Food Processing: Techniques and Technology Food Processing: Techniques and Technology Техника и технология пищевых производств 2074-9414 2313-1748 100432 10.21603/2074-9414-2025-2-2573 ОРИГИНАЛЬНАЯ СТАТЬЯ ORIGINAL ARTICLE ОРИГИНАЛЬНАЯ СТАТЬЯ Activity and Composition of Hydrolases Secreted by Thermophilic Strain Bacillus sp. Grown on Plant Flour Media Активность и состав секретируемых гидролаз термофильного штамма Bacillus sp. при культивировании на средах c растительной мукой https://orcid.org/0000-0003-3109-8445 Романова Мария Васильевна Romanova Mariia V. romanova.m.v@muctr.ru https://orcid.org/0009-0008-9627-7499 Макеев Фёдор Дмитриевич Makeev Fedor D. https://orcid.org/0009-0000-7076-1794 Мирзалиева Наргиз Ахмедовна Mirzalieva Nargiz A. https://orcid.org/0000-0002-4808-5002 Евдокимова Светлана Александровна Evdokimova Svetlana A. https://orcid.org/0000-0002-4425-8068 Белодед Андрей Васильевич Beloded Andrey V. Российский химико-технологический университет имени Д. И. Менделеева Москва Россия Dmitry Mendeleev University of Chemical Technology of Russia Moscow Russian Federation Российский химико-технологический университет имени Д. И. Менделеева Москва Россия Dmitry Mendeleev University of Chemical Technology of Russia Moscow Russian Federation Российский химико-технологический университет имени Д. И. Менделеева Москва Россия Dmitry Mendeleev University of Chemical Technology of Russia Moscow Russian Federation Российский химико-технологический университет имени Д. И. Менделеева Москва Россия Dmitry Mendeleev University of Chemical Technology of Russia Moscow Russian Federation Российский химико-технологический университет имени Д. И. Менделеева Москва Россия Dmitry Mendeleev University of Chemical Technology of Russia Moscow Russian Federation 23 06 2025 23 06 2025 55 2 300 314 03 07 2024 01 04 2025 https://fptt.ru/en/issues/23587/23620/

Растительное сырье – доступный и перспективный субстрат для получения индивидуальных ферментов и мультиэнзимных композиций разного назначения. Эффективными продуцентами для промышленного производства ферментных препаратов могут стать термофильные бактерии, поскольку их ферменты обладают рядом преимуществ – термоустойчивостью и химической стабильностью. Актуальным является поиск термофильных штаммов, способных продуцировать промышленно ценные ферменты с высоким выходом при использовании недорогих растительных субстратов. Цель исследования – изучить состав комплекса гидролитических ферментов термофильного штамма-продуцента протеаз Bacillus subtilis Кб.12.Гл.35 при культивировании на средах с компонентами растительного происхождения и выбрать перспективное сырье для синтеза термостабильных гидролаз, в частности протеаз, амилаз и гемицеллюлаз. Объект исследования – термофильный штамм Кб.12.Гл.35, выделенный из образца компоста и идентифицированный методом секвенирования гена 16S рРНК как Bacillus subtilis. Для исследования состава комплекса гидролаз штамм выращивали на средах с растительной мукой в комбинации с дрожжевым или кукурузным экстрактом, оценивали активность внеклеточных ферментов. В качестве растительной муки в количестве 1 % использовали гороховую, льняную, нутовую, овсяную, рисовую, амарантовую, соевую, а также смесь на основе амарантовой и соевой муки. Активность протеаз определяли по казеину и на основе зимографического анализа, активность карбогидраз – методом с 3,5-динитросалициловой кислотой. При культивировании продуцента гидролаз на питательных средах с растительной мукой (1 %) и кукурузным экстрактом (0,5 %) максимальный уровень синтеза протеаз (более 400 ед/мл) выявлен при использовании амарантовой, овсяной и рисовой муки; амилаз (более 1300 ед/мл) – нутовой, овсяной и рисовой муки; галактоманнаназ (более 200 ед/мл) и ксиланаз (более 60 ед/мл) – амарантовой, гороховой и нутовой; арабиногалактаназ (более 35 ед/мл) – амарантовой, нутовой и рисовой муки. По результатам зимографического анализа можно предположить, что наибольшую роль в протеолитической активности играют ферменты массой 24,7–28,2 кДа, а также протеазы массой 62,7–75,0 кДа, т. к. их активность ярко выражена при культивировании на растительном сырье, в отличие от стандартной среды LB. Секреция протеазы массой 15,1 кДа на сырье практически не отличалась от ее уровня на стандартной среде. Термофильный штамм Кб.12.Гл.35 можно рассматривать в качестве перспективного продуцента для получения протеаз, амилаз и гемицеллюлаз при использовании в составе среды растительной муки и кукурузного экстракта. Дальнейшие исследования могут быть направлены на оптимизацию состава питательных сред и условий культивирования для повышения выхода целевой группы гидролитических ферментов.

Plant raw materials are an accessible and efficient substrate for multipurpose enzymes and multi-enzyme complexes obtaining. Thermophilic bacteria are reliable producer strains for industrial enzymes with valuable physicochemical properties, e.g., heat resistance, stability at extreme pH, and chemical stability. Food scientists are on the lookout for new thermophilic strains capable of producing high yields of industrially valuable enzymes from cheap plant raw materials. In this research, various plant cultivation media affected the complex hydrolytic enzymes produced by a thermophilic strain of Bacillus subtilis Kb.12.Gl.35. The research objective was to select the optimal raw materials for the synthesis of thermostable hydrolases, i.e., proteases, amylases, and hemicellulases. The thermophilic strain was isolated from a compost sample, identified by 16S rRNA sequencing, and tested using standard microbiological and biochemical methods. The strain grew on different media with plant flour combined with yeast or corn extract. The activity of extracellular enzymes made it possible to study the composition of the hydrolase complex. The proteolytic activity was determined using casein as the substrate and by zymography. The 3,5-dinitrosalicylic acid (DNS) method revealed the carbohydrase activity. When culturing the hydrolase producer on nutrient media with plant flour (1%) and corn extract (0.5%), the highest level of protease synthesis (≥ 40 units/mL) was detected in the samples with amaranth, oat, and rice flour. The highest level of amylase (≥ 1,300 units/mL) belonged to the chickpea, oat, and rice flour samples. The biggest yields of galactomannanase (≥ 200 units/mL) and xylanases (≥ 60 units/mL) was found in the samples with amaranth, pea, and chickpea samples. The biggest amount of arabinogalactanase (≥ 35 units/mL) belonged to the amaranth, chickpea, and rice flour. Based on the zymographic analysis, the enzymes of 24.7–28.2 kDa and the proteases of 62.7–75.0 kDa appeared to be the most efficient proteolytic agents. Their activity was obvious on plant raw materials, in contrast to the standard LB medium. The secretion level of 15.1 kDa proteases was the same on the plant media as on the standard medium. Bacillus subtilis Kb.12.Gl.35 proved to be an effective producer of proteases, amylases, and hemicellulases with plant flour and corn extract in the medium. Further studies are needed to optimize the composition of nutrient media and cultivation conditions to increase the yield of the target hydrolytic enzymes.

 Термофильные бактерии Bacillus протеазы амилазы растительное сырье зимография Thermophilic bacteria, Bacillus, proteases, amylases, plant raw materials, zymography

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