tatpipТехника и технология пищевых производствFood Processing: Techniques and Technology2074-94142313-1748Кемеровский государственный университет10.21603/2074-9414-2023-1-2422tatpip-43Research ArticleСтатьиПептиды трипсинового гидролизата молозива коровPeptides of Trypsin Hydrolyzate in Bovine Colostrumhttps://orcid.org/0000-0003-4863-9834ТихоновС. Л.TikhonovS. L.

Екатеринбург

Sergei L. Tikhonov

Yekaterinburg

tihonov75@bk.ruhttps://orcid.org/0000-0001-5841-1791ТихоноваН. В.TikhonovaN. V.

Екатеринбург

Natalia V. Tikhonova

Yekaterinburg

ТурсуновХ. Х.TursunovKh. Kh.

Андижан

Khatam Kh. Tursunov

Andijan

https://orcid.org/0000-0001-6841-1197ДаниловаИ. Г.DanilovaI. G.

Екатеринбург

Irina G. Danilova

Yekaterinburg

https://orcid.org/0000-0002-0470-7324ЛазаревВ. А.LazarevV. A.

Екатеринбург

Vladimir A. Lazarev

Yekaterinburg

Уральский государственный экономический университетРоссияUral State University of EconomicsRussian FederationАндижанский государственный медицинский институтУзбекистанAndijan State Medical InstituteUzbekistanИнститут иммунологии и физиологии Уральского отделения Российской академии наукРоссияInstitute of Immunology and Physiology of the Ural Branch of the Russian Academy of SciencesRussian Federation202317082026531150158Copyright © Тихонов С.Л., Тихонова Н.В., Турсунов Х.Х., Данилова И.Г., Лазарев В.А., 20262026Тихонов С.Л., Тихонова Н.В., Турсунов Х.Х., Данилова И.Г., Лазарев В.А.Tikhonov S.L., Tikhonova N.V., Tursunov K.K., Danilova I.G., Lazarev V.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.fptt.ru/jour/article/view/43

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.

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.

молозивомолочный белокферментгидролизантимикробная активностьпротивогрибковая активностьбиологически активные веществаcolostrummilk proteinenzymehydrolysisantimicrobial activityantifungal activitybiologically active substancesReferencesGłówka N, Woźniewicz M. Potential use of Colostrum bovinum supplementation in athletes – A review. Acta Scientiarum Polonorum Technologia Alimentaria. 2019;18(2):115–123. https://doi.org/10.17306/J.AFS.2019.0654Główka N, Woźniewicz M. Potential use of Colostrum bovinum supplementation in athletes – A review. Acta Scientiarum Polonorum Technologia Alimentaria. 2019;18(2):115–123. https://doi.org/10.17306/J.AFS.2019.0654Van Hese I, Goossens K, Vandaele L, Opsomer G. Invited review: MicroRNAs in bovine colostrum – Focus on their origin and potential health benefits for the calf. Journal of Dairy Science. 2020;103(1):1–15. https://doi.org/10.3168/jds.2019-16959Van Hese I, Goossens K, Vandaele L, Opsomer G. Invited review: MicroRNAs in bovine colostrum – Focus on their origin and potential health benefits for the calf. Journal of Dairy Science. 2020;103(1):1–15. https://doi.org/10.3168/jds.2019-16959Sukhikh SA, Astakhova LA, Golubcova YuV, Lukin AA, Prosekova EA, Milent`eva IS, et al. Functional dairy products enriched with plant ingredients. Foods and Raw Materials. 2019;7(2):428–438. http://doi.org/10.21603/2308-4057-2019-2-428-438Sukhikh SA, Astakhova LA, Golubcova YuV, Lukin AA, Prosekova EA, Milent`eva IS, et al. Functional dairy products enriched with plant ingredients. Foods and Raw Materials. 2019;7(2):428–438. http://doi.org/10.21603/2308-4057-2019-2-428-438Kharitonov VD, Asafov VA, Iskakova EL, Tankova NL, Halavach TM, Kurchenko VP. Quality control of colostrum and protein calf milk replacers. Food Processing: Techniques and Technology. 2021;51(1):188–195. https://doi.org/10.21603/2074-9414-2021-1-188-195.Kharitonov VD, Asafov VA, Iskakova EL, Tankova NL, Halavach TM, Kurchenko VP. Quality control of colostrum and protein calf milk replacers. Food Processing: Techniques and Technology. 2021;51(1):188–195. https://doi.org/10.21603/2074-9414-2021-1-188-195.Chandwe K, Kelly P. Colostrum therapy for human gastrointestinal health and disease. Nutrients. 2021;13(6). https://doi.org/10.3390/nu13061956Chandwe K, Kelly P. Colostrum therapy for human gastrointestinal health and disease. Nutrients. 2021;13(6). https://doi.org/10.3390/nu13061956Menchetti L, Traina G, Tomasello G, Casagrande-Proietti P, Leonardi L, Barbato O, et al. Potential benefits of colostrum in gastrointestinal diseases. Frontiers in Bioscience-Scholar. 2016;8(2):331–351. https://doi.org/10.2741/S467Menchetti L, Traina G, Tomasello G, Casagrande-Proietti P, Leonardi L, Barbato O, et al. Potential benefits of colostrum in gastrointestinal diseases. Frontiers in Bioscience-Scholar. 2016;8(2):331–351. https://doi.org/10.2741/S467Playford RJ, Weiser MJ. Bovine colostrum: Its constituents and uses. Nutrients. 2021;13(1). https://doi.org/10.3390/nu13010265Playford RJ, Weiser MJ. Bovine colostrum: Its constituents and uses. Nutrients. 2021;13(1). https://doi.org/10.3390/nu13010265Sanctuary MR, Kain JN, Chen SY, Kalanetra K, Lemay DG, Rose DR, et al. Pilot study of probiotic/colostrum supplementation on gut function in children with autism and gastrointestinal symptoms. PLoS ONE. 2019;14(1). https://doi.org/10.1371/journal.pone.0210064Sanctuary MR, Kain JN, Chen SY, Kalanetra K, Lemay DG, Rose DR, et al. Pilot study of probiotic/colostrum supplementation on gut function in children with autism and gastrointestinal symptoms. PLoS ONE. 2019;14(1). https://doi.org/10.1371/journal.pone.0210064Jørgensen ALW, Juul-Madsen HR, Stagsted J. Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells. Journal of Peptide Science. 2010;16(1):21–30. https://doi.org/10.1002/psc.1190Jørgensen ALW, Juul-Madsen HR, Stagsted J. Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells. Journal of Peptide Science. 2010;16(1):21–30. https://doi.org/10.1002/psc.1190Korhonen HJ. Production and properties of health-promoting proteins and peptides from bovine colostrum and milk. Cellular and Molecular Biology. 2013;59(1):12–24.Korhonen HJ. Production and properties of health-promoting proteins and peptides from bovine colostrum and milk. Cellular and Molecular Biology. 2013;59(1):12–24.Novoselova MV, Prosekov AYu. Technological options for the production of lactoferrin. Foods and Raw Materials. 2016;4(1):90–101. http://doi.org/10.21179/2308-4057-2016-1-90-101Novoselova MV, Prosekov AYu. Technological options for the production of lactoferrin. Foods and Raw Materials. 2016;4(1):90–101. http://doi.org/10.21179/2308-4057-2016-1-90-101Функциональные свойства кисломолочных продуктов с гидролизатами сывороточных белков / О. В. Королёва [и др.] // Молочная промышленность. 2013. № 11. С. 52–55.Koroleva OV, Agarkova EYu, Botina SG, Nikolaev IV, Ponomareva NV, Melnikova EI, et al. Functional proper ties of fermented milk products with whey protein hydrolysates. Dairy Industry. 2013;(11):52–55. (In Russ.).Pradeep H, Najma U, Aparna HS. Milk peptides as novel multi-targeted therapeutic candidates for SARS-CoV2. The Protein Journal. 2021;40(3):310–327. https://doi.org/10.1007/s10930-021-09983-8Pradeep H, Najma U, Aparna HS. Milk peptides as novel multi-targeted therapeutic candidates for SARS-CoV2. The Protein Journal. 2021;40(3):310–327. https://doi.org/10.1007/s10930-021-09983-8Rezaei Ahvanooei MR, Norouzian MA, Vahmani P. Beneficial Effects of vitamins, minerals, and bioactive peptides on strengthening the immune system against COVID-19 and the role of cow’s milk in the supply of these nutrients. Biological Trace Element Research. 2021;200(11):4664–4677. https://doi.org/10.1007/s12011-021-03045-xRezaei Ahvanooei MR, Norouzian MA, Vahmani P. Beneficial Effects of vitamins, minerals, and bioactive peptides on strengthening the immune system against COVID-19 and the role of cow’s milk in the supply of these nutrients. Biological Trace Element Research. 2021;200(11):4664–4677. https://doi.org/10.1007/s12011-021-03045-xKütt M-L, Stagsted J. Caseins from bovine colostrum and milk strongly bind piscidin-1, an antimicrobial peptide from fish. International Journal of Biological Macromolecules. 2014;70:364–372. https://doi.org/10.1016/j.ijbiomac.2014.06.063Kütt M-L, Stagsted J. Caseins from bovine colostrum and milk strongly bind piscidin-1, an antimicrobial peptide from fish. International Journal of Biological Macromolecules. 2014;70:364–372. https://doi.org/10.1016/j.ijbiomac.2014.06.063Shteinfer-Kuzmine A, Amsalem Z, Arif T, Zooravlov A, Shoshan-Barmatz V. Selective induction of cancer cell death by VDAC1-based peptides and their potential use in cancer therapy. Molecular Oncology. 2018;12(7):1077–1103. https://doi.org/10.1002/1878-0261.12313Shteinfer-Kuzmine A, Amsalem Z, Arif T, Zooravlov A, Shoshan-Barmatz V. Selective induction of cancer cell death by VDAC1-based peptides and their potential use in cancer therapy. Molecular Oncology. 2018;12(7):1077–1103. https://doi.org/10.1002/1878-0261.12313Ko S-C, Kim D, Jeon Y-J. Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology. 2012;50(7):2294–2302. https://doi.org/10.1016/j.fct.2012.04.022Ko S-C, Kim D, Jeon Y-J. Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology. 2012;50(7):2294–2302. https://doi.org/10.1016/j.fct.2012.04.022Poirier K, Van Esch H, Friocourt G, Saillour Y, Bahi N, Backer S, et al. Neuroanatomical distribution of ARX in brain and its localisation in GABAergic neurons. Molecular Brain Research. 2004;122(1):35–46. https://doi.org/10.1016/j.molbrainres.2003.11.021Poirier K, Van Esch H, Friocourt G, Saillour Y, Bahi N, Backer S, et al. Neuroanatomical distribution of ARX in brain and its localisation in GABAergic neurons. Molecular Brain Research. 2004;122(1):35–46. https://doi.org/10.1016/j.molbrainres.2003.11.021Morgan AJ, Riley LG, Sheehy PA, Wynn PC. The influence of protein fractions from bovine colostrum digested in vivo and in vitro on human intestinal epithelial cell proliferation. Journal of Dairy Research. 2014;81(1):73–81. https://doi.org/10.1017/S0022029913000654Morgan AJ, Riley LG, Sheehy PA, Wynn PC. The influence of protein fractions from bovine colostrum digested in vivo and in vitro on human intestinal epithelial cell proliferation. Journal of Dairy Research. 2014;81(1):73–81. https://doi.org/10.1017/S0022029913000654Birkemo GA, O’Sullivan O, Ross RP, Hill C. Antimicrobial activity of two peptides casecidin 15 and 17, found naturally in bovine colostrum. Journal of Applied Microbiology. 2009;106(1):233–240. https://doi.org/10.1111/j.1365-2672.2008.03996.xBirkemo GA, O’Sullivan O, Ross RP, Hill C. Antimicrobial activity of two peptides casecidin 15 and 17, found naturally in bovine colostrum. Journal of Applied Microbiology. 2009;106(1):233–240. https://doi.org/10.1111/j.1365-2672.2008.03996.xSears KT, Tennant SM, Reymann MK, Simon R, Konstantopoulos N, Blackwelder WC, et al. Bioactive immune components of anti-diarrheagenic enterotoxigenic Escherichia coli hyperimmune bovine colostrum products. Clinical and Vaccine Immunology. 2017;24(8). https://doi.org/10.1128/CVI.00186-16Sears KT, Tennant SM, Reymann MK, Simon R, Konstantopoulos N, Blackwelder WC, et al. Bioactive immune components of anti-diarrheagenic enterotoxigenic Escherichia coli hyperimmune bovine colostrum products. Clinical and Vaccine Immunology. 2017;24(8). https://doi.org/10.1128/CVI.00186-16Anderson RC, Dalziel JE, Haggarty NW, Dunstan KE, Gopal PK, Roy NC. Short communication: Processed bovine colostrum milk protein concentrate increases epithelial barrier integrity of Caco-2 cell layers. Journal of Dairy Science. 2019;102(12):10772–10778. https://doi.org/10.3168/jds.2019-16951Anderson RC, Dalziel JE, Haggarty NW, Dunstan KE, Gopal PK, Roy NC. Short communication: Processed bovine colostrum milk protein concentrate increases epithelial barrier integrity of Caco-2 cell layers. Journal of Dairy Science. 2019;102(12):10772–10778. https://doi.org/10.3168/jds.2019-16951Playford RJ, Weiser MJ, Marchbank T. Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar. PLoS ONE. 2021;16(6). https://doi.org/10.1371/journal.pone.0253422Playford RJ, Weiser MJ, Marchbank T. Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar. PLoS ONE. 2021;16(6). https://doi.org/10.1371/journal.pone.0253422

The authors declare that there are no conflicts of interest present.