897 0.730769
Observations 13 13 Observations 13 13
Pearson Correlation ‐0.01903 Pearson Correlation 0.720865Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 4.898979 t Stat 0.805387
P(T<=t) one‐tail 0.000183 P(T<=t) one‐tail 0.218132
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.000367 P(T<=t) two‐tail 0.436265
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PM1 PK2 PK1 PK2
Mean 2.692308 4.615385 Mean 4.307692 4.615385
Variance 0.897436 1.589744 Variance 1.397436 1.589744
Observations 13 13 Observations 13 13
Pearson Correlation ‐0.3864 Pearson Correlation 0.756935
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Judith Salim
Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat ‐3.7547 t Stat ‐1.29777
P(T<=t) one‐tail 0.001374 P(T<=t) one‐tail 0.109379
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.002747 P(T<=t) two‐tail 0.218758
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means t‐Test: Paired Two Sample for Means
PM1 PM2 PK1 PM2
Mean 2.692308 2.461538 Mean 4.307692 2.461538
Variance 0.897436 0.935897 Variance 1.397436 0.935897
Observations 13 13 Observations 13 13
Pearson Correlation 0.622515 Pearson Correlation ‐0.13453Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 12 df 12
t Stat 1 t Stat 4.095937
P(T<=t) one‐tail 0.168525 P(T<=t) one‐tail 0.000742
t Critical one‐tail 1.782288 t Critical one‐tail 1.782288
P(T<=t) two‐tail 0.337049 P(T<=t) two‐tail 0.001483
t Critical two‐tail 2.178813 t Critical two‐tail 2.178813
t‐Test: Paired Two Sample for Means
PM1 PK3
Mean 2.692308 5.153846
Variance 0.897436 0.641026
Observations 13 13
Pearson Correlation 0.067612Hypothesized Mean Difference 0
df 12
t Stat ‐7.40656
P(T<=t) one‐tail 4.1E‐06
t Critical one‐tail 1.782288
P(T<=t) two‐tail 8.2E‐06
t Critical two‐tail 2.178813
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head 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)
2004 – 2007 SMA Labschool Kebayoran, Jakarta
2001 – 2004 SMP Labschool Kebayoran, Jakarta
Courses : English Course at EF, Jakarta
Piano Course at Yamaha
German Course at Goethe Institute, Jakarta
Work Experience : March 2010 – August 2010, Internship Program in
Kattendorfer Hof, Germany.
December 2008 – January 2009, Internship Program at
PT. Frisian Flag Indonesia, Jakarta.
September 2008 – November 2008, Internship Program
PT. Multi Bintang Indonesia, Tbk., Jakarta.
Seminars and Workshop : 2007, Robotics and Neuroprothesis in Theurapeutic
Science – Innovative Biomedical Engineering
Solutions to Improve Human Functioning, SGU.
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 100 of 100
Judith Salim
2008, Cross Transfer Effects on Muscular Training,
SGU.
2008, Bio – reaction Modelling, SGU.
2009, Management of Obesity, SGU.
2010, Plant Biotechnology, SGU.
Skills/Interests : Computer Ms. Office,
Language Indonesian (native speaker)
English (Intermediate)
German (Basics)
Hobbies Travelling, Cooking, Singing, Playing
piano, Swimming, and Jogging