International Journal of Design & Nature and Ecodynamics

Vol. 16, No. 4, August, 2021, pp. 445-450

Journal homepage: http://iieta.org/journals/ijdne

Effect of Different Papain Concentrations on the Properties of Chicken Skin Protein

Hydrolysates
Ni Made Puspawati1*, Sri Wahjuni1, Ni Kadek Inda Kusmaning Ayu1, Ahmad Fudholi2,3
1
Faculty of Mathematics and Natural Sciences, Udayana University, Jl. Raya Kampus Unud, Bukit Jimbaran, Badung, Bali
80361, Indonesia
2
Universiti Kebangsaan Malaysia, Bangi 43600, Selangor, Malaysia
3
Indonesian Institute of Sciences (LIPI), Bandung 40135, Indonesia

Corresponding Author Email: made_puspawati@unud.ac.id

https://doi.org/10.18280/ijdne.160411 ABSTRACT

Received: 23 May 2021 Chicken skin is a source of animal protein hydrolysate, which has a potential as an
Accepted: 10 August 2021 antioxidant. This study aimed to determine the effect of different concentrations of the
enzyme papain on the degree of hydrolysis, antioxidant capacity, percentage of reducing
Keywords: power (%RP) and amino acid composition of chicken skin protein hydrolysate.
antioxidant, amino acid, FRAP, protein Hydrolysis was carried out using papain with various concentrations (3%, 4% and 5%,
hydrolysate, chicken skin w/w protein substrate) at pH 7 and 50℃ for 6 h. The degree of hydrolysis was determined
by spectrophotometry. Antioxidant capacity and %RP was determined by ferric reducing
antioxidant power method and expressed in milligram of ascorbic acid equivalent per
gram of sample, and amino acid composition were determined using high-performance
liquid chromatography. The results showed that variations in papain concentration had a
significant effect (P<0.05) on the degree of hydrolysis, antioxidant capacity and %RP of
chicken skin hydrolysate. The papain concentration of 4% resulted in the optimum
protein hydrolysate with a degree of hydrolysis of 61.68%±0.64%, an antioxidant
capacity of 8.72±0.30 mg AAE/g sample and a %RP of 54.12%±1.78%. The protein
hydrolysates of the treated chicken skin showed a high content of amino acids, namely,
glycine, glutamic acid, proline, arginine, alanine, and aspartic acid.

1. INTRODUCTION and chicken liver protein hydrolysate [9]. Current explorations

on bioactive peptides are carried out on the by-products of fish
Oxidation reactions by free radicals are one of the main processing, such as skin, eyes and fish bones, and poultry
causes of pathogenesis and diseases in humans, such as cell processing, such as claws, liver, bones, skin and other parts
damage, which plays a role in the ageing process, Alzheimer's that are rich in protein [4, 5, 8].
disease, inflammation and cancer. The normal state of cellular Chicken skin is one of the waste from processing chicken
health is maintained through a balance between endogenous meat that has not been further process into added-value
antioxidants and oxidants. However, this balance can be products. Locally, chicken skin is consumed as fry crackers
disrupted by factors, such as age, environmental conditions which are considered a high-fat food though effort has been
and genetic susceptibility [1]. Human need uptake of natural done to utilize chicken skin as an alternative source of halal
dietary antioxidants such as vitamin C, E, polyphenol, a gelatin [12]. Several convenient chicken skin-based food
carotenoid which have been well-known, and also dietary products are currently being developed to meet the increasing
natural antioxidant peptides derived from food protein demand, reduced cost, and protect the environment against
hydrolysis [2]. Recent studies have shown that peptides with waste and pollution. However, research on the development of
antioxidant properties can be released from food sources such chicken-based food with functional and health-promoting
as milk, whey protein, soy protein, egg, meat/fish [3-5], and values is still unexplored [13]. Chicken skin with high protein
meat/fish processing waste such as bone, skin, blood, eye, content is a good source for protein hydrolysates. It has been
head, and liver by process hydrolysis [6-9]. Therefore, natural reported that protein hydrolysates obtained from enzymatic
antioxidant peptides derived from protein hydrolysis are now hydrolysis of chicken skin using Alcalase and Gastrointestinal
being studied as novel sources of potential dietary ingredients enzymes exhibited antioxidant and anti-hypertensive activity
to promote human health. [14, 15]. The properties of peptides in the protein hydrolysates
Peptides with functional properties can be produced by depend on the nature of the substrate, the enzyme used for
enzymatic hydrolysis using protease enzymes, and this method hydrolysis, and the process of hydrolysis.
is more effective compared with chemical methods, such as The antioxidant activity of protein hydrolysates is
the use acids and bases. Papain is a protease enzyme that is influenced by the degree of hydrolysis and amino acid
commonly used for protein hydrolysis, such as the production composition. The degree of hydrolysis is an indicator of the
of protein hydrolysate from the bones and heads of catfish [10], success of the hydrolysis process and is influenced by the
tuna fish eyes [7], chicken claws [8], white snapper offal [11] characteristics of the substrate, the type and concentration of

445
enzymes, hydrolysis time, temperature and pH [16-18]. Epi et nitrogen was determined using Eq. (1):
al. [9] reported that the optimum enzyme concentration of
chicken liver protein hydrolysate is 6% with a degree of 𝑁 (%)
hydrolysis of 68.28% and an antioxidant activity of 876.913 =
( sample − HCl blanko)ml × N HCl × 14,007 × 100% (1)
weight of sample (mg)
ppm. According to He et al. [19], the hydrophobic properties
and electron transfer ability of aspartate and glutamate are able
2.1 Hydrolysis of chicken skin protein with papain
to provide antioxidant properties.
The measurement of antioxidant capacity by
The materials used in this study were gambier granules. Ten
spectrophotometric methods has been developed, but none is
grams of fat-free chicken skin powder was mixed with distilled
ideal. According to Niken [20], the measurement of
water to obtain 5% w/v of the total protein. The condition of
antioxidant capacity using cupric reducing antioxidant
the mixture of chicken skin powder with distilled water was
capacity, and ferric reducing antioxidant power (FRAP)
adjusted to the active conditions of the papain: pH 7 (pH
showed statistically different results. FRAP has advantages
adjusted using 2 M NaOH) and a temperature of 50℃. The
over other methods because FRAP reagents are easy to prepare,
pre-hydrolysis process was carried out using a thermostat
and the process is quite simple and can determine the ability
magnetic stirrer at a speed of 4.5 m/s for 20 min. Different of
of antioxidant compounds to reduce Fe3+ ions to Fe2+
papain concentrations (3%, 4% and 5% w/w protein substrate)
complexes [21]. Based on the above background, this study
were added at the same quantity in each treatment. The
was conducted to determine the amino acid composition and
hydrolysis process was carried out using a thermostat stirrer
the effect of enzyme (papain) concentration on the degree of
with a speed of 4.5 m/s at 50℃ and pH 7 for 6 h after
hydrolysis, antioxidant capacity and the percentage of
incubation, and the reaction was stopped by heating at 90℃
reducing power (%RP) of chicken skin protein hydrolysate
for 15 min. The hydrolysed protein was centrifuged, and the
produced through enzymatic hydrolysis using the FRAP
supernatant was collected and stored in a dark glass bottle at
method.
−15℃. The protein hydrolysate was dried using a freeze dryer.

2. MATERIALS AND METHODS 2.2 Determination of degree of hydrolysis

The chicken skins used were purchased randomly at the The standard solution of leucine (5 mg/mL) was prepared in
Ciomas Adisatwa Chicken Slaughterhouse, Tabanan Regency, various concentrations (1, 2, 3 and 4 mg/mL). Then, 0.6 mL of
Bali. The other materials used include n-hexane, aquades, 0.35% ninhydrin was added to 3 mL of each standard leucine
NaOH, K2SO4, MgO, H2SO4, H3BO3, 0.2% methyl red, 0.2% solution. Two millilitres of the protein hydrolysate sample to
methylene blue, 95% alcohol, ninhydrin, leucine, papain be tested and 2 mL of blank solution were separately added
(activity, 100000 U/g), aluminium foil, HCl, acetonitrile, 0.2 with 0.4 mL of ninhydrin and heated at 85℃ for 5 min. The
M phosphate buffer, K2HPO4, KH2PO4, 1% K3Fe(CN)6, 0.1% absorbance of the standard solution and protein hydrolysate
FeCl3, 10% trichloroacetic acid (TCA), ascorbic acid and samples were read at a wavelength of 570 nm using the UV–
demineralised aqua. Vis spectrophotometer. A calibration curve was made by
The equipment used include beakers, measuring cups, plotting the absorbance value versus the standard amino acid
analytical scale (Kern Alj), cutters, freeze dryer (Labconco), concentration. The degree of hydrolysis can be calculated
blenders, Soxhlet apparatus, oven (Memmert), thermostat using Eq. (2):
magnetic stirrer (DF-101S), vortex mixer (Labnet), dark glass
bottles, volume pipettes, volumetric flasks, stirrers, dropper ℎ
𝐷𝐻 = × 100% (2)
pipette, hot plate (MS 79-1), centrifuge (Kubota), centrifuge ℎ 𝑡𝑜𝑡𝑎𝑙
tube, pH metre (Activon Model 209 pH/mV metre), Kjeldahl
flask, UV–Vis spectrophotometer (Shimadzhu UV-1800) and where, h is the total number of peptide bonds (obtained from
high-performance liquid chromatograph. reconstitution of protein samples (w/v). h is the number of
Chicken skins were cleaned with water, cut into small amino groups released (determined by plugging the
pieces and dried in a freeze dryer for 2 days. The dried chicken absorbance of the sample into the equation obtained on the
skins were then blended. The chicken skin powder was standard curve).
extracted by Soxhlet extraction using n-hexane solvent for 6 h
to remove the fat. The chicken skin residue was then heated in 2.3 Measurement of antioxidant capacity using the FRAP
an oven at 40 °C for 4 h to remove any residual solvent. method
The protein contents of the fat-free chicken skin samples
were determined using the Kjeldahl digestion method. A 0.2 g The measurement of antioxidant capacity using the FRAP
sample was placed into a kjeldahl flask and added with 1.9 g method was based on the steps of Vijayalakshmi and
K2SO4, 40 mg MgO and 2.5 mL H2SO4. The solution was Ruckmani [22] with modification. The following processes
boiled until clear. The clear solution was transferred to a were used to manufacture the reagents: (i) phosphate buffer
distillation device. The kjeldahl flask was washed with 2 mL solution (0.2 M, pH 6.6): 2.7216 g KH2PO4 and 3.4834 g
of water, and the washing water was placed into a distillation K2HPO4 were dissolved with demineralised aqua to 100 mL in
device and added with 8 mL of 40% NaOH solution. An a volumetric flask, then 64.52 mL of 0.2 M KH 2PO4 solution
Erlenmeyer flask with 5 mL of H3BO3 solution and 2–4 drops and 35.48 mL of 0.2 M K2HPO4 solution were collected, and
of the indicator (a mixture of 2 parts 0.2% methyl red and 1 the pH of the solution was adjusted to 6.6 by measuring with
part 0.2% methylene blue in alcohol) was placed in the a pH metre; (ii) 1% potassium ferricyanide (K3Fe[CN]6)
condenser. The contents of the Erlenmeyer flask were diluted solution: 1 g K3Fe(CN)6 was weighed and dissolved with
to 50 mL and titrated with 0.02 N HCl until the colour changed demineralised aqua to 100 mL in a volumetric flask; (iii) 0.1%
to grey. The same process was carried out for blanks. Total ferric chloride (FeCl3) solution: 0.1 g FeCl3 was weighed and

446
dissolved with demineralised aqua to 100 mL in a volumetric where, %RP is the reduction power of Fe3+ to Fe2+, As is
flask; (iv) 10% TCA solution: 10 g TCA was weighed and absorbance of the sample [highest], and Ac is the highest
dissolved with demineralised aqua to 100 mL in a volumetric standard absorbance
flask.
A standard solution of ascorbic acid was prepared by 2.6 Amino acid composition analysis
dissolving 10 mg ascorbic acid with demineralised aqua to 10
mL in a volumetric flask to obtain a concentration of 1000 ppm. The amino acid composition of the peptide fraction was
Then, 50, 500, 1250 and 2500 μL of the stock solution were analysed by hydrolysing the sample using 6 N HCl. The results
collected using pipettes and dissolved with demineralised aqua of the hydrolysis were derivatised using the AccQ•Fluor
to 5 mL in volumetric flasks to obtain the concentrations of 10, Reagent Kit. Five millilitres of the derivatised sample were
100, 250 and 500 ppm. Two millilitres of each solution with injected into the HPLC system (Alliance) using two mobile
various concentrations were collected using a pipette, then phases (buffer A and acetonitrile), an AccQ-Tag Waters
added with 2 mL of phosphate buffer (0.2 M, pH 6.6) and 2 column and a fluorescence detector. The HPLC measurement
mL of 1% K3Fe(CN)6 and mixed in a vortex mixer. conditions are: temperature, 37°C; column length, 250–395
Subsequently, the solutions were incubated for 20 min at 50°C, nm; mobile phase flow rate, 1.05 mL/min.
then added with 2 mL of 10% TCA solution and centrifuged The measurement data were analysed using SPSS. One-way
at a rate of 9 m/s for 10 min. Two millilitres of the top solution ANOVA was used to determine the significance of the effect
were pipetted and added with 2 mL of demineralised aqua and of each variation in papain concentration on the degree of
0.4 mL of 0.1% FeCl3. Finally, the absorbance was measured, hydrolysis, antioxidant capacity and %RP of the hydrolysates.
and the linear regression equation was determined. P<0.05 was considered a significant effect. Post hoc test (least
Sample solutions were prepared by dissolving 50 mg of significant difference) was further carried out to see the
each sample with demineralised aqua to 5 mL in a volumetric magnitude of the difference or effect in each variation.
flask to obtain a stock concentration of 10000 ppm. Different
volumes of the stock solution (250, 500, 1000 and 2000 μL)
were pipetted and dissolved with demineralised aqua to 5 mL 3. RESULT AND DISCUSSION
in a volumetric flask to obtain the concentrations of 250, 500,
1000 and 2000 ppm, respectively. Two millilitres of each The defatted chicken skin samples were analyzed to
sample were added with 2 mL of phosphate buffer 0.2 M (pH determine the chemical composition such as protein, moisture
6.6) and 2 mL of 1% K3Fe(CN)6 and then vortexed. (water), fat, and ash before enzymatic hydrolysis. The result
Subsequently, the solutions were incubated for 20 min at 50°C, of the proximate analysis are shown in Table 1.
then added with 2 mL of 10% TCA solution and centrifuged
at a rate of 9 m/s for 10 min. Two millilitres of the top solution Table 1. Proximate composition of fat-free dry chicken skin
were pipetted added with 2 mL of demineralised aqua and 0.4
mL of 0.1% FeCl3. Finally, the absorbance of the sample Parameter (%w/w) Value (%)
solutions was measured at 700 nm. Water 13.16
Protein 68.27
2.4 Determination of antioxidant capacity Fat 5.10
Ash 7.11
Antioxidant capacity is expressed as the weight equivalent
to ascorbic acid per gram of protein hydrolysate and Table 1 shows the moisture and ash content of defatted
determined by Eq. (3) [23]: chicken skin samples was 13.16% and 7.11% respectively,
higher than reported by Puspawati [24], 7.84% and 2.56%, and
𝑚𝑔𝐴𝐴 𝑉(𝑚𝐿) × 𝐹𝑝 × 𝐶 × 10−3 Onuh [15] which were 5.84 and 4.06% for thigh skin 6.64 and
AA ( )= (3)
𝑔 𝑔 𝑠𝑎𝑚𝑝𝑙𝑒 2.43 for breast skin). The fat content was 5.10%, lower than of
6.45% [24], 25.84% and 25.71% for thigh skin, and 29.21 %
where, AA is the ascorbic acid equivalent, V is the volume of for breast skin [15]. This result indicating that the fat content
the sample (mL), mgAA is the mass of ascorbic acid (mg), C of chicken skin sample in this experiment has been reduced
is the sample concentration (mg/L), Fp is the dilution factor, effectively.
and g is weight of the sample used (g). C can be calculated The protein content of the defatted chicken skin sample was
from the standard linear regression equation for ascorbic acid 68.27% which is considered high, though lower than the
as shown in Eq. (4): protein content of defatted chicken skin samples reported by
Puspawati et al. [24] (75.48%) but comparable to the protein
𝑦 = 𝑏𝑥 + 𝑎 (4)
content of the defatted chicken skin sample reported by Onuh
where, y is the absorbance of the sample, b is the regression [15] which was 68.84% for chicken thigh and 65.05% for
coefficient or slope, a is the intercept, r is the correlation breast skin. The difference in the chemical composition of the
coefficient, x is the value of the sample's antioxidant defatted chicken skin sample is may due to the age difference,
concentration or content. the fed of the chicken or cultivar, and the methods used for the
preparation of the samples. The high protein content of the
2.5 Determination of %RP sample is a good source of protein for producing protein
hydrolysates.
%RP was determined by comparing the sample and the Indicators of the occurrence of hydrolysis and results are
standard using Eq. (5): described by general parameters, such as the degree of
hydrolysis. The degree of hydrolysis indicates the percentage
𝐴𝑠 of peptide bonds released in the form of dissolved proteins or
%𝑅𝑃 = [1 − (1 − )] × 100 (5) amino acids. The degree of hydrolysis is influenced by the
𝐴𝑐

447
characteristics of the substrate, the type and concentration of the protein hydrolysate samples at various enzyme
enzymes, pH, temperature and time used in the hydrolysis concentrations was obtained by comparing the absorbance at
process. The hydrolysis degree of chicken skin protein was the highest concentration of the sample and that of ascorbic
determined using a spectrophotometric method. The samples acid. %RP is shown in Table 4.
were reacted with ninhydrin before the analysis. The
qualitative test results of the samples with ninhydrin show a
purple colour with a strong intensity, which indicates the
presence of amino acids in the samples.
Ninhydrin causes the oxidative decarboxylation of α-amino
acids to give CO2, NH3 and aldehydes. Reduced ninhydrin
(hydrindantin) reacts with NH3 and ninhydrin to form complex
blue-purple compounds [25]. The difference in amino acid
levels in the protein hydrolysate sample at 3%, 4% and 5%
papain concentrations (w/w protein substrate) was determined
by spectrophotometery UV-Vis at a wavelength of 570 nm.
The degrees of hydrolysis determined by the
spectrophotometric method using the leucine standard are
Figure 1. Graph of absorbance versus concentration
shown in Table 2.

Table 2. Degree of hydrolysis of chicken skin protein Table 3. Antioxidant capacity of chicken skin protein
hydrolysis hydrolysate

Protein hydrolyzate Antioxidant capacity

Enzyme concentration (% w/w) Hydrolysis Degree (%)
(mg AAE/g sample)
3 42.70 ±2.83a
Enzyme concentration 3% w/w 5.82 ±0.27a
4 6168 ±0.64b
Enzyme concentration 4% w/w 8.72 ±0.30b
5 73.43 ±1.95c
Enzyme concentration 5% w/w 8.52 ±0.15b
Note: Numbers accompanied by different letters indicate a significantly
The degree of hydrolysis increased with the increase in different value at 5% level.
papain concentration, because the high concentration of the
enzyme on the substrate can increase the speed of the Table 4. %RP of chicken skin protein hydrolysate samples
hydrolysis reaction hence more peptide bond cleaved result in
the increased production of dissolved protein. Epi et al. [9] Protein hydrolyzate Percentage reduction power
produced a similar degree of hydrolysis (68.82%) in chicken (%)
liver protein hydrolysate. Ratih [26] reported that the Enzyme concentration 3% 36.96 ±1.58a
hydrolysis of the same substrate with the same enzyme and w/w
enzyme concentration results in different degrees of hydrolysis Enzyme concentration 4% 54.12 ±1.78b
because of differences in the protein content and type of the w/w
Enzyme concentration 5% 52.88 ±0.89b
substrate, the activity of the enzymes used and the presence of
w/w
inhibitors that inhibit the reaction between the enzyme and the Note: Numbers accompanied by different letters indicate a significantly
substrate. different value at 5% level.
The antioxidant capacity of chicken skin protein
hydrolysate at various papain concentrations (3%, 4% and 5% The results of the antioxidant capacity and %RP of the
w/w protein substrate) was determined by the FRAP method protein hydrolysate samples at different papain concentrations
using ascorbic acid standards, because various free radicals (3%, 4% and 5% w/w protein substrate) showed the highest
can be suppressed by ascorbic acid. The FRAP method can antioxidant capacity and %RP at 4% enzyme concentration.
determine the ability of the sample to reduce Fe 3+ to Fe2+. Different papain concentrations resulted in different
Increasing the sample concentration can increase the antioxidant capacity and %RP, which were influenced not only
absorbance, as well as antioxidant capacity and reduction by the degree of hydrolysis produced but also the amino acid
power, but the increase is not constant because of differences composition. Antioxidant capacity and %RP decreased at 5%
in the antioxidant content of the sample. The curves of the enzyme concentration (w/w protein substrate), but the
concentration variation with the absorbance of the protein decrease was not substantial. This may because of the
hydrolysate samples at the enzyme concentrations of 3%, 4% dissolved peptides or amino acids formed from hydrolysis did
and 5% (w/w protein substrate) are shown in Figure 1. not donate many electrons. The optimum concentration of
The concentration and absorbance curves of the protein papain in producing the highest capacity and %RP was 4%.
hydrolysate samples show that the absorbance increases with Research on catfish protein hydrolysate by Baehaki [27]
increasing sample concentration and enzyme concentration. reported the same results, that is, antioxidant activity initially
The highest absorbance value was obtained in the samples increases along with the increase in enzyme concentration and
hydrolysed with 4% papain (w/w protein substrate). The decreases after the optimum enzyme concentration is reached
antioxidant capacity of chicken skin protein hydrolysate at a even when the degree of hydrolysis is increased. Barlett and
sample concentration of 2000 ppm is presented in Table 3. Levine [28] stated that the degree of hydrolysis, amino acid
Based on Table 3, the antioxidant capacity of the sample is composition and sequence, peptide size and solubility will
expressed as mg AA/ g of sample, which means that the affect antioxidant capacity and %RP. Besides, the FRAP
amount of ascorbic acid that is equivalent to the mass of the method is sensitive to pH and incubation time; accurate results
sample in reducing Fe3+ to Fe2+. The %RP of Fe3+ to Fe2+ by

448
will only be generated on samples that have thermodynamic [31] also reported that lower reducing power activity of
reaction conditions and a fairly fast reaction rate [28]. Phaseolin and Bean protein hydrolysates after fractionation
Amino acid composition has a major effect on the due to low amount of Tyr, Cys, His, Met, Lys, and Trp residue.
bioactivity of protein hydrolysates [19]. This study only Tjay et al. [32] and Lagowski [33] states that Ser and Lys
analysed 15 amino acids, namely Gly, Glu, Pro, Arg, Ala, Asp, contain O and N atoms, which have lone pairs and can form
Leu, Lys, Ser, Val, Thr, Phe, Ile, Tyr, And His, using the complexes with Fe to reduce Fe3+ to Fe2+. The unremarkable
HPLC method. The results are presented in Table 5. The difference in amino acid levels between the protein
amino acid composition of the protein and peptides has a great hydrolysate sample and the negative control indicated that the
influence on functionality. Due to some limitations, in this dry, fat-free chicken skin that was not hydrolysed contained
stage of the research, three amino acids i.e methionine, high levels of amino acids and has a potential as an antioxidant.
cysteine, and tryptophan have not been analyzed. The amino
acid composition of protein hydrolysates produced using 3, 4,
and 5% of papain showed a similar profile which is rich in 4. CONCLUSIONS
hydrophobic amino acids residue and poor in tyrosine and
histidine residue as summarised in Table 3. Different papain concentrations (3%, 4% and 5% w/w
protein substrate) had different effects (P<0.05) on the degree
Table 5. Amino acid composition of chicken skin protein of hydrolysis, antioxidant capacity and %RP, but antioxidant
hydrolysate capacity and %RP between 4% and 5% papain concentrations
were not significantly different (P>0.05). The papain
Amino Level (%) concentration of 4% produced optimum protein hydrolysate
acid 3% 4% 5% (-) with a degree of hydrolysis of 61.68%±0.64%, an antioxidant
Gly 16.02 15.37 14.98 13.26 capacity of 8.72±0.30 mg AA/g sample and a %RP of
Glu 9.85 10.33 10.29 9.49 54.12%±1.78%. The protein hydrolysates of the treated
Pro 9.12 8.79 8.59 7.79 chicken skin showed a high content of amino acids, namely,
Arg 6.74 6.88 6.67 6.25 glycine, glutamic acid, proline, arginine, alanine, and aspartic
Ala 6.73 6.65 6.57 6.11 acid.
Asp 5.13 5.65 5.59 5.46
Leu 4.13 4.45 4.21 4.54
Lys 4.18 4.31 4.25 4.27
Ser 3.18 3.29 3.18 3.02 ACKNOWLEDGMENT
Val 2.84 3.04 2.91 3.24
Thr 2.74 2.93 2.82 2.74 This work was funded by Udayana University through
Phe 2.31 2.63 2.47 2.70 Udayana Flagship Research Grant, year 2021.
Ile 2.07 2.29 2.16 2.37
Tyr 1.30 1.53 1.45 1.58
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