УДК 57.085.2:577.112:612.3
Табл. 2. Ил. 4. Библ. 40.
DOI: 10.21323/2071-2499-2023-4-40-45

Комплексная модель in vitro и ex vivo для изучения трансформации белков в процессе пищеварения

Чернуха И.М.¹, академик РАН, Василевская Е.Р.¹, канд. техн. наук, Арюзина М.А.¹, Кусай А.², Полищук Е.К.^1
1ФНЦ пищевых систем им. В.М. Горбатова
²Российский биотехнологический университет (РОСБИОТЕХ)
Ключевые слова: ферментолиз, электрофорез, перевариваемость, белок, изолированный кишечник,
Реферат:
Существующие способы оценки степени и механизмов деградации белковых соединений, которые попадают в организм человека с пищей, не отличаются комплексностью. С их помощью оценивается воздействие единичных ферментов на отдельные нутриенты, что не позволяет оценить реальный потенциал биоконверсии. В статье освещены многомерный процесс пищеварения in vitro и всасывания ex vivo кишечным эпителием белковых соединений, содержащихся в варёной индейке и в мясном продукте на её основе. Исследуемые объекты первоначально были подвергнуты двухстадийному ферментативному гидролизу в модели пищеварения по Покровскому и Ертанову (с использованием пепсина и панкреатина). В рамках оценки полноты биоконверсии белков в процессе гидролиза контролировались концентрация белка (Лоури, Бредфорд), свободных пептидов и аминокислот. Также был использован метод одномерного электрофореза для контроля процесса переваривания. После завершения моделирования этапа пищеварения мы погружали в полученные гидролизаты изолированный вывернутый тонкий кишечник крысы для оценки проницаемости продуктов гидролиза (белковых и пептидных соединений, аминокислот) стенками кишечника и оставляли для инкубации. После инкубации отобранная инкубационная среда и серозная жидкость из кишечного «мешка» была исследована на содержание пептидных соединений и белка. Было показано, что пептидные соединения мясного продукта и индейки подвергались дополнительному кишечному пищеварению средой кишечника. В результате показана эффективность апробируемой модели пищеварения, позволяющая оценивать переваримость пищевых продуктов, максимально приближенную к механизму in vivo; определять уровень образования потенциальных биоактивных соединений и отслеживать их дальнейший путь в организме.

Complex in vitro and ex vivo model to study protein transformation during digestion

Chernukha I.M.¹, Vasilevskaya E.R.¹, Aryuzina M.A.¹, Qusay A.², Polishchuk E.K.^1
1Gorbatov Research Center for Food Systems
²Russian Biotechnological University "Rosbiotech"
Key words: fermentolysis, electrophoresis, digestibility, protein, isolated intestine
Summary:
Existing methods for assessing a degree and mechanisms of degradation of protein compounds that enter the human body with food are not complex. They make it possible to assess the effect of single enzymes on individual nutrients, which does not allow assessing the real potential for bioconversion. In this article, we highlight the multidimensional process of in vitro digestion and ex vivo absorption by the intestinal epithelium of protein compounds contained in cooked turkey and a meat product based on it. The studied objects were initially subjected to two-stage enzymatic hydrolysis in the digestion model according to Pokrovsky and Ertanov (using pepsin and pancreatin). As part of assessing the completeness of protein bioconversion during hydrolysis, the concentrations of protein (Lowry, Bradford), free peptides, and amino acids were controlled. The method of one-dimensional electrophoresis was also used to control the digestion process. After completing the modeling of the digestion stage, we immersed the isolated everted rat small intestine into the obtained hydrolysates to assess the permeability of hydrolysis products (protein and peptide compounds, amino acids) by the intestinal walls and left for incubation. After incubation, the selected incubation medium and serous fluid from the gut sac were examined for the content of peptide compounds and protein. It was shown that the peptide compounds of the meat product and turkey were subjected to additional intestinal digestion by the environment of the intestine. As a result, the effectiveness of the tested model of digestion has been shown, which makes it possible to evaluate the digestibility of food products, as close as possible to the in vivo mechanism, determine the level of formation of potential bioactive compounds and track their further path in the body.

СПИСОК ЛИТЕРАТУРЫ / REFERENCES:

Fu, Y. Exploration of collagen recovered from animal by-products as a precursor of bioactive peptides: Successes and challenges. / Y. Fu, M. Therkildsen, R.E. Aluko [et al.] // Critical Reviews in Food Science and Nutrition. – 2019. – V. 59. – № 13. P. 2011-2027. DOI: 10.1080/10408398.2018.1436038.

Horner, K. Bioavailability of milk protein-derived bioactive peptides: A glycaemic management perspective / K. Horner, E. Drummond, L. Brennan // Nutrition Research Reviews. – 2016. – V. 29. – № 1. – P. 91-101. DOI: 10.1017/S0954422416000032.

Górska-Warsewicz, H. Food Products as Sources of Protein and Amino Acids-The Case of Poland / H. Górska-Warsewicz, W. Laskowski, O. Kulykovets, [et al.] // Nutrients. – 2018. – V. 10. – № 12. – P.1977. DOI: 10.3390/nu10121977.

Schutz, Y. Protein turnover, ureagenesis and gluconeogenesis // International journal for vitamin and nutrition research. – 2011. – V. 81. – № 2-3. – P. 101-107. DOI: 10.1024/0300-9831/a000064.

Gorissen, S. Protein Type, Protein Dose, and Age Modulate Dietary Protein Digestion and Phenylalanine Absorption Kinetics and Plasma Phenylalanine Availability in Humans / S. Gorissen, J. Trommelen, I. Kouw [et al.] // The Journal of nutrition. – 2020. – V. 150. – № 8. – Р. 2041-2050. DOI: 10.1093/jn/nxaa024.

Santana Luz, A.B. What are the digestion and absorption models used to reproduce gastrointestinal protein processes? A protocol for systematic review / A.B. Santana Luz, R.O. de Araújo Costa, G. de Medeiros [et al.] // Medicine. – 2021. – V. 100. – № 30. – P. e26697. DOI: 10.1097%2FMD.0000000000026697.

Kim, D.Y. A neural circuit mechanism for mechanosensory feedback control of ingestion / D.Y. Kim, G. Heo, M. Kim [et al.] // Nature. – 2020. – V. 580. – № 7803. – P. 376-380. DOI: 10.1038/s41586-020-2167-2.

Yılmaz, C. Neuroactive compounds in foods: Occurrence, mechanism and potential health effects / C. Yılmaz, V. Gökmen // Food Research International. – 2020. – V. 128. – P. 108744. DOI: 10.1016/j.foodres.2019.108744.

Bertechini, A.G. Apparent total tract and ileal amino acids digestibility values of vegetal protein meals with dietary protease to broiler diet / A.G. Bertechini, F.S. Dalolio, J.C. Carvalho [et al.] // Translational Animal Science. – 2020. – V. 4. – № 4. – P. txaa219. DOI: 10.1093/tas/txaa219.

Ravindran, V. Progress in ileal endogenous amino acid flow research in poultry // Journal of Animal Science and Biotechnology. – 2021. – V. 12. – P. 1-11.

Chernukha, I.M. Evolution of in vitro digestibility techniques: a systematic review / I.M. Chernukha, A.V. Meliashchenia, I.V. Kaltovich E.R. [et al.] // Theory and practice of meat processing. – 2021. – V. 6. – № 4. – P. 300-310. DOI: 10.21323/2414-438X-2021-6-4-300-310.

Покровский, А.А. Атакуемость белков пищевых продуктов протеолитическими ферментами in vitro / А.А. Покровский, И.Д. Ертанов // Вопросы питания. – 1965. – № 3. – С. 38-44.

Pokrovskii, А.А. Pokrovskiy, A.A. Atakuyemost' belkov pishchevykh produktov proteoliticheskimi fermentami in vitro [Susceptibility of nutritional proteins to proteolytic enzymes in vitro] / А.А. Pokrovskii, I.D. Ertanov // Voprosy Pitaniia. – 1965. – № 3. – P. 38-44.

Василевская, Е.Р. Биоконверсия in vitro: сравнение мультиферментных систем INFOGEST 2.0 и протокола Покровского-Ертанова / Е.Р. Василевская, М.А. Арюзина, А.Г. Ахремко // Все о мясе. – 2022. – № 6. – С. 44-49. DOI: 10.21323/2071-2499-2022-6-44-49.

Biokonversiya in vitro: sravneniye mul'tifermentnykh sistem INFOGEST 2.0 i protokola Pokrovskogo-Yertanova [In vitro bioconversion: comparison of multi-enzyme systems INFOGEST 2.0 and Pokrovkiy-Ertanov protocol] / E.R. Vasilevskaya, М.А. Aruzina, А.G. Аkhremko // Vsyo o myase. – 2022. – № 6. – P. 44-49. DOI: 10.21323/2071-2499-2022-6-44-49.

Sousa, R. Protein digestion of different protein sources using the INFOGEST static digestion model / R. Sousa, R. Portmann, S. Dubois [et al.] // Food research international. – 2020. – V. 130. – P. 108996.

Vasilevskaya, E.R. Study of the functional product’s protein compounds digestion features / E.R. Vasilevskaya, A.G. Akhremko, E.K. Polishchuk [et al.] // Theory and Practice of Meat Processing. – 2020. – V. 5. – № 3. – P. 18-21. DOI: 10.21323/2414-438X-2020-5-3-18-21.

Ansia, I. Nutrient digestibility and endogenous protein losses in the foregut and small intestine of weaned dairy calves fed calf starters with conventional or enzyme-treated soybean meal / I. Ansia, H.H. Stein, C. Brøkner [et al.] // Journal of Dairy Science. – 2021. – V. 104. – № 3. – P. 2979-2995. DOI: 10.3168/jds.2020-18776.

Moughan, P.J. Amino acid digestibility and availability in foods and feedstuffs / P.J. Moughan // Proceedings of the 9th international symposium on digestive physiology in pigs, 2003. – P. 199-221.

Gelberg, H.B. Comparative anatomy, physiology, and mechanisms of disease production of the esophagus, stomach, and small intestine / H.B. Gelberg // Toxicologic Pathology. – 2014. – V. 42. – № 1. – P. 54-66. DOI: 10.1177/0192623313518113.

Kaminsky, L.S. The small intestine as a xenobiotic-metabolizing organ / L.S. Kaminsky, Q.-Y. Zhang // Drug Metabolism and Disposition. – 2003. – V. 31. – № 12. – P. 1520-1525. DOI: 10.1124/dmd.31.12.1520.

Reed, K.K. Review of the gastrointestinal tract: from macro to micro / K.K. Reed, R. Wickham // Seminars in Oncology Nursing. – 2019. – V. 25 – № 1. – P. 3-14. DOI: 10.1016/j.soncn.2008.10.002.

Thummel, K.E. Enzyme-catalyzed processes of firstpass hepatic and intestinal drug extraction / K.E. Thummel, K.L. Kunze, D.D. Shen // Advanced Drug Delivery Reviews. – 1997. – V. 27. – № 23. – P. 99-127. DOI: 10.1016/S0169-409X(97)00039-2.

Rao, J.N. Intestinal Architecture and Development / J.N. Rao, J.-Y. Wang // In Regulation of Gastrointestinal Mucosal Growth. – San Rafael (CA): Morgan & Claypool Life Sciences, 2010. – 220 p.

Collins, J.T. Anatomy, Abdomen, Small Intestine / J.T. Collins, S.S. Bhimji // Treasure Island (FL): StatPearls Publishing, 2017. – 540 p.

Tortora, G.J. Principles of anatomy and physiology / G.J. Tortora, B.H. Derrickson // Hoboken (NJ): John Wiley & Sons, 2008. – 87 p.

Reinus, J.F. Gastrointestinal anatomy and physiology: the essentials / J.F. Reinus, D. Simon // Oxford, UK: John Wiley & Sons, 2014. – 60 p.

Shroyer, N.F. Anatomy and physiology of the small and large intestines / N.F. Shroyer, S.A. Kocoshis // In Pediatric Gastrointestinal and Liver Disease. – Philadelphia, PA: Saunders Elsevier, 2011. – Р. 324-336.

Krüger, P. Metabolism of boswellic acids in vitro and in vivo / P. Krüger, R. Daneshfar, G.P. Eckert [et al.] // Drug metabolism and disposition. – 2008. – V. 36. – № 6. – P. 1135-1142. DOI: 10.1124/dmd.107.018424.

Luo, Z. Ex vivo and in situ approaches used to study intestinal absorption / Z. Luo, Y. Liu, B. Zhao [et al.] // Journal of pharmacological and toxicological methods. – 2013. – V. 68. – № 2. – P. 208-216. DOI: 10.1016/j.vascn.2013.06.001.

Jung, S.M. In vitro models of the small intestine for studying intestinal diseases / S.M. Jung, S. Kim // Frontiers in Microbiology. – 2022. – V. 12. – P. 4102. DOI: 10.3389/fmicb.2021.767038.

Vasilevskaya, E.R. Research methodology of Sus scrofa tissue extracts protein-peptide components / E.R. Vasilevskaya, E.A. Kotenkova, E.A. Lukinova [et al.] // Theory and practice of meat processing. – 2017. – V. 2. – № 3. – P. 79-85. DOI: 10.21323/2414-438X-2017-2-3-79-85.

Li, L. Proteome profiles of digested products of commercial meat sources / L. Li, Y. Liu, G. Zhou [et al.] // Frontiers in Nutrition. – 2017. – V. 4. – P. 8. DOI: 10.3389/fnut.2017.00008.

Wen, S. Discrimination of in vitro and in vivo digestion products of meat proteins from pork, beef, chicken, and fish / S. Wen, G. Zhou, S. Song [et al.] // Proteomics. – 2015. – V. 15. – № 21. – P. 3688-3698. DOI: 10.1002/pmic.201500179.

Mészáros, P. Investigation of Intestinal Absorption and Excretion of Paracetamol in Streptozotocin-Induced Hyperglycemia / P. Mészáros, S. Kovács, G. Kulcsár [et al.] // International Journal of Molecular Sciences. – 2022. – V. 23. – № 19. P. 11913. DOI: 10.3390/ijms231911913.

Kiela, P.R. Physiology of Intestinal Absorption and Secretion / P.R. Kiela, F.K. Ghishan // Best Practice & Research Clinical Gastroenterology. – 2016. – V. 30. – № 2. – P. 145-159. DOI: 10.1016/j.bpg.2016.02.007.

Boyd, B.J. Successful oral delivery of poorly water-soluble drugs both depends on the intraluminal behavior of drugs and of appropriate advanced drug delivery systems / B.J. Boyd, C.A.S. Bergström, Z. Vinarov [et al.] // European Journal of Pharmaceutical Sciences. – 2019. – V. 137. – P. 104967. DOI: 10.1016/j.ejps.2019.104967.

Nunes, R. Tissue-based in vitro and ex vivo models for intestinal permeability studies / R. Nunes, C. Silva, L. Chaves // In Concepts and Models for Drug Permeability Studies. – Elsevier. – 2016. – P. 203-236.

Chen, M.-Ch. A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery / M.-Ch. Chen, K. Sonaje, K.-J. Chen [et al.] // Biomaterials. – 2011. – V. 32. – № 36. – P. 9826-9838. DOI: 10.1016/j.biomaterials.2011.08.087.

Renukuntla, J. Approaches for enhancing oral bioavailability of peptides and proteins / J. Renukuntla, A.D. Vadlapudi, A. Patel [et al.] // International Journal of Pharmaceutics. – 2013. – V. 447. – № 1-2. – P. 75-93. DOI: 10.1016/j.ijpharm.2013.02.030.

Patel, G. Oral Delivery of Proteins and Peptides / G. Patel, A. Misra // In Challenges in Delivery of Therapeutic Genomics and Proteomics. – Elsevier. – 2011. – P. 481-529.

40. Xie, Y. Real meat and plant-based meat analogues have different in vitro protein digestibility properties / Y. Xie, L. Cai, D. Zhao, H. Liu, X. Xu, G. Zhou, C. Li // Food Chemistry. – 2022. – V. 387. – Article № 132917. DOI: 10.1016/j.foodchem.2022.132917.

Контакты:

Чернуха Ирина Михайловна
imcher@inbox.ru
Василевская Екатерина Романовна
e.vasilevskaya@fncps.ru
Арюзина Марина Александровна
m.aryuzina@fncps.ru
Кусай Абутраби
qusay2077@gmail.com
Полищук Екатерина Константиновна
e.politchuk@fncps.ru

Для цитирования:

Чернуха, И.М. Комплексная модель in vitro и ex vivo для изучения трансформации белков в процессе пищеварения / И.М. Чернуха, Е.Р. Василевская, М.А. Арюзина, А. Кусай, Е.К. Полищук // Все о мясе. – 2023. – № 4. – С. 40-45. DOI: 10.21323/2071-2499-2023-4-40-45.

For citation:

Chernukha, I.M. Complex in vitro and ex vivo model to study protein transformation during digestion / I.M. Chernukha, E.R. Vasilevskaya, M.A. Aryuzina, A. Qusay, E.K. Polishchuk // Vsyo o myase. – 2023. – № 4. – Р. 40-45. DOI: 10.21323/2071-2499-2023-4-40-45.