IDENTIFYING BIOACTIVE PEPTIDES FROM POULTRY BY-PRODUCTS
Abstract and keywords
Abstract (English):
Bioactive peptides derived from food proteins are becoming increasingly popular ingredients due to their beneficial effect on the immune system and other functional properties. We aimed to develop a technology for obtaining peptides from poultry by-products and identify their bioactivity. Pepsin was the main reagent for the in vitro enzymatic hydrolysis. Specialized equipment and methods were used to determine the key indicators. The molecular weight and bioactivity of the resulting peptides were calculated by using the Peptide Mass Calculator and PeptideRanker online resources. First, we developed a flow chart for obtaining bioactive peptides and produced hydrolysates from poultry by-products. The hydrolysates had identical physicochemical parameters, with no significant differences. The molecular weight distribution revealed that most protein fractions were represented by peptides with a molecular weight below 20 kDa. Then, we evaluated the bioactivity of the peptides. The hydrolysate obtained using pepsin with an activity of 30 units per 100 g of material showed higher bioactivity in the FD peptides (0.922094). The hydrolysate obtained using pepsin with an activity of 45 units per 100 g of material had greater bioactive properties in the CYG p eptides (0.947378). Based on the results, we designed a flow chart for obtaining hydrolysates from poultry by-products and evaluated the bioactive properties of the peptides obtained. For further work, these properties should be confirmed by in vitro experiments to determine the reliability of our data and identify specific bioactive properties of the peptides.

Keywords:
Peptides, hydrolysis, hydrolysates, waste-free technologies, in vitro, bioactive properties
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Introduction
Meat is a source of complete protein in the human
diet. Meat proteins have a high nutritional value
and therefore contribute to the normal physiological
status. They are converted into various forms
during cooking, processing, and digestion. For
example, bioactive peptides obtained from meat
proteinsthrough an enzymatic reaction can help
maintain the immune status of the human body. Not only
muscle meat is a good source of protein and bioactive
peptides, but offal is as well. Therefore, meat by-products
have increasingly been studied and used to produce
functional ingredients and bioactive peptides [1–6].
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Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554
Consumers are becoming increasingly aware of
the immune-boosting properties of bioactive peptides
derived from dietary proteins. Bioactive peptides are
small fragments of dietary proteins, mostly consisting
of 2–20 amino acid residues. They can be ligands
and therefore have many targets in the human body,
such as the immune, cardiovascular, digestive, and
endocrine systems [7–11].
Hydrolysis is the main process of isolating peptides.
Many studies confirm that the proteins of meat byproducts
are hydrolyzed by proteolytic enzymes
under controlled conditions, enabling bioactive peptides
to form. The main proteolytic enzymes that can
be used to hydrolyze proteins are pepsin, trypsin,
chymotrypsin, corolase, papain, as well as enzymes
of microbial origin, such as Neutrase from Bacillus
amyloliquefaciens and Alcalase from Bacillus licheniformis
[12–15].
The efficacy and safety of bioactive peptides must
be proven by clinical studies on living organisms before
they can be approved for use by regulatory and
supervisory authorities [16–18].
Some food-derived peptides have antioxidant,
immunomodulatory, antihypertensive, anticancerous,
anti-inflammatory, antimicrobial, hypocholesterolemic,
intestine-modulatory, antidiabetic, opioid, and metalchelating
properties [19–23]. Their biological activity
mainly depends on their amino acid composition,
sequence, length, and charge [24, 25].
Currently, hundreds of peptides with different
biological action have been identified and isolated from
various food sources, including milk, whey, eggs, fish,
rice, soybeans, peanuts, chickpeas, corn, and algae [24].
However, only a few of them are marketed as functional
nutraceutical products. For example, bioactive peptides
derived from milk and fish are more commonly used
as food ingredients than peptides from other food
sources. Table 1 presents some bioactive peptides
obtained from various protein sources and lists their
properties [26].
Antioxidant peptides usually contain hydrophobic
amino acids and residues of histidine, phenylalanine,
tryptophan, or tyrosine.
Thus, the food industry needs to develop technologies
for isolating bioactive peptides from by-products,
including meat by-products.
We aimed to develop a technology for obtaining
peptides from poultry by-products and study the
bioactivity of some peptide sequences.
To achieve this aim, we set to:
– select enzymes and develop a scheme for hydrolyzing
a homogeneous mass of chicken skin;
– develop two alternative schemes for producing bioactive
peptides through the hydrolysis of poultry by-products;
– conduct an electron microscopy of the obtained
hydrolysates;
– determine the main physicochemical parameters of
the hydrolysates; and
– perform a comparative analysis of the peptides.
Study objects and methods
We studied broiler chicken skin in a homogeneous
state. First, we selected proteolytic enzymes and
hydrolysis conditions that could increase the bioactivity
and yield of hydrolysates. In particular, we chose a
Table 1. Some bioactive peptides and their properties
Таблица 1. Некоторые биоактивные пептиды и их свойства
Protein source Peptide sequence Bioactive properties
Cow’s milk whey lactoferrin Glu-Asn-Leu-Pro-Glu-Lys-Ala-Asp-Arg-Asp-Gln-Tyr-Glu-Leu Osteoblast-proliferating
Beans (Phaseolus vulgari) Gly-Leu-Thr-Ser-Lys, Leu-Ser-Gly-Asn-Lys, Gly-Glu-Gly-Ser-Gly-Ala,
Met-Pro-Ala-Cys-Gly-Ser-Ser, and Met-Thr-Glu-Glu-Tyr
Anticancerous
Soy Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg Immunomodulatory
Chicken egg
lysozyme
Val-Ala-Trp-Arg-Asn-Arg-Cys-Lys-Gly-Thr-Asp, Trp-Arg-Asn-Arg-Cys-
Lys-Gly-Thr-Asp, Ala-Trp-Ile-Arg-Gly-Cys-Arg-Leu, Trp-Ile-Arg-Gly-
Cys-Arg-Leu, and Ile-Arg-Gly-Cys-Arg-Leu
Antioxidant
Egg white Ala-Glu-Glu-Arg-Tyr-Pro, Asp-Glu-Asp-Thr-Gln-Ala-Met-Pro, Pro-Val-
Asp-Glu-Asn-Asp-Glu-Gly, Gln-Pro-Ser-Ser-Val-Asp-Ser-Gln-Thr-Ala-
Met, and Glu-Glu-Arg-Tyr-Pro
Antioxidant
Casein Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu-Gln-Tyr Antihypertensive
Egg yolk Ile-Arg-Trp and Ile-Gln-Trp Antidiabetic
Tuna Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-
Try-Lys-Asp-Thr-Pro
Antihypertensive
Freshwater clam
(Corbicula fluminea)
Val-Lys-Pro and Val-Lys-Lys Hypocholesterolemic
Soy Tyr-Val-Val-Asn-Pro-Asp-Asn-Asp-Glu-Asn and Tyr-Val-Val-Asn-Pro-
Asp-Asn-Asn-Glu-Asn
Hypocholesterolemic
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Voroshilin R.A. et al. Food Processing: Techniques and Technology. 2022;52(3):545–554
pepsin produced by Meito Sangyo Co. (Japan) for our
experiments as a potentially more suitable enzyme for
obtaining peptides from poultry by-products (Table 2).
As can be seen in Table 2, the enzyme has high
proteolytic properties and is activated at temperatures
that are favorable for protein structures.
Table 2. Characteristics of the enzyme used in the study
Таблица 2. Характеристика фермента, используемого в исследовани и
Indicator Description
Composition Pepsin based on Rhyzomucor miehei (CAS: 9001-92-7)
Origin Microbial
Assumed splitting Phe1 + Val, Gln4 + His, Glu13 + Ala, Ala14 + Leu, Leu15 + Tyr,
Tyr16 + Leu, Gly23 + Phe, Phe24 + Phe, and Phe25
Form, color White powder
Activity, units per 1 g At least 300 000
Activation temperature, °С 30 ± 2
Producer Meito Sangyo Co., Ltd., Japan
Figure 1. Flow chart for producing bioactive peptides from poul try by-products using hydrolysis (two variants)
Рисунок 1. Схема производства биоактивных пептидов с применение м гидролиза (в двух вариациях) из вторичных сырьевых
ресурсов переработки птицы
Homogeneous mass of chicken skin
Hydrolysis parameters:
Hydrolysis 1:
Enzyme: pepsin
30 units per 100 g of material
Time: 12 h
t = 28 ± 2 оС
pH 3.0 ± 0.2
Enzyme neutralization with weak alkali and inactivation
at t = 45 ± 2 оС
Fractionation
by centrifugation at 3000 rpm
Aqueous extraction of the protein fraction
at t = 70 ± 5 °С
Concentration by ultrafiltration
at a controlled pressure of 3.0 bar
Spray drying
at 90°C and a solution supply rate of 3.0 ± 0.2 mL/min
Protein hydrolysates (bioactive peptides)
from poultry by-products
Hydrolysis 2:
Enzyme: pepsin
45 units per 100 g of material
Time: 12 h
t = 28 ± 2 оС
pH 3.0 ± 0.2
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Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554
Enzymatic hydrolysis was carried out in vitro. MM-5
laboratory magnetic stirrers were used to ensure a
uniform treatment of the materials with an enzyme
solution throughout the experiment (100 rpm, 28 ± 2°C).
The acidity was maintained with 1 M hydrochloric acid.
The hydrolysates were dried in a B-290 Mini Spray
Dryer (Buchi, Sweden) at 100 ± 2°C and at a solution
supply rate of 3.5–4.0 mL/min. The dried hydrolysates
were microphotographed with a JEOL JED-2300
electronic microscope (Japan). The mass fraction
of protein was determined on a RapidN Elementar
nitrogen (protein) analyzer. This analyzer uses the
Dumas method that involves combusting the samples
and registering total nitrogen on a thermal conductivity
detector. Molecular weight distribution was performed
by polyacrylamide gel electrophoresis in the presence
of an anionic detergent (sodium dodecyl sulfate).
The amino acid sequence of the peptides was determined
by matrix-activated laser desorption/ionization on a
MALDI Biotyper (Bruker). The molecular weight
was calculated by using the Peptide Mass Calculator.
The bioactivity of the peptides was assessed in silico
using the PeptideRanker online server that ranks
peptides by the predicted probability of their bioactivity.
The structure of the peptides was modeled by using the
PepDraw online tool.
The experiments were performed at the Department
of Animal Origin Food Technology and the Scientific
and Educational Center of the Research and Innovation
Department, Kemerovo State University.
Results and discussion
The flow chart for producing bioactive peptides from
poultry by-products is presented in Fig. 1. It includes
two sets of parameters for enzymatic hydrolysis of a
homogeneous mass of chicken skin.
Chicken skin was homogenized with a laboratory
homogenizer. The homogeneous mass was hydrolyzed
with pepsin in two variations during 12 h at 28 ± 2°C.
The enzyme was then thermally inactivated at 45 ± 2°C
and neutralized with a weak alkali to pH 7.0 ± 2. Next,
the hydrolysate was fractionated into protein and fat
parts using a CM 6 M Multi centrifuge at 3000 rpm.
The protein part was then subjected to thermal water
extraction in order to dissolve the protein fractions at
70 ± 5°C. The resulting solution was filtered using a
MFU-R-45-300 laboratory ultrafiltration unit (Russia)
at a controlled pressure of 3.0 bar and a difference
of 0.2–0.5 kgf/cm2 in the discharge and return
headers. The protein part of the solution went into the
retentate, while the water and minerals went into the
permeate. Further, protein hydrolysate samples were
obtained by spray drying at 90 ± 2°C and a solution
supply rate of 3.0 ± 0.2 mL/min. The samples were then
microphotographed with an electronic microscope
(magnified 500 times), as can be seen in Fig. 2.
First, we evaluated the color and particle size of
the hydrolysates. As we can see in Fig. 2, the samples
differed in color, with sample 1 having a darker creamy
color and sample 2 having a whitish color. Also, the
hydrolysates differed in particle size, although they
were dried under the same conditions. In particular,
sample 2 had a more finely dispersed structure,
which can be explained by a deeper hydrolysis of this
sample.
Next, we determined the main physicochemical
parameters of the hydrolysates for further studies
(Table 3).
As can be seen in Table 3, the samples had no
significant differences in physicochemical parameters.
Since the hydrolysates had a high protein content
(over 90%), they can be classified as a high-protein
product.
Next, we analyzed the distribution of protein
fractions by polyacrylamide gel electrophoresis in the
Figure 2. Protein hydrolysates from poultry by-products:
a – Sample 1 based on pepsin with an activity of 30 units
per 100 g of material; b – Sample 2 based on pepsin with
an activity of 45 units per 100 g of material
Рисунок 2. Белковые гидролизаты из вторичных сырьевых
ресурсов переработки птицы: a – образец № 1, полученный
с применением пепсина активностью 30 ед. на 100 г сырья;
b – образец № 2, полученный с применением пепсина
активностью 45 ед. на 100 г сырья
Table 3. Physicochemical parameters of the hydrolysates
Таблица 3. Основные физико-химические показатели
гидролизатов
Parameters, % Sample 1 Sample 2
Protein 90.7 ± 0.1 91.4 ± 0.2
Fat 0.60 ± 0.03 0.40 ± 0.06
Moisture 8.1 ± 0.1 7.8 ± 0.2
Ash 0.60 ± 0.04 0.40 ± 0.03
a b
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presence of an anionic detergent, sodium dodecyl sulfate.
Table 4 shows the molecular weight distribution of the
hydrolysates.
As can be seen in Table 4, most peptide fractions
had a molecular weight below 20 kDa, especially those
in sample 2. Thus, we can assume that the hydrolysates
contained peptides with bioactive properties, especially
sample 2.
Then, we determined the amino acid sequence of
the hydrolysates under study (Table 5).
According to Table 5, sample 1 had a chain of 41 peptides,
while sample 2 was represented by 27 peptides.
We can also see that the first and the second variants
of hydrolysis, which used pepsin with lower and higher
activity, respectively (Fig. 1), split the protein into
14 and 8 fragments of amino acids (peptide sequences),
respectively.
Next, we used online resources to determine
the molecular weight and bioactivity of individual
peptide sequences, as well as modelled their structure
(Tables 6 and 7).
The PeptideRanker service has a threshold value
of 0.5 for peptides’ bioactive properties, i.e. any peptide
with an estimated value above 0.5 is ranked as
bioactive. However, literature shows that using a higher
threshold, particularly 0.8, reduces the number of
false positive results. Therefore, we assessed the
bioactive properties of peptides based on a threshold
bioactivity value of 0.8 and the maximum
value of 1.
According to Table 6, the FD peptides (38–39 in
the sequence) and the NW peptides (40–41 in the
sequence) had greater bioactivity values of 0.922094
and 0.934148, respectively. Their structure shows
aromatic rings which are mainly represented by
phenylalanine and tryptophan.
As can be seen in Table 7, sample 2 had three
peptide sequences with high bioactive properties. They
were the CYG peptides (25–27 in the sequence), the
GHG peptides (13–15), and the AYG peptides (22–24),
with bioactivity values of 0.947378, 0.839383, and
0.815664, respectively. Structurally, these bioactive
peptides had aromatic rings represented by the aromatic
α-amino acid of tyrosine.
We found no correlation between the bioactivity
values of the peptides and their molecular weight.
Table 4. Molecular weight distribution of the hydrolysates
Таблица 4. Молекулярно-массовое распределение образцов гидролиз атов
Molecular weight, kDa Distribution of peptide fractions by molecular weight, %
Sample 1 Sample 2
200 1.06376 2.194861
150 4.905115 2.328694
100 2.186618 4.007342
85 14.60372 0.948302
60 6.369427 3.001682
50 4.813185 0.776231
40 3.224112 1.697767
30 0.965264 2.011318
25 1.267319 5.766289
20 4.392935 11.56317
Less than 20 56.20855 65.70435
Table 5. Amino acid sequence of the hydrolysates
Таблица 5. Аминокислотная последовательность исследуемых гидрол изатов
Parameter Sample 1 Sample 2
Amino acid sequence in a
one-letter code*
PILG/PILV/ILA/ILG/ILV/
ILT/PSV/GVS/HGL/HVI/SVP/SV/FD/NW
ILKH/PSPV/PSVP/GHG/SPV/SVP/AYG/
CYG
Peptide chain length 41 27
Isoelectric point (pI) 6.05 8.84
* A – alanine; C – cysteine; D – aspartic acid; E – glutamic acid; F – phenylalanine; G – glycine; H – histidine; I – isoleucine; K – lysine;
L – leucine; M – methionine; N – asparagine; P – proline; Q – g lutamine; R – arginine; S – serine; T – threonine; V – valine; W – tryptophan;
Y –tyrosine.
* A – аланин; C – цистеин; D – аспарагиновая кислота; E – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин;
I – изолейцин; K – лизин; L – лейцин; M – метионин; N – аспа рагин; P – пролин; Q – глутамин; R – аргинин; S – серин; T – треонин;
V – валин; W – триптофан; Y – тирозин.
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Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554
Table 6. Individual peptide sequences for hydrolysate Sample 1
Таблица 6. Характеристика отдельных пептидных последовательност ей гидролизата образца № 1
The number of peptide
in the chain
Peptide sequence
in a one-letter code*
Molecular weight,
g/mol
Bioactivity Peptide structure
1–4 PILG 398 0.492674
5–8 PILV 440 0.291507
9–11 ILA 315 0.237443
12–14 ILG 301 0.480965
15–17 ILV 343 0.123381
18–20 ILT 345 0.154222
21–23 PSV 301 0.166459
24–26 GVS 261 0.109533
27–29 HGL 325 0.446783
30–32 HVI 367 0.0887486
33–35 SVP 301 0.194373
36–37 SV 204 0.0523218
38–39 FD 280 0.922094
40–41 NW 318 0.934148
* A – alanine; C – cysteine; D – aspartic acid; E – glutamic acid; F – phenylalanine; G – glycine; H – histidine; I – isoleucine; K – lysine; L – leucine; M –
methionine; N – asparagine; P – proline; Q – glutamine; R – arg inine; S – serine; T – threonine; V – valine; W – tryptophan; Y –tyrosine.
* A – аланин; C – цистеин; D – аспарагиновая кислота; E – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин; I – изолейцин; K – лизин;
L – лейцин; M – метионин; N – аспарагин; P – пролин; Q – глутамин; R – аргинин; S – серин; T – треонин; V – валин; W – триптофан; Y – тирозин.
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Voroshilin R.A. et al. Food Processing: Techniques and Technology. 2022;52(3):545–554
products. Then, we evaluated the bioactive properties
of the peptides obtained. The hydrolysate sample
obtained with a lower pepsin activity (30 units per 100 g
of material) had greater bioactivity values in the FD and
NW peptides (0.922094 and 0.934148, respectively).
The sample obtained with a higher pepsin activity
(45 units per 100 g of material) had higher bioactivity
in the CYG, GHG, and AYG peptides (0.947378 units,
0.839383, and 0.815664, respectively). However, these
data obtained in silico need to be confirmed by in vitro
experiments for further work. Such experiments will
determine their reliability and identify specific bioactive
properties of the peptides.
We should note that the detection of more bioactive
peptides by the in silico method should be confirmed
by further in vitro experiments with various
modifications of peptides. Based on literature, we
can assume that some peptide sequences obtained in
our study may have antimicrobial and antioxidant
activity due to the presence of proline and leucine
that have such properties [26]. Further in vitro
experiments can determine the reliability of our
results and identify specific bioactive properties of the
studied peptides.
Conclusion
Based on the research results, we designed a flow
chart for obtaining hydrolysates from poultry by-
Table 7. Individual peptide sequences for hydrolysate sample 2
Таблица 7. Характеристика отдельных пептидных последовательност ей гидролизата опытного образца № 2
The number of peptide
in the chain
Peptide sequence
in a one-letter code*
Molecular weight,
g/mol
Bioactivity Peptide structure
1–4 ILKH 509 0.162711
5–8 PSPV 398 0.390668
9–12 PSVP 398 0.327229
13–15 GHG 269 0.839383
16–18 SPV 301 0.222562
19–21 SVP 301 0.194373
22–24 AYG 309 0.815664
25–27 CYG 341 0.947378
* A – alanine; C – cysteine; D – aspartic acid; E – glutamic acid; F – phenylalanine; G – glycine; H – histidine; I – isoleucine; K – lysine;
L – leucine; M – methionine; N – asparagine; P – proline; Q – g lutamine; R – arginine; S – serine; T – threonine; V – valine; W – tryptophan;
Y –tyrosine.
* A – аланин; C – цистеин; D – аспарагиновая кислота; E – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин;
I – изолейцин; K – лизин; L – лейцин; M – метионин; N – аспа рагин; P – пролин; Q – глутамин; R – аргинин; S – серин; T – треонин;
V – валин; W – триптофан; Y – тирозин.
553
Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554
Contribution
The authors were equally involved in writing
the manuscript and are equally responsible for
plagiarism.
Conflict of interest
The authors declare that there is no conflict of interest
regarding the publication of this article.
Критерии авторства
Авторы были в равной степени вовлечены в на-
писание рукописи и несут равную ответственность
за плагиат.
Конфликт интересов
Авторы заявляют, что конфликта интересов
нет.

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