spfpХранение и переработка сельхозсырьяStorage and Processing of Farm Products2072-96692658-767XРОСБИОТЕХ10.36107/spfp.2022.341spfp-341Research ArticleБИОТЕХНОЛОГИЧЕСКИЕ И МИКРОБИОЛОГИЧЕСКИЕ АСПЕКТЫBIOTECHNOLOGICAL AND MICROBIOLOGICAL ASPECTSОпределение протеолитической активности молочнокислых бактерий и выявление генов протеиназDetermination of the proteolytic activity of lactic acid bacteria and identification of proteinase geneshttps://orcid.org/0000-0002-8864-602XКоврижныхАнна ВладиславовнаКоvrijhnykhАnna V.anya-1998@mail.ruhttps://orcid.org/0000-0002-7463-4503АфанасьевДмитрий АлексеевичAfanasyevDmitry A.dmitr.afanasjew2010@yandex.ruhttps://orcid.org/0000-0003-4796-2066АхангаранМахбубехAhangaranMahboobehahangaran@hotmail.comhttps://orcid.org/0000-0002-4318-7077ГаравириМахмудGharaviriMahmoodgharaviri@hotmail.comhttps://orcid.org/0000-0003-4298-0927ЧернухаИрина МихайловнаChernukhaIrina M.imcher@inbox.ruhttps://orcid.org/0000-0002-9287-0585МашенцеваНаталья ГеннадьевнаMashentsevaNatalya G.natali-mng@yandex.ruhttps://orcid.org/0000-0003-4184-3306ВасилиевичНаталья ВладимировнаVasilievichNatalya V.vasilievich_n@mail.ru20220412202204102112Copyright © Коврижных А.В., Афанасьев Д.А., Ахангаран М., Гаравири М., Чернуха И.М., Машенцева Н.Г., Василиевич Н.В., 20222022Коврижных А.В., Афанасьев Д.А., Ахангаран М., Гаравири М., Чернуха И.М., Машенцева Н.Г., Василиевич Н.В.Коvrijhnykh А.V., Afanasyev D.A., Ahangaran M., Gharaviri M., Chernukha I.M., Mashentseva N.G., Vasilievich N.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.spfp-mgupp.ru/jour/article/view/341Введение

Введение. Биоактивные пептиды описываются как аминокислотные последовательности белков, оказывающие положительное влияние на биологические процессы и/или здоровье человека в целом. Они могут образовываться в высокобелковом животном и растительном сырье под действием ферментов стартовых и заквасочных культур in situ. Данные ферменты представляют собой протеиназы с молекулярной массой около 200 кДа, относящиеся к семейству PrtP. Цель. Целью исследования являлось определение генов протеиназ у молочнокислых и денитрифицирующих микроорганизмов, оценка их протеолитической активности и определение взаимосвязи между протеолитической активностью и генетическими детерминантами.Материалы и методы. Был оценен протеолитический потенциал микроорганизмов родов Latilactobacillus и Pediococcus, а также рода Staphylococcus из коллекции университета. Метод ТНБС использовался для измерения высвобождаемых аминогрупп. На агаровой среде с обезжиренным молоком были определены размеры зон просветления вокруг колоний микроорганизмов. Для определения наличия генов PRTP, PRTM, PRTB, PRTH и PRTR, отвечающих за протеолитическую активность, использовали 5 пар праймеров геномной ДНК (PrtP700/PrtM700; P15C/P06C; PRTB10/PRTB20; Jp23/Jp25; prti2/IP6Xba).Результаты. На молочном агаре все исследуемые микроорганизмы образовывали зоны просветления вокруг колоний, что указывает на их протеолитическую активность. Самые большие зоны задержки роста были вокруг колоний штамма Latilactobacillus curvatus 2, а также Pediococcus acidilactici 28 и Pediococcus acidilactici 38. По данным метода ТНБС, Latilactobacillus curvatus 1 и Staphylococcus carnosus 108 оказались наименее продуктивными по накоплению аминного азота. Результаты ПЦР показали, что максимальное количество генов протеиназ присутствует у штамма L. sakei 105. Но данный штамм не проявил высокой протеолитической активности на среде качественным и количественным методами. Поэтому для отбора штаммов с высокой протеолитической активностью нужен комплексный подход. Выводы. С помощью комплексного подхода были отобраны штаммы с высокой протеолитической активностью, потенциально способные к образования биоактивных пептидов в ферментированных пищевых продуктах.

 

 

Background

Background. Bioactive peptides are described as amino acid sequences of proteins that have a positive effect on biological processes and/or human health in general. They can be formed in high-protein animal and vegetable raw materials under the action of enzymes of starter and starter cultures in situ. These enzymes are proteinases with a molecular weight of about 200 kDa belonging to the PrtP family.Purpose. The aim of the study was to determine the genes of proteinases in lactic acid and denitrifying microorganisms, to evaluate their proteolytic activity and to determine the relationship between proteolytic activity and genetic determinants.Materials and methods. The proteolytic potential of microorganisms of the genera Latilactobacillus and Pediococcus, as well as the genus Staphylococcus from the university collection was evaluated. The TNBS method was used to measure the released amino groups. The sizes of the enlightenment zones around the colonies of microorganisms were determined by the cup method on milk agar. 5 pairs of genomic DNA primers (PrtP700/PrtM700; P15C/P06C; PRTB10/PRTB20; Jp23/Jp25; prti2/IP6Xba) were used to determine the presence of PRTP, PRTM, PRTB, PRTH and PRTR genes responsible for proteolytic activity.Results. On milk agar, all the studied microorganisms formed zones of enlightenment around the colonies, which indicates their proteolytic activity. The largest growth retardation zones were around colonies of the strain Latilactobacillus curvatus 2, as well as Pediococcus acidilactici 28 and Pediococcus acidilactici 38. According to the TNBS method, Latilactobacillus curvatus 1 and Staphylococcus carnosus 108 were the least productive in amine nitrogen accumulation. PCR results showed that the maximum number of proteinase genes is present in the strain L. sakei 105. But this strain did not show high proteolytic activity on the medium by qualitative and quantitative methods. Therefore, an integrated approach is needed to select strains with high proteolytic activity.Conclusions. Using an integrated approach, strains with high proteolytic activity, potentially capable of forming bioactive peptides in fermented foods, were selected.

 

 

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