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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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
2.4. Protein hydrolysis ........................................................................................ 27 2.4.1. Chemical hydrolysis ............................................................................... 27
3.1. Time and Venue .......................................................................................... 35 3.2. Materials ...................................................................................................... 35
3.2.1. Raw Materials ........................................................................................ 35
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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
LIST OF REFERENCES ............................................................................................. 67 APPENDICES ............................................................................................................. 72 CURRICULUM VITAE .............................................................................................. 99
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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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Judith Salim
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
Production of Protein Concentrate by Enzymatic Hydrolysis of Shrimp (L.vannamei) Head Page 78 of 100
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
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