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2023 Т. 53 № 1 / Техника и технология пищевых производств / Food Processing: Techniques and. Technology
ISSN 2074-9414 (Print) ISSN 2313-1748 (Online)
https://doi.org/10.21603/2074-9414-2023-1-2422 https://elibrary.ru/GXNVFU
Original article Available online at https://fptt.ru/en
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Peptides of Trypsin Hydrolyzate in Bovine Colostrum
Sergei L. Tikhonov1* , Natalia V. Tikhonova1 , Khatam Kh. Tursunov2, Irina G. Danilova3 , Vladimir A. Lazarev1
1 Ural State University of Economics^**, Yekaterinburg, Russia
2 Andijan State Medical InstituteRt-**J Andijan, Uzbekistan
3 Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences"0",
Yekaterinburg, Russia
*Sergei L. Tikhonov: tihonov75@bk.ru, https://orcid.org/0000-0003-4863-9834 Natalia V. Tikhonova: https://orcid.org/0000-0001-5841-1791 Irina G. Danilova: https://orcid.org/0000-0001-6841-1197 Vladimir A. Lazarev: https://orcid.org/0000-0002-0470-7324
© S.L. Tikhonov, N.V. Tikhonova, Kh.Kh. Tursunov, I.G. Danilova, V.A. Lazarev, 2023
Abstract.
Bovine colostrum contains biologically active substances, e.g., immunoglobulins, peptides, and cytokines, which makes it a logical component of numerous functional products. Colostrum peptides also possess antimicrobial activity. This bioavailability increases during colostrum fermentation with proteolytic enzymes. The research objective was to describe peptides isolated from the trypsic hydrolyzate supernatant of bovine colostrum and to evaluate their antimicrobial and antifungal properties. The supernatant of trypsin hydrolyzate of bovine colostrum was isolated by centrifugation at 3900 rpm for 7 min. The supernatant was separated by preparative chromatography. Its peptide composition was determined on a MALDI-TOF mass spectrometer, while the protein sequences were deciphered using the Mascot database. Proteins were precipitated with ammonium sulfate, and the antimicrobial activity was measured by the disk-diffusion method against gram-positive and gram-negative bacteria and dipoloid fungi. Strains were cultivated on a thick LB nutrient medium at 37°C. The antimicrobial activity was defined experimentally on Wistar rats infected intraperitoneally with Salmonella enteritidis 92.
The trypsin hydrolyzate supernatant of bovine colostrum revealed four peptides, one of which belonged to short peptides, while the remaining three belonged to polypeptides. The isolated peptides had different molecular weights of 8.4, 6.5, 13.0, and 8 kDa. The enzymatic hydrolyzate proved bactericidal against Escherichia coli and Bacillus subtilis and demonstrated antifungal activity against Candida albicans. When rats infected with S. enteritidis 92 were administered with trypsin hydrolysate, it promoted their survival, decreased LD50, and increased the mean day of death period from 2 to 4 days. The research proved the antimicrobial effect of colostrum peptides and suggested their immunotropic properties. The peptides obtained from the trypsin hydrolyzate supernatant of bovine colostrum can be recommended for functional food industry as part of antimicrobial products.
Keywords. Colostrum, milk protein, enzyme, hydrolysis, antimicrobial activity, antifungal activity, biologically active substances
For citation: Tikhonov SL, Tikhonova NV, Tursunov KhKh, Danilova IG, Lazarev VA. Peptides of Trypsin Hydrolyzate in Bovine Colostrum. Food Processing: Techniques and Technology. 2023;53(1):150-158. https://doi.org/10.21603/2074-9414-2023-1-2422
Received: 30.06.2022 Revised: 08.09.2022 Accepted: 04.10.2022
https://doi.org/10.21603/2074-9414-2023-1-2422 Оригинальная статья
https://elibrary.ru/GXNVFU https://fptt.ru
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Пептиды трипсинового гидролизата молозива коров
С. Л. Тихонов1'* , Н. В. Тихонова1 , X. X. Турсунов2, И. Г. Данилова3 , В. А. Лазарев1
1 Уральский государственный экономический университет^**, Екатеринбург, Россия
2 Андижанский государственный медицинский институтАндижан, Узбекистан
: Институт иммунологии и физиологии Уральского отделения Российской академии наук
Екатеринбург, Россия
*С. Л. Тихонов: tihonov75@bk.ru, https://orcid.org/0000-0003-4863-9834 Н. В. Тихонова: https://orcid.org/0000-0001-5841-1791 И. Г. Данилова: https://orcid.org/0000-0001-6841-1197 В. А. Лазарев: https://orcid.org/0000-0002-0470-7324
© С. Л. Тихонов, Н. В. Тихонова, Х. Х. Турсунов, И. Г. Данилова, В. А. Лазарев, 2023
Аннотация.
Молозиво коров из-за содержания биологически активных веществ, в частности иммуноглобулинов, пептидов и цитокинов, является перспективным сырьем для производства продуктов функциональной направленности. Пептиды молозива обладают антимикробным действием. Биодоступность действующих начал молозива повышается при его ферментации протеолитическими ферментами. Цель исследования - выделение и характеристика пептидов надосадочной жидкости трипсиного гидролизата молозива коров, а также оценка их антимикробной и противогрибковой активностей. Для эксперимента использовали надосадочную жидкость трипсинового гидролизата молозива коров, выделенную методом центрифугирования при 3900 об/мин в течение 7 мин. Надосадочную жидкость разделяли методом препаративной хроматографии. Пептидный состав надосадочной жидкости ферментативного гидролизата определяли на МАЛДИ-ТОФ масс-спектрометре, расшифровку белковых последовательностей проводили с помощью базы данных Mascot. Для изучения белкового состава надосадочной жидкости гидролизата проводили осаждение белков сульфатом аммония. Антимикробную активность определяли диско-диффузионным методом. Культивирование штаммов бактерий проводили на плотной питательной среде LB при температуре 37 °C. Для оценки противомикробного действия пептидов провели эксперимент на крысах линии Вистар, инфицированных внутрибрюшинно Salmonella enteritidis 92. В надосадочном трипсиновом гидролизате молозива коров выделили 4 пептида, один из которых относится к коротким пептидам, три - к полипептидам. Выделенные пептиды имели различную молекулярную массу - 8,4, 6,5, 13,0 и 18 кДа. Установлено, что ферментативный гидролизат надосадочной жидкости молозива коров обладал бактерицидным действием к грамотрицательной бактерии Escherichia coli и грамположительной бактерии Bacillus subtilis, а также антигрибковой активностью против Candida albicans. Введение крысам, инфицированным S. enteritidis 92, внутрь трипсинового гидролизата надосадочной жидкости молозива коров способствовало их выживаемости, снижению ЛД50 и увеличению среднего срока гибели животных с 2 до 4 суток.
Полученные данные свидетельствуют об антимикробном действии пептидов молозива и возможных иммуннотропных свойствах. Практическая значимость проведенного исследования заключается в перспективности использования пептидов надосадочной жидкости трипсиного гидролизата молозива коров для производства продуктов функциональной направленности с антимикробными свойствами.
Поступила в редакцию: 30.06.2022 Принята после рецензирования: 08.09.2022 Принята к публикации: 04.10.2022
Ключевые слова. Молозиво, молочный белок, фермент, гидролиз, антимикробная активность, противогрибковая активность, биологически активные вещества
Для цитирования: Пептиды трипсинового гидролизата молозива коров / С. Л. Тихонов [и др.] // Техника и технология пищевых производств. 2023. Т. 53. № 1. С. 150-158. (На англ.). https://doi.org/10.21603/2074-9414-2023-1-2422
Introduction
Colostrum is a complex biological fluid. Glowka & Wozniewicz believe that bovine colostrum is beneficial for the digestive, immune, and neuroendocrine systems and can boost physical performance in general
because it contains growth factors, immunoglobulins, peptides, cytokines, lactoferrin, and hormones [1]. Athletes consume colostrum during high-intensity training because it is rich in immunoglobulins and can increase the buffer capacity of muscles. However,
no scientific publications feature the exact recommended doses of colostrum that can increase physical performance.
Van Hese et al., Sukhikh et al., and Kharitonov et al. proved that dairy products and colostrum have a high content of immunoglobulins, minerals, vitamins, growth factors, and immune cells, as well as microRNAs [2-4]. A microRNA is a short non-coding RNA molecule that regulates post-transcriptional gene expression. MicroRNAs act as key regulators of various biological and developmental processes. Bovine colostrum MicroRNAs are signaling molecules. They are located inside extracellular vesicles that protect them from the harsh conditions of the gastrointestinal tract. As a result, they can get into the small intestine, where they are absorbed and enter the bloodstream. MicroRNAs stimulate the viability, proliferation, and activity of intestinal epithelial stem cells. In addition, they affect the entire immune system by differentiating B- and T-cells and controlling the production of interleukin by macrophages [2].
Chandwe & Kelly reported the anti-inflammatory activity of bovine colostrum in bowel diseases and infectious diarrhea [5].
Menchetti et al. and Playford et al. published in vitro and in vivo evidence of the beneficial effect of bovine colostrum on gastrointestinal diseases [6, 7].
More than half of all children with autism spectrum disorder have gastrointestinal comorbidities, e.g., chronic constipation, diarrhea, irritable bowel syndrome, etc. The severity of these symptoms correlates with the degree of gastrointestinal dysbacteriosis in the patient. Sanctuary et al. combined a probiotic (Bifidobacterium infantis) with a bovine colostrum product as a source of prebiotic oligosaccharides [8]. They assessed the efficacy of this preparation and the state of the gastrointestinal tract, microbiome, and immune factors in young gastrointestinal patients with autism spectrum disorder. The patients that received both preparations demonstrated a lower incidence of irritable gastrointestinal tract disorder, dysbacteriosis, and certain behavioral abnormalities. This improvement probably resulted from a lower production rate of IL-13 and TNF-a.
Bovine colostrum was reported to have a greater bioavailability when fermented with proteolytic enzymes, which increase the peptide yield. Jorgensen et al. tested the biological activity of peptides isolated from bovine colostrum on mouse intestinal cells (mIC(c12)) [9]. After protease treatment, colostrum peptides became more bioactive. They detected bioactive colostrum peptides mainly in the casein fraction (MALDI MS/MS).
Jorgensen et al. explained the beneficial effect of colostrum consumption by the presence of biologically active peptides obtained from intact proteins [9]. According to Korhonen, these peptides can be relea-
sed during gastrointestinal digestion or colostrum fermentation [10]. Therefore, enzymatic hydrolysates of bovine colostrum are a potential source of biologically active native proteins and peptide fractions to be included in functional foods [11, 12].
Nowadays, dairy peptides are more than a quintessence of healthy nutrition: they have gained therapeutic value as well. For instance, colostrum peptides appeared to inhibit SARS-CoV2 [13]. Fermented colostrum, raw milk, and microfiltered milk from cows vaccinated against SARS-CoV-2 may even provide a short-term protection against SARS-CoV-2 [14]. Colostrum peptides also demonstrated a strong antimicrobial effect [15].
Enzymatic protein hydrolysis based on proteolytic enzyme preparations can be an effective production method for obtaining bioactive peptides [13]. Animal enzymes, e.g., trypsin, often participate in hydrolysis [14].
This research isolated peptides from the trypsic hydrolyzate supernatant of bovine colostrum to be described and tested for antimicrobial and antifungal activities.
Study objects and methods
We obtained trypsin hydrolyzate of bovine colostrum by the following technology. First, we removed the fat fraction by centrifugation at 3900 rpm for 10 min in a SM-12-06 centrifuge (TAGLER, Russia). After that, we introduced trypsin (Samson-Med, Russia) in the ratio of 0.15% to the colostrum weight into a phosphate buffer solution at pH 7.4. The solution consisted of disodium hydrogen phosphate dodecahyd-rate (Rosspolymer, Russia). The hydrolysis lasted for 12 h at 36°C. Eventually, the temperature was brought up to 75°C to inactivate the enzyme. Table 1 summarizes the physicochemical parameters of trypsin hydrolyzate of bovine colostrum.
The trypsin hydrolyzate supernatant of bovine colostrum was isolated by centrifugation at 3900 rpm for 7 min.
The supernatant was separated by preparative chromatography on silica gel, and the isocratic ratio of eluent PBS to EtOH was 9:1. The peptide composition of
Table 1. Physicochemical parameters of trypsin hydrolyzate of bovine colostrum
Таблица 1. Физико-химические показатели трипсинового гидролизата молозива коров
Indicator Property
Mass fraction of protein, % 13.18 ± 0.09
Mass fraction of fat, % 0.15 ± 0.01
Mass fraction of ash, % 9.50 ± 0.02
Mass fraction of solids, % 24.17 ± 0.42
Density, g/cm3 1.05 ± 0.03
Acidity, 0T 4.78 ± 0.12
the trypsin hydro lyzate supernatant was determined on a MALDI-TOF mass spectrometer, while the protein sequences were deciphered using the Mascot database.
Proteins were precipitated with ammonium sulfate to allow for a more complete study of the protein composition of the hydrolyzate supernatant. After sedimentation, the samples were centrifuged at 3900 rpm for 7 min to collect the protein precipitate. The protein precipitate was purified from salts and inorganic impurities on a column with Amberlit XAD2 with eluent for buffer A of 10 mM (CH3COONa), pH = 6, 10 mM (CH3COONa) pH = 4, 10 mM (KCl/HCl), pH = 1.5. The salt gradient for buffer A+ was 0.2, 0.4, and 1% NaCl. We used the Bradford method to test fractions of each sample for protein. The resulting peptide fractions were separated by preparative chromatography on silica gel. The isocratic ratio of eluent PBS and EtOH was 9:1. The separation resulted in the mpT fraction, which was studied using the MALDI-TOF method.
We used the disk-diffusion method to define the antimicrobial activity of the trypsin hydrolyzate supernatant on gram-positive Bacillus subtilis and gramnegative Escherichia coli.
The strains were cultivated on solid and liquid media at 37°C. The solid LB nutrient medium consisted of 1.5% agar, 1% tryptone, 0.5% yeast extract, and 1% NaCl. The liquid LB nutrient medium included 1% tryptone, 0.5% yeast extract, and 1% NaCl.
The diffusion method was applied to determine the antimicrobial activity of the hydrolyzate. The test strain was sown on a lawn plate with agar nutrient medium, and the supernatant was placed on the lawn. A paper disk with a nutrient medium served as a control, and a disk with a standard antibiotic (Kanamycin) served as reference. The petri dishes were incubated at the optimal temperature for each test strain for
24.0 ± 0.5 h. The results depended on the presence and size (mm) of a microorganism-free area around the disk.
The antimicrobial activity of the peptides was tested on nine groups of Wistar rats. Each group included four three-month-old males. Group 1 consisted of control animals that received 0.4 mL of water per day intragastrically for 7 days. Groups 2, 3, 4, and 5 received 0.3 mL of the trypsin hydrolyzate supernatant of bovine colostrum every day for 7 days. One day after the introduction, groups 2-9 were injected intraperitoneally with a daily culture of Salmonella enteritidis 92. Groups 2 and 5 received 5 CFU, groups 3 and 6 - 100 CFU, groups 4 and 8 - 500 CFU, groups 5 and 9 - 5000 CFU. The rats were observed for 21 days after the infection. The efficacy of the preparation was defined based on the LD50 number of survivors and the mean day pf death period.
All animal procedures complied with the European Communities Council Directive 2010/63/EU on the welfare of experimental animals and were approved by the Ethics Committee of the Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences.
Results and discussion
The fractionated supernatant of trypsin hydroly-zate (T) yielded three peptide fractions with different molecular weights: TT1, TT2, and TT3. Figure 1 shows a chromatogram for peptides.
Figures 2 show the mass spectra of peptide fractions TT1, TT2, and TT3 of the trypsin hydrolyzate supernatant of bovine colostrum.
Figures 2 demonstrate quite clearly that the pep-tides differed in molecular weight.
The molecular weight differed as follows: TT1 -8.4 kDa, TT2 - 6.5 kDa, and TT3 - 13.0 kDa.
x1
o4
Й
О j>
*o
m
100 80 60 40 20
5 6 7 8 9 10 11 12
V
ТТ2
ТТ3
AB
1
8 12 Time, min Solvent, % "UV1 ■ UV2
16
1.18 0.94 0.70 0.47 0.23 0.00
20
>
>
>1
UV3
Figure 1. Supernatant of trypsin hydrolyzate of bovine colostrum: peptides T Рисунок 1. Хроматограмма образца T надосадочной жидкости трипсинового гидролизата молозива коров
2
4
3
0
0
4
50 100 150 200 250 300 350 400 450 500
m/z
a
50 100 150 200 250 300 350
m/z
b
50 100 150 200 250 300 350
m/z
c
Figure 2. Mass spectrum: Samples TT1 (a), TT (b), and TT3 (c) Рисунок 2. Масс-спектры образцов ТТ1 (a), TT (b) и TT3 (c)
Table 2 shows the amino acid sequence for each sample. Peptide TT3 consisted of nine amino acids (nonapeptides) and belonged to short peptides. Peptides TT1 and TT2 had 17 amino acids and belonged to polypeptides. Ko et al. identified TT1 peptide as the NCI_CGAP_Brn23 peptide Homo sapiens c DNA clone [17]. In fact, it is similar to TR: 035085 035085 ARX HOMEOPROTEIN. This peptide is believed to
affect the prenatal development of the central nervous system. Peptides TT2 and TT3 have never been identified.
This research featured the peptide composition of proteins precipitated with ammonium sulfate in the trypisine hydrolyzate supernatant of bovinecolostrum.
Figure 3 demonstrates a chromatogram of the peptide composition of proteins precipitated by ammonium sulfate of hydrolysate trypisine supernatant.
Table 2. Protein sequences of peptide samples TT1, TT2, and TT3 of trypsin hydrolyzate supernatant of bovine colostrum
Таблица 2. Белковые последовательности пептидов ТТ1, ТТ2 и ТТ3 надосадочной жидкости трипсинового гидролизата молозива коров
Peptide sample Amino acid sequence
ТТ1 EGKSPRQ CLK SR G RK GY
ТТ2 PK CD YKRRS GP ALR TAK
ТТ3 LARKTSK IK
Note: A - alanine; D - aspartic acid; Q - glutamine; E - glutamic acid; G - glycine; I - isoleucine; L - leucine; K - lysine; P - proline; S - serine; T - threonine; Y - tyrosine; R - arginine; D - aspartic acid. Примечание: А - аланин; D - аспарагиновая кислота; Q - глутамин; Е - глутаминовая кислота; G - глицин; I - изолейцин; L - лейцин; K - лизин; P - пролин; S - серин; T - треонин; Y - тирозин; R - аргинин; D - аспаргиновая кислота.
When we precipitated supernatant proteins of bovine trypisine hydrolysate with ammonium sulfate,
it yielded a TT4 peptide with the following amino acid sequence: EK LAKNK LAR GLK RK. The peptide had a molecular weight of 18.0 kDa and was identified as C0950255 protein (sus scrofa) with unknown functions.
We isolated four peptides: one short peptide and three polypeptides. All the samples had different molecular weights.
Our data were consistent with those obtained by Poirier et al., who detected biologically active peptides in the trypsin hydrolyzate of bovine colostrum [18]. They stimulated the proliferative activity of the T84 human intestinal epithelial cell line with pepsin and trypsin hydrolyzate of bovine colostrum. Therefore, bovine colostrum peptides are potentially
bioactive substances that are able to restore the gastrointestinal tract during infections [19].
Birkemo et al. were the first to describe peptides with antimicrobial activity in fresh bovine colostrum [20]. They used chromatography to isolate three peptides of fresh colostrum and described their antimicrobial activity against Escherichia coli DH5alpha. The first two peptides were casecidin 17 and casecidin 15. They were identical to the sequences at the C-terminus of bovine beta-casein (YQEPVLGPVRGPFPIIV and YQEPVLGPVRGPFPI). Casecidin 17 and casecidin 15 had molecular weights of 1881.00 and 1669.06 Da, respectively. The third peptide was isracidin with a mass of 2763.80 Da and the sequence of RPKHPIKHQGLPQEVLNENLLRF. Casecidin 17 and casecidin 15 shared the same minimal inhibition concentrations against E. coli DPC6053: 0.4 mg/mL. Structural modeling showed that the amphiphilic structures also had identical inhibitory and structural properties. The minimal inhibition concentration of isracidin against E. coli DPC6053 was 0.2 mg/mL [20].
In this research, we studied the antimicrobial properties of the trypsin hydrolyzate supernatant of bovine colostrum on gram-positive and gram-negative bacteria, as well as on the Candida albicans diploid fungus. E. coli and Bacillus subtilis were selected as test bacterial strains.
Table 3 summarizes the antimicrobial and antifungal activity of the trypsin hydrolyzate supernatant of bovine colostrum.
Table 3 illustrates the effect of trypsin hydrolyzate on the survival of Wistar rats infected with Salmonella enteritidis 92.
According to Table 4, the trypsin hydrolyzate had a bactericidal effect against gram-negative E. coli (lysis
o4
nt
e lv ol
m
100 80 60 40 20 0
1 1
mpT
\
---- — *__ KAB - .j ____
1
8 12 Time, min Solvent, % "UV1 ■ UV2 ■ UV3
16
0.50 0.40 0.29 0.19 0.09
20
>
> y
Figure 3. Peptides during the precipitation of proteins from trypisine hydrolysate supernatant of bovine colostrum
with ammonium sulfate
Рисунок 3. Хроматограмма пептидов при осаждении сульфатом аммония белков надосадочной жидкости триписинового
гидролизатата молозива коров
5
2
4
0
4
zone diameter - 3 mm) and gram-positive B. subtilis (lysis zone diameter - 5 mm), as well as antifungal activity against C. albicans.
Table 4 demonstrates the effect of trypsin hydrolyzate on the survival of rats infected with S. enteritidis 92.
Rats that received trypsin hydrolyzate supernatant of bovine colostrum had a better survival rate against S. enteritidis 92. Therefore, the colostrum peptides possessed antimicrobial properties. Group 4 rats, which received 500 CFU of S. enteritidis, had two survivors (50%), while Group 7 had only one (25%). The LD50 for Wistar rats with S. enteritidis was 100 CFU, but those injected with trypsin hydrolyzate had a LD50 of 500 CFU of S. enteritidis. The mean day of death value was lower by 50% in the infected animals treated with hydrolysate. It was two days in Group 9 infected with 5000 CFU of S. enteritidis. In Group 5, where the rats received the same dose of S. enteritidis and were treated with hydrolysate, it was three days.
Our results were consistent with the data obtained by Sears et al., who proved that bovine colostrum is a practical and effective prophylactic against gastrointestinal diseases [21]. Travelan is a commercial drug known to prevent diarrhea caused by enterotoxigenic E. coli. It possesses official clinical efficacy against Salmonella. However, its immune components and antimicrobial activity are yet to be identified [21].
Our research revealed four antimicrobial peptides in the trypsin hydrolyzate supernatant of bovine
colostrum. They possessed bioprotective properties against pathogens, which was consistent with other studies on the antimicrobial and immunotropic effects of bovine colostrum. Anderson et al. argue that bovine colostrum increases the protective barrier of the small intestine and positively affects colon peristalsis [22]. However, for colostrum to be used as a functional product, it must remain bioactive after processing. The integrity of the intestinal protective barrier and the antimicrobial properties of colostrum increased because of the short peptides that were released during enzymatic processing. In [22], proteolytic enzymes had a beneficial effect on the integrity of the small intestine barrier, which is consistent with our research results. Indeed, the peptides can be used to develop functional foods aimed at improving intestinal health.
Playford et al. reported that orally-administered bioactive peptides have potential clinical benefits [23]. However, their applicability is limited by the proteolysis with gastric and pancreatic enzymes. As oral antimicrobial agents, bioactive pep-tides have to be combined with substances that ensure their proper digestion. In [23], the authors turned to casein and/or soy flour with high protease inhibiting activity to provide the stability of bovine colostrum peptides in laboratory rats. Soy and, to a lesser extent, casein increased the biostabi-lity of colostrum peptides in relation to digestive enzymes.
Table 3. Antimicrobial and antifungal activity of the trypsin hydrolyzate supernatant of bovine colostrum
Таблица 3. Антимикробная и противогрибковая активность надосадочной жидкости трипсинового гидролизата
молозива коров
Sample Lysis diameter, mm
Escherichia coli ATCC 25922 Bacillus subtilis Candida albicans
Trypsin hydrolyzate supernatant of bovine colostrum 3 5 7
Control 0 0 0
Antibiotic 25 26 0
Group Surviving animals, g Mean day of death, days
Group 1 (control) 4 -
Group 2 (5 CFU Salmonella enteritidis + hydrolyzate) 4 -
Group 3 (100 CFU Salmonella enteritidis + hydrolyzate) 4 -
Group 4 (500 CFU Salmonella enteritidis + hydrolyzate) 2 4
Group 5 (5000 CFU Salmonella enteritidis + hydrolyzate) 0 3
Group 6 (5 CFU Salmonella enteritidis) 4 -
Group 7 (100 CFU Salmonella enteritidis) 2 2
Group 8 (500 CFU Salmonella enteritidis) 1 2
Group 9 (5000 CFU Salmonella enteritidis) 0 2
Table 4. Effect of trypsin hydrolyzate on the survival of rats infected with Salmonella enteritidis 92
Таблица 4. Влияние трипсинового гидролизата надосадочной жидкости молозива коров на выживаемость крыс линии Вистар,
зараженных Salmonella enteritidis 92
Conclusion
We isolated peptides from the trypsin hydrolyzate supernatant of bovine colostrum and investigated their antimicrobial and antifungal activities. The tryp-sin hydrolyzate was preliminarily fractionated by centrifugation. Three peptide fractions (TT1, TT2, and TT3) differed in amino acid sequences and molecular weights. One of the selected peptides was classified as short because it had nine amino acids in its composition, while the other two belonged to polypeptides and consisted of 17 amino acids. Sample TT1 was identified as the NCI_CGAP_Brn23 peptide, which is known to affect prenatal development of the central nervous system. Peptides TT2 and TT3 were not identified. We isolated a polypeptide with a molecular weight of 18.0 kDa from the ammonium sulfate-precipitated trypsin hydrolyzate supernatant of bovine colostrum. The peptide was identified as CO950255 protein sus scrofa, but its functions remained unknown. The peptides demonstrated activity against gram-positive bacteria Escherichia coli, gram-negative bacteria Bacillus subtilis, and diploid fungus Candida albicans. We infected laboratory Wistar rats with various doses of
Salmonella enteritidis 92, and the orally-administered trypsin hydrolyzate promoted their survival, as well as reduced the LD50 and the mean day of death period. The research proved the antimicrobial and immunotropic properties of the peptides obtained from the trypsin hydrolyzate of bovine colostrum.
Contribution
All authors provided critical feedback and helped shape the research, analysis and manuscript.
Conflict of interest
The authors declared no conflict of interests regarding the publication of this article.
Критерии авторства
Авторы в равной степени принимали участие в исследовании и оформлении рукописи.
Конфликт интересов
Авторы заявляют об отсутствии конфликта интересов.
References/Список литературы
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