WO2012060729A1 - Bacteria strain bacillius sp. - relic microorganisms isolated from permafrost rocks and having immuno-modulating and gero-protective activity - Google Patents

Bacteria strain bacillius sp. - relic microorganisms isolated from permafrost rocks and having immuno-modulating and gero-protective activity Download PDF

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WO2012060729A1
WO2012060729A1 PCT/RU2010/000654 RU2010000654W WO2012060729A1 WO 2012060729 A1 WO2012060729 A1 WO 2012060729A1 RU 2010000654 W RU2010000654 W RU 2010000654W WO 2012060729 A1 WO2012060729 A1 WO 2012060729A1
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bacillus
activity
permafrost
gero
relic
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Анатолий Викторович БРУШКОВ
Геннадий Иванович ГРИВА
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Brushkov Anatoly Viktorovich
Griva Gennady Ivanovich
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

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  • the invention relates to biotechnology, in particular, to strains of microorganisms and can be used to stimulate the activity of the immune system and increase the life expectancy of animals and humans.
  • the present invention relates to bacteria of the genus Bacillus, found in ancient permafrost of Yakutia.
  • a known bacterial strain Bacillus subtilis 1719 isolated in vivo and exhibiting a wide spectrum of antagonistic activity, low adhesive activity and immunomodulating activity (patent RU 2298032 C2, published October 27, 2007).
  • the patented strain of Bacillus sp. Unlike the known ones, was isolated from permafrost, which is many thousands of years old, and therefore is characterized by exceptional viability and ability to survive for a long time at low temperatures. In experiments with his culture, an increase in muscle strength, an increase in physical and mental activity, immunomodulation and a slowdown in aging are observed.
  • the specified technical result is achieved by a microorganism - a strain of Bacillus sp., Isolated from permafrost and possessing immunomodulating and geroprotective activity.
  • VKPM Industrial Microorganisms
  • samples were taken from natural outcrops. They are located on the left bank of the Aldan, 325 kilometers upstream from its confluence with the Lena, on Mammoth Mountain. Samples were selected in 0.9-1 m deeper than the seasonal thawing layer. The outcrop is destroyed by the river (more than a meter per year), so that the sediments from which the samples were taken were obviously in a permafrost state. In this case, the annual spring flushing of caving occurs, preventing blockages and mixing of rocks.
  • the latter are fine-grained sands and siltstones; their age corresponds to the Middle Miocene (Baranova Yu.P., Ilyinskaya I.A., Nikitin V.P., Pneva G.N., Fradkina A.F., Shvareva N.Ya. Miocene of the Mammoth Mountain. Proceedings of the GIN SB AS USSR Science, Moscow, 1976.284 s). Cooling began here at the end of the Pliocene, about 3 - 3.5 million years ago. The temperature in January for that time was estimated from -12 to -32 ° C, and in July from +12 to + 16 ° C.
  • Samples were taken using metal instruments sterilized with alcohol and burned in a flame (drills, tweezers, scalpels). In order to surface sterilize the samples, a sample weighing about 50 g was placed in a glass with 96% ethanol solution for one minute, and then in the burner flame for about 2 seconds and, finally, in a sterile tube. The selected rocks were stored at a temperature of -5 ° C, which was close to natural conditions. Samples were transported in refrigerated containers with frozen refrigerants.
  • Samples of various dilutions under sterile conditions were added to Petri dishes containing YPD, MRS and NA media (see Manual of environmental microbiology. 1997. Ed. Hurst C.J. FSM Press. Washington DC. 894 p.). Samples were also added to liquid meat-peptone broth under anaerobic and aerobic conditions.
  • Ribosomal DNA of the microbial culture was extracted using the Fast DNA kit for soil (BIO 101 Inc., Vista, CA), where a method based on the destruction of cells by glass beads is used.
  • Nucleic acids were precipitated from the solution using a solution consisting of 0.1 parts of 3 M sodium acetate (pH 5.2) and 2.5 parts of ethanol, incubated on ice, and then centrifuged for 30 minutes at a speed of 12,000 rpm. Precipitated nucleic acids were then dissolved in distilled water (free of RNase and DNase), and stored at -20 ° C. Fragments of 16S rRNA were amplified by NDP carried out with bacterial primers (27F; 5'-AGAGTTTGATCCTGGCTCAG-3 ', 1492R;
  • PCR was performed in a 20- ⁇ 1 volume using a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) as follows: 4 min at 94 ° C, then 30 cycles of 1 min at 94 ° C, 1 min at 50 ° C, and 1.5 min at 72 ° C, then 7 min at 72 ° C.
  • PCR amplicons were subjected electrophoresis and purification using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Purified amplicons were cloned using pCR2.1 vector, E.
  • plasmid DNA containing 16S rDNA was obtained using a Mini prep spin kit (Quiagen, Crawley, UK). The purified plasmid DNA was sequenced on an ABI PRISM 3100 Genetic Analyzer using the Big Dye Terminator cycle-sequencing kit (Applied Biosystems).
  • the amplified 27F-1492R products were sequenced in both directions with primers 27F, 357F (5'-CTACGGGAGGCAGCAG-3 '), 520R (5'-ACCGCGGGGTGCTGGC-3'), 920F (5 '- AAACTC AAAGGAATTGACG 5'- CCCAACATCTCACGAC-3 ') and 1492R as described by Mori et al. (1997).
  • the length of the sequence was 1488 bp.
  • the resulting sequence was compared with others using BLAST (Altschul et al., 1997).
  • the phylogenetic tree was constructed according to the method of Saitou and Nei (1987) using the CLUSTAL W software package (Thompson et al., 1994).
  • the nucleotide sequence of 16S rRNA was deposited in DDBJ / EMBL / GeneBank under the number AB 178889, identification number 20040510203204.24251.
  • a cultured bacterium capable of aerobic and anaerobic growth in YPD, MRS, and NA was found in frozen Miocene sediments on Mammoth Mountain; optimal growth temperature is determined at + 37 ° C.
  • the microorganism is psychrotolerant, because capable of metabolic activity at -5 ° C.
  • the bacillus is a relatively large ((1-1.5) x (3-6) micron) rod, which in culture combines in a chain and is able to form spores of a round shape. She is weakly mobile, gram-positive, non-pathogenic.
  • the microorganism belongs to the genus Bacillus and is a new species. The greatest species similarity of the isolated bacillus was noted with B. cereus and B. macroides., The homology with 16S rRNA of which is 99 and 97%, respectively.
  • Strain Bacillus sp. VKPM B-10130 is characterized by the following features.
  • Morphological straight rods with rounded ends, weakly mobile 1-1, 2 x 3-10 microns, 1-2, chains up to 7. Forms oval endospores located centrally and terminally, not exceeding the size of vegetative cells. Gram-positive.
  • the strain is undemanding to growth factors, grows well on ordinary nutrient media (SPA, MPA, MPB).
  • VKPM B-10130 used the following medium, g / l: peptone 10, yeast extract 5; distilled water the rest. Cultivation is carried out at 37 ° C and intensive aeration until the growth retardation phase is reached.
  • the culture is maintained by reseeding on solid media, stored in a solution of glycerol at -70 ° C or in a freeze-dried state.
  • FIG. 1 shows the effect of a culture of Bacillus sp. on the life span of Drosophila melanogaster
  • FIG. 2 the influence of the culture of Bacillus sp. with parenteral administration of 5000 cells for the life expectancy of laboratory mice of 17 months of age.
  • FIG. 3 levels of IFN- ⁇ after 6 days (A) and TNF- ⁇ after 90 minutes (B) in the blood serum of mice after intraperitoneal injection of Bacillus sp.
  • test tubes For the experiment, individuals of Drosophila melanogaster of the same age (1 day) were selected. They were placed in test tubes with a nutrient medium (5-7 ml) of 5 pairs. The sample size for each variant was 100 flies. Individuals for the experiment were selected by etherification; dead and surviving flies were counted every third day. The experiment was conducted with a daily culture of Bacillus sp. MOH grown on meat and peptone broth. A culture of 20 ⁇ l was added to the test tube.
  • the experiment included a control variant in which the flies were kept on medium supplemented with yeast; and the experimental version, when the first 5 days of flies were kept on the medium with the addition of yeast, then 1 day on the medium with the bacillus (alternating the entire observation period). Virgin flies were selected on the day of departure to determine fecundity. Females and males were placed separately and kept for 5 days after they reached maximum fecundity. Then placed in pairs in test tubes with removable caps. The bottom of the lids was poured with sugar agar confectionery agar. Fertility accounting was carried out daily for 6 days. The number of eggs laid was taken into account, and the number of undeveloped eggs was determined after 24 hours.
  • the preparation of the bacterial culture was carried out similarly to the testing method on Drosophila; The diurnal culture of Bacillus sp. was used, however, before administration, it was frozen and thawed. The experiments were conducted on Fl CBA / B1ask-6 mice, on average 15 individuals in each group. In the first series of experiments, the effect of culture doses on the parameters of the immune system of young animals (age 3-4 months) was studied. Two groups of animals were used in the control, one of which was intact, and physiological saline was administered to the second group. Bacterial culture of Bacillus sp. 5,000 animals were administered once intraperitoneally; 50,000; 500,000; 5,000,000 and 50,000,000 microbial bodies (bw) per animal.
  • the standard methods were used to evaluate the morphophysiological activity of the thymus and spleen by the organ index (ratio of organ weight to body weight,%), the activity of non-specific immunoresistance factors by the level of phagocytic (AF,%) and metabolic (HCT test,%) activity of splenic macrophages, cellular immunity in HRT in vivo, the activity of humoral immunity by the number of antibody-forming cells (AOK) in 1 million nucleated cells in the spleen, the muscle strength of animals in the load-lifting test, behavioral reactions in the test Open field ", as well as life expectancy.
  • Bacillus sp. at a dose of 5,000 microbial bodies, it increases the thymus and spleen indices.
  • the culture of bacilli in a small dose (5000 mt) is stimulated, and in medium doses (500,000 and 5,000,000 mt) they inhibit the phagocytic activity of splenic macrophages.
  • Bacillus culture Bacillus sp. in almost all doses increases the activity of humoral immunity, and a dose of 5000 mt contributes to an increase in functional activity and cellular and humoral immunity.
  • mice from the control group were 589 days, and the maximum was 833 days.
  • Minimum life mice from the experimental group amounted to 836 days, and the maximum - 897 (Fig. 2).
  • Body weight in animals of the experimental group was higher than in animals of the control group 2 months after the introduction of the culture.
  • Muscle strength in experimental animals increased (about 60%) relative to peers from the control group.
  • An increase in the ability of animals to orient in space and research activity was evidenced by their more frequent visits to the internal sectors of the open field, an increase in the number of uprights and visits to minks.
  • a parenteral bacterial culture stimulates the immune system and improves the emotional stability of laboratory animals.
  • An increase in the life span of mice indicates the possible presence of Bacillus sp. geroprotectors.
  • the strain was investigated as immunomodulating biocorrectors capable of triggering an innate immune response in animal models.
  • the activation of immunity was studied in experiments on changes in the metabolic activity of macrophages, interferon-gamma levels (IFN- ⁇ ), tumor necrosis factor (TNF- ⁇ ) and the non-specific cytotoxic effect of T-lymphocytes under the influence of Bacillus sp.
  • Cells introduced into C57B1 / 6 mice . It was found that intraperitoneal injections of the strain in the dose range of 5,000 - 5,000,000 mt per animal lead to a dose-dependent activation of the metabolism of intraperitoneal macrophages, estimated by the level of chemically active aerobic metabolism intermediates (ROIs) more than 2 times.
  • ROIs chemically active aerobic metabolism intermediates
  • T lymphocytes Single intraperitoneal injections at a dose of 5,000 mt lead to an increase in levels of IFN- ⁇ by 2.6 times, and also contribute to a significant decrease in TNF- ⁇ (Fig. 3).
  • the cytotoxic effect of T lymphocytes obtained from animal spleen after injection of Bacillus sp. was studied in vitro against RLS mouse lymphosarcoma cells. It was established that reliable activation of a non-specific clone of T-lymphocytes occurs at a dose of 5 LLC LLC bw, while the cytotoxic effect of such T-lymphocytes against lymphosarcoma cells was 1.8 times higher than their effect without activation by strain.

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Abstract

The invention relates to biotechnology, in particular to strains of microorganisms, and can be used for stimulating immune system activity and increasing animal and human life expectancy. The relic microorganism - a bacteria strain Bacillus sp. VKPM (Russian National Collection of Industrial Microorganisms) В-10130 - is isolated from permafrost rocks and has immuno-stimulating and gero-protective activity.

Description

ШТАММ БАКТЕРИЙ Bacillius sp. - РЕЛИКТОВЫЕ  BACTERIA STRAIN Bacillius sp. - RELICT
МИКРООРГАНИЗМЫ, ВЫДЕЛЕННЫЕ ИЗ MICROORGANISMS ALLOCATED FROM
МНОГОЛЕТНЕМЕРЗЛЫХ ПОРОД И ОБЛАДАЮЩИЕPERMANENT FROZEN BREEDS AND OWNERS
ИММУНОМОДУЛИРУЮЩЕЙ И ГЕРОПРОТЕКТОРНОЙ АКТИВНОСТЬЮIMMUNOMODULATING AND HEROPROTECTIVE ACTIVITY
ОБЛАСТЬ ТЕХНИКИ FIELD OF TECHNOLOGY
Изобретение относится к биотехнологии, в частности, к штаммам микроорганизмов и может быть использовано для стимулирования деятельности иммунной системы и увеличения продолжительности жизни животных и человека.  The invention relates to biotechnology, in particular, to strains of microorganisms and can be used to stimulate the activity of the immune system and increase the life expectancy of animals and humans.
УРОВЕНЬ ТЕХНИКИ  BACKGROUND
В условиях, когда фундаментальные механизмы старения по- прежнему представляют собой актуальную проблему, изучение клеток, способных выживать в течение тысячелетий вполне может представлять интерес для геронтологии. Свидетельства жизнеспособности микроорганизмов в мерзлоте появились еще в девятнадцатом столетии. С.С. Абызов в 1979 году обнаружил во льду на Антарктической станции Восток бактерии, грибы, диатомеи и другие микроорганизмы (Абызов С.С, Бобин Н.Е., Кудряшов Б.Б., 1979. Микробиологические исследования ледника в Центральной Антарктиде. Известия АН СССР, серия Биология, 6, с. 828-836). Не отрицая вероятности развития микроорганизмов в мерзлых породах, отметим, что их рост, вероятно, затруднен. Даже в лабораторных условиях стареющие культуры, как известно, прекращают расти. Кристаллизация воды и остановка внешнего обмена веществ уменьшает способность к росту. Поэтому можно считать, что бактерии в многолетнемерзлых породах представляют собой ископаемые, реликтовые организмы. Их возраст подтверждается геологическими условия местонахождения (Баранова Ю.П., Ильинская И.А., Никитин В.П., Пнева Г.Н., Фрадкина А.Ф., Шварева Н.Я. Миоцен Мамонтовой горы. Труды ГИН СО АН СССР. Наука. Москва. 1976. 284 с), радиоуглеродными датировками (Katayama Т., Tanaka М., Moriizumi J., Nakamura Т., Brouchkov A., Douglas Т.А., Fukuda M., Tomita R, Asano K. Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years. Appl. Environ. Microbiol., Apr. 2007: 2360-2363), изучением оптических изомеров аминокислот (Brinton K.L.F., Tsapin A.I., Gilichinsky D., McDonald GD. Aspartic Acid Racemization and Age-Depth Relationships for Organic Carbon in Siberian Permafrost. Astrobiology, Volume 2, Number 1, 2002, p.77-82), косвенно, биоразнообразием встречаемых видов (Friedmann EI. 1994. Permafrost as microbial habitat. In Viable Microorganisms in Permafrost. Russian Academy of Sciences: Pushchino, Russia; 21-26). In conditions when the fundamental mechanisms of aging are still an urgent problem, the study of cells capable of surviving for millennia may well be of interest to gerontology. Evidence of the viability of microorganisms in permafrost appeared back in the nineteenth century. S.S. In 1979, Abyzov discovered bacteria, fungi, diatoms, and other microorganisms in ice on the Vostok Antarctic Station (Abyzov S.S., Bobin N.E., Kudryashov B.B., 1979. Microbiological studies of the glacier in Central Antarctica. Biology series, 6, pp. 828-836). Without denying the likelihood of the development of microorganisms in frozen rocks, we note that their growth is probably difficult. Even under laboratory conditions, aging crops are known to stop growing. Crystallization of water and stopping of external metabolism reduces the ability to grow. Therefore, we can assume that bacteria in permafrost are fossil, relict organisms. Their age is confirmed by geological conditions of location (Baranova Yu.P., Ilyinskaya I.A., Nikitin V.P., Pneva G.N., Fradkina A.F., Shvareva N.Ya. Miocene of the Mammoth Mountain. Proceedings of the GIN SB AS USSR. Science. Moscow. 1976.284 s), radiocarbon dating (Katayama T., Tanaka M., Moriizumi J., Nakamura T., Brouchkov A., Douglas T.A., Fukuda M., Tomita R, Asano K. Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years. Appl. Environ. Microbiol., Apr. 2007: 2360-2363) by studying optical amino acid isomers (Brinton KLF, Tsapin AI, Gilichinsky D., McDonald GD. Aspartic Acid Racemization and Age-Depth Relationships for Organic Carbon in Siberian Permafrost. Astrobiology, Volume 2, Number 1, 2002, p.77-82), indirectly, by the biodiversity of the species found (Friedmann EI. 1994. Permafrost as microbial habitat. In Vi able Microorganisms in Permafrost. Russian Academy of Sciences: Pushchino, Russia; 21-26).
Природа длительной жизнеспособности микроорганизмов в древней мерзлоте не имеет исчерпывающего объяснения. Способность реликтовых микроорганизмов сохранять жизнеспособность длительное время предполагает существование механизма, предотвращающего накопление повреждений. Мы считаем возможным использование этого механизма для лечения болезней и продления жизни животных и человека.  The nature of the long-term viability of microorganisms in ancient permafrost does not have an exhaustive explanation. The ability of relic microorganisms to maintain viability for a long time suggests the existence of a mechanism that prevents the accumulation of damage. We consider it possible to use this mechanism to treat diseases and prolong the life of animals and humans.
Настоящее изобретение относится к бактериям рода Bacillus, найденным в древних многолетнемерзлых породах Якутии.  The present invention relates to bacteria of the genus Bacillus, found in ancient permafrost of Yakutia.
Известен штамм бактерий Bacillus subtilis 1719, выделенный в естественных условиях и проявляющий широкий спектр антагонистической активности, низкую адгезивную активность и иммуномодулирующую активность (патент RU 2298032 С2, опубликовано 27.10.2007). Известен штамм бактерий Bacillus macroides Excel 00, выделенный из мицелия лекарственного гриба Agaricus Blazei Murill , проявляющий иммунную активность и способность препятствовать старению и болезням (заявка JP 2008-005702 А, опубликовано 17.01.2008). A known bacterial strain Bacillus subtilis 1719, isolated in vivo and exhibiting a wide spectrum of antagonistic activity, low adhesive activity and immunomodulating activity (patent RU 2298032 C2, published October 27, 2007). A known bacterial strain Bacillus macroides Excel 00 isolated from the mycelium of the medicinal fungus Agaricus Blazei Murill, showing immune activity and the ability to prevent aging and disease (application JP 2008-005702 A, published 01/17/2008).
СУЩНОСТЬ ИЗОБРЕТЕНИЯ  SUMMARY OF THE INVENTION
Патентуемый штамм Bacillus sp., в отличие от известных, выделен из вечной мерзлоты, возраст которой составляет многие тысячи лет, и поэтому характеризуется исключительной жизнеспособностью и способностью выживать в течение длительного времени при низких температурах. В экспериментах с его культурой наблюдается увеличение мышечной силы, увеличение физической и умственной активности, иммуномодулирование и замедление старения.  The patented strain of Bacillus sp., Unlike the known ones, was isolated from permafrost, which is many thousands of years old, and therefore is characterized by exceptional viability and ability to survive for a long time at low temperatures. In experiments with his culture, an increase in muscle strength, an increase in physical and mental activity, immunomodulation and a slowdown in aging are observed.
Указанный технический результат достигается микроорганизмом - штаммом Bacillus sp., выделенным из многолетнемерзлых пород и обладающим иммуномодулирующей и геропротекторной активностью.  The specified technical result is achieved by a microorganism - a strain of Bacillus sp., Isolated from permafrost and possessing immunomodulating and geroprotective activity.
Штамм Bacillus sp. задепонирован во Всероссийской Коллекции Промышленных Микроорганизмов (ВКПМ) ФГУШ осНИИГенетика 30.01.2009, регистрационный номер ВКПМ: В-10130.  Strain Bacillus sp. deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) FGUS osNII Genetika 01/30/2009, registration number VKPM: B-10130.
1. МАТЕРИАЛЫ И МЕТОДЫ ВЫДЕЛЕНИЯ МИКРООРГАНИЗМОВ  1. MATERIALS AND METHODS OF ISSUING MICRO-ORGANISMS
Для получения микроорганизмов из мерзлых пород были отобраны образцы из естественных обнажений. Они расположены на левом берегу Алдана, в 325 километрах вверх по течению от его впадения в Лену, на Мамонтовой горе. Образцы были отобраны в 0.9-1 м глубже слоя сезонного оттаивания. Обнажение разрушается рекой (более метра в год), так что отложения, из которых отбирались образцы, находились, очевидно, в многолетнемерзлом состоянии. При этом происходит ежегодное весенние смывание обрушений, предотвращающее завалы и смешения пород. Последние представляют собой тонкозернистые пески и алевролиты; их возраст соответствует среднему миоцену (Баранова Ю.П., Ильинская И.А., Никитин В.П., Пнева Г.Н., Фрадкина А.Ф., Шварева Н.Я. Миоцен Мамонтовой горы. Труды ГИН СО АН СССР. Наука. Москва. 1976. 284 с). Похолодание началось здесь в конце плиоцена, около 3 - 3.5 миллионов лет тому назад. Температура в январе для того времени была оценена от -12 до -32°С, а в июле от +12 до +16°С. Отложения, по-видимому, не оттаивали в плейстоцене из-за холодного климата Якутии. Таким образом, возраст мерзлоты на Мамонтовой горе, вероятно, может достигать 3.5 миллионов лет. Кроме того, были отобраны образцы из повторно-жильных льдов ледяного комплекса в Якутии и на Аляске: в тоннеле Фокс и на золотом руднике вблизи Фербенкса, а также из стенок подземелья Института Мерзлотоведения им. П.И.Мельникова в Якутске. To obtain microorganisms from frozen rocks, samples were taken from natural outcrops. They are located on the left bank of the Aldan, 325 kilometers upstream from its confluence with the Lena, on Mammoth Mountain. Samples were selected in 0.9-1 m deeper than the seasonal thawing layer. The outcrop is destroyed by the river (more than a meter per year), so that the sediments from which the samples were taken were obviously in a permafrost state. In this case, the annual spring flushing of caving occurs, preventing blockages and mixing of rocks. The latter are fine-grained sands and siltstones; their age corresponds to the Middle Miocene (Baranova Yu.P., Ilyinskaya I.A., Nikitin V.P., Pneva G.N., Fradkina A.F., Shvareva N.Ya. Miocene of the Mammoth Mountain. Proceedings of the GIN SB AS USSR Science, Moscow, 1976.284 s). Cooling began here at the end of the Pliocene, about 3 - 3.5 million years ago. The temperature in January for that time was estimated from -12 to -32 ° C, and in July from +12 to + 16 ° C. The deposits did not seem to thaw in the Pleistocene due to the cold climate of Yakutia. Thus, the permafrost age on Mammoth Mountain can probably reach 3.5 million years. In addition, samples were taken from the re-vein ices of the ice complex in Yakutia and Alaska: in the Fox tunnel and at the gold mine near Fairbanks, as well as from the walls of the underground of the Institute of Permafrost them. P.I. Melnikova in Yakutsk.
1.1. Отбор образцов  1.1. Sampling
Пробы отбирались с использованием стерилизованных спиртом и обожженных в пламени металлические инструменты (буры, пинцеты, скальпели). Для того, чтобы провести поверхностную стерилизацию образцов, проба весом около 50 г помещалась в стакан с 96% раствором этанола на одну минуту, и затем в пламя горелки на примерно 2 сек и, наконец, в стерильную пробирку. Отобранные породы хранились при температуре -5°С, что было близко к естественным условиям. Транспортировка проб осуществлялась в термоконтейнерах с хладагентами в мерзлом состоянии. Samples were taken using metal instruments sterilized with alcohol and burned in a flame (drills, tweezers, scalpels). In order to surface sterilize the samples, a sample weighing about 50 g was placed in a glass with 96% ethanol solution for one minute, and then in the burner flame for about 2 seconds and, finally, in a sterile tube. The selected rocks were stored at a temperature of -5 ° C, which was close to natural conditions. Samples were transported in refrigerated containers with frozen refrigerants.
1.2. Рост на искусственных средах  1.2. Artificial Growth
Образцы различного разведения в стерильных условиях добавлялись в чашки Петри, содержащие среды YPD, MRS и NA (см инструкцию Manual of environmental microbiology. 1997. Ed. Hurst C.J. FSM Press. Washington DC. 894 p.). Образцы добавлялись также в жидкий мясо-пептонный бульон в анаэробных и аэробных условиях.  Samples of various dilutions under sterile conditions were added to Petri dishes containing YPD, MRS and NA media (see Manual of environmental microbiology. 1997. Ed. Hurst C.J. FSM Press. Washington DC. 894 p.). Samples were also added to liquid meat-peptone broth under anaerobic and aerobic conditions.
1.3. Получение и секвенирование ДНК  1.3. DNA production and sequencing
Рибосомальная ДНК микробной культуры извлекалась с помощью Fast DNA kit for soil (BIO 101 Inc., Vista, CA), где применяется метод, основанный на разрушении клетки стеклянными шариками. Нуклеиновые кислоты осаждались из раствора с использованием раствора, состоящего из 0.1 части 3 М ацетата натрия (рН 5.2) и 2.5 частей этанола, инкубировались на льду, а затем центрифугировались в течение 30 минут на скорости 12,000 об./мин. Осажденные нуклеиновые кислоты растворялись затем в дистиллированной воде (свободной от RN-аз и DN-аз), и хранились при -20°С. Фрагменты 16S rRNA были амплифицированы ПНР, проводимой с бактериальными праймерами (27F; 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R;  Ribosomal DNA of the microbial culture was extracted using the Fast DNA kit for soil (BIO 101 Inc., Vista, CA), where a method based on the destruction of cells by glass beads is used. Nucleic acids were precipitated from the solution using a solution consisting of 0.1 parts of 3 M sodium acetate (pH 5.2) and 2.5 parts of ethanol, incubated on ice, and then centrifuged for 30 minutes at a speed of 12,000 rpm. Precipitated nucleic acids were then dissolved in distilled water (free of RNase and DNase), and stored at -20 ° C. Fragments of 16S rRNA were amplified by NDP carried out with bacterial primers (27F; 5'-AGAGTTTGATCCTGGCTCAG-3 ', 1492R;
5 ' -TG ACTG ACTGAGG YTACCTTGTTACGACTT-3 ' ) . ПЦР проводилась в объеме 20-μ1 с помощью GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) следующим образом: 4 мин при 94°С, затем 30 циклов по 1 мин при 94°С, 1 мин при 50°С, и 1.5 мин при 72°С, затем 7 мин при 72°С. ПЦР ампликоны подвергались электрофорезу и очищению с помощью Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Очищенные ампликоны были клонированы с использованием pCR2.1 вектора, культуры Е. coli, а также ТА cloning kit (Invitrogen) в соответствии с рекомендациями производителя. Из суточной культуры ДНК плазмид, содержащая 16S rDNA, получена с помощью Mini prep spin kit (Quiagen, Crawley, UK). Очищенные ДНК плазмид секвенировали на ABI PRISM 3100 Genetic Analyzer с помощью Big Dye Terminator cycle-sequencing kit (Applied Biosystems). Амплицицированные продукты 27F-1492R были секвенированы в обоих направлениях с праймерами 27F, 357F (5'- CTACGGGAGGCAGCAG -3'), 520R (5'-ACCGCGGGGTGCTGGC- 3'), 920F (5 '- AAACTC AAAGGAATTGACGG-3 '), 1080R (5'- CCCAACATCTCACGAC-3') и 1492R как описано Mori et al. (1997). Длина последовательности составила 1488 bp. Полученная последовательность сравнивалась с другими, используя BLAST (Altschul et al., 1997). Филогенетическое дерево строилось по методу Saitou и Nei (1987), используя CLUSTAL W software package (Thompson et al., 1994). Нуклеотидная последовательность 16S rRNA была депонирована в DDBJ/EMBL/GeneBank под номером АВ 178889, идентификационный номер 20040510203204.24251. 5 '-TG ACTG ACTGAGG YTACCTTGTTACGACTT-3'). PCR was performed in a 20-μ1 volume using a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) as follows: 4 min at 94 ° C, then 30 cycles of 1 min at 94 ° C, 1 min at 50 ° C, and 1.5 min at 72 ° C, then 7 min at 72 ° C. PCR amplicons were subjected electrophoresis and purification using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Purified amplicons were cloned using pCR2.1 vector, E. coli culture, and a TA cloning kit (Invitrogen) in accordance with the manufacturer's recommendations. From a 24-hour culture of plasmid DNA containing 16S rDNA was obtained using a Mini prep spin kit (Quiagen, Crawley, UK). The purified plasmid DNA was sequenced on an ABI PRISM 3100 Genetic Analyzer using the Big Dye Terminator cycle-sequencing kit (Applied Biosystems). The amplified 27F-1492R products were sequenced in both directions with primers 27F, 357F (5'-CTACGGGAGGCAGCAG-3 '), 520R (5'-ACCGCGGGGTGCTGGC-3'), 920F (5 '- AAACTC AAAGGAATTGACG 5'- CCCAACATCTCACGAC-3 ') and 1492R as described by Mori et al. (1997). The length of the sequence was 1488 bp. The resulting sequence was compared with others using BLAST (Altschul et al., 1997). The phylogenetic tree was constructed according to the method of Saitou and Nei (1987) using the CLUSTAL W software package (Thompson et al., 1994). The nucleotide sequence of 16S rRNA was deposited in DDBJ / EMBL / GeneBank under the number AB 178889, identification number 20040510203204.24251.
2. РЕЗУЛЬТАТЫ ВЫДЕЛЕНИЯ, ИЗУЧЕНИЯ РОСТА И ИДЕНТИФИКАЦИИ МИКРООРГАНИЗМОВ  2. RESULTS OF ISOLATION, STUDY OF GROWTH AND IDENTIFICATION OF MICRO-ORGANISMS
В мерзлых миоценовых отложениях на Мамонтовой горе было обнаружена культивируемая бактерия, способная к аэробному и анаэробному росту в средах YPD, MRS и NA; оптимальная температура роста определена в +37°С. Микроорганизм является психротолерантным, т.к. способен к метаболической активности при -5°C. Бацилла представляет собой сравнительно большую ((1- 1.5) х (3-6) микрон) палочку, которая в культуре соединяется в цепи, и способна образовывать споры круглой формы. Она слабоподвижна, грамположительна, непатогенна. Микроорганизм принадлежит роду Bacillus и является новым видом. Наибольшее видовое подобие выделенной бациллы отмечено с В. cereus и В. macroides., гомология с 16S rRNA которых составляет 99 и 97% соответственно . A cultured bacterium capable of aerobic and anaerobic growth in YPD, MRS, and NA was found in frozen Miocene sediments on Mammoth Mountain; optimal growth temperature is determined at + 37 ° C. The microorganism is psychrotolerant, because capable of metabolic activity at -5 ° C. The bacillus is a relatively large ((1-1.5) x (3-6) micron) rod, which in culture combines in a chain and is able to form spores of a round shape. She is weakly mobile, gram-positive, non-pathogenic. The microorganism belongs to the genus Bacillus and is a new species. The greatest species similarity of the isolated bacillus was noted with B. cereus and B. macroides., The homology with 16S rRNA of which is 99 and 97%, respectively.
Выживание и рост бацилл при низких температурах наблюдались ранее; известно, например, что Bacillus anthracis легко переносит замораживание (Luyet B.J., Gehenio P.M. 1940. Life and death at low temperatures. Biodynamica: Normany, Missouri; 99 p., a также Baross J.A., Morita R.Y. Life at Low Temperatures: Ecological Aspects. In Microbial Life in Extreme Environments, Kushner DJ (ed.). Academic Press: London 1978; 9 - 71). Однако оптимальная температура роста найденной бациллы довольно высока. Несмотря на то, что она оказалась способной на искусственной среде расти и ниже нуля, видимых колоний на мерзлых образцах при этом не наблюдалось. Насколько активна ее жизнь в мерзлоте, неясно; это относится и к микроорганизмам, выделенным из льдов Центральной Якутии и Аляски (Katayama Т., Tanaka М., Moriizumi J., Nakamura Т., Brouchkov A., Douglas Т.А., Fukuda M., Tomita R, Asano K. Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years. Appl. Environ. Microbiol., Apr. 2007: 2360-2363).  Survival and growth of bacilli at low temperatures were previously observed; it is known, for example, that Bacillus anthracis can easily tolerate freezing (Luyet BJ, Gehenio PM 1940. Life and death at low temperatures. Biodynamica: Normany, Missouri; 99 p., as well as Baross JA, Morita RY Life at Low Temperatures: Ecological Aspects. In Microbial Life in Extreme Environments, Kushner DJ (ed.) Academic Press: London 1978; 9-71). However, the optimum growth temperature of the found bacillus is quite high. Despite the fact that it turned out to be capable of growing below zero on an artificial environment, no visible colonies on frozen samples were observed. How active her life in permafrost is is unclear; this applies to microorganisms isolated from the ices of Central Yakutia and Alaska (Katayama T., Tanaka M., Moriizumi J., Nakamura T., Brouchkov A., Douglas T.A., Fukuda M., Tomita R, Asano K. Phylogenetic Analysis of Bacteria Preserved in a Permafrost Ice Wedge for 25,000 Years. Appl. Environ. Microbiol., Apr. 2007: 2360-2363).
Штамм Bacillus sp. ВКПМ B-10130 характеризуется следующими признаками.  Strain Bacillus sp. VKPM B-10130 is characterized by the following features.
Культурально-морфологические особенности штамма: Морфологические: прямые палочки с закругленными концами, слабоподвижные 1-1 ,2 х 3-10 мкм, по 1-2, цепочки до 7. Образует эндоспоры овальной формы, расположенные центрально и терминально, не превышающие размер вегететивных клеток. Грамположительные. Cultural and morphological features of the strain: Morphological: straight rods with rounded ends, weakly mobile 1-1, 2 x 3-10 microns, 1-2, chains up to 7. Forms oval endospores located centrally and terminally, not exceeding the size of vegetative cells. Gram-positive.
Культуральные: Оптимальные условия (температура + 30- 37°С, аэробные условия): однако способен расти и при температурах от +5°С и до 43 °С и в широком разбросе значений рН.  Cultural: Optimal conditions (temperature + 30-37 ° С, aerobic conditions): however, it can grow at temperatures from + 5 ° С to 43 ° С and in a wide range of pH values.
На плотных агаризованных питательных средах образуют полиморфные, непрозрачные, блестящие, слегка шероховатые, мягкие колонии слабо желтоватого оттенка, с волнистым краем.  On dense agarized nutrient media form polymorphic, opaque, shiny, slightly roughened, soft colonies of a slightly yellowish hue, with a wavy edge.
Штамм нетребователен к факторам роста, хорошо растет на обычных питательных средах (СПА, МПА, МПБ).  The strain is undemanding to growth factors, grows well on ordinary nutrient media (SPA, MPA, MPB).
Для культивирования Bacillus sp. ВКПМ В- 10130 применяют среду следующего состава, г/л: пептон 10, дрожжевой экстракт 5; вода дистиллированная остальное. Культивирование проводят при 37°С и интенсивной аэрации до достижения фазы замедления роста.  For cultivation of Bacillus sp. VKPM B-10130 used the following medium, g / l: peptone 10, yeast extract 5; distilled water the rest. Cultivation is carried out at 37 ° C and intensive aeration until the growth retardation phase is reached.
Культуру поддерживают пересевом на плотных средах, хранят в растворе глицерина при -70°С или в лиофильно-высушенном состоянии.  The culture is maintained by reseeding on solid media, stored in a solution of glycerol at -70 ° C or in a freeze-dried state.
Физиологические признаки.  Physiological signs.
Не обладает гемолитической активностью, не подавляет рост типовых штаммов Bacillus cereus, Escherichia coli, Candida albicans, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia, каталазоположителен, растет при концентрации NaCI до 5%, но не на 10% , устойчив к рифампицину, оксациллину, полимиксину, цефтазидиму, цефепиму; чуствителен к стрептомицину, бензилпенициллину, канамицину, гентамицину, амикоцину, эритромицину, меропенему, ампициллину, тетрациклину, левомицитину при 37°С, а при 10°С даже минимальная концентрация антибиотиков препятствует росту бактерии. It does not have hemolytic activity, does not inhibit the growth of typical strains of Bacillus cereus, Escherichia coli, Candida albicans, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia, catalase-positive, grows at a concentration of NaCI up to 5%, but not 10%, resistant to rifampicin, oxacillin, polymyxin, ceftazidime, cefepime; it is sensitive to streptomycin, benzylpenicillin, kanamycin, gentamicin, amikocin, erythromycin, meropenem, ampicillin, tetracycline, levomycin, at 37 ° C, and even at 10 ° C, even a minimal concentration of antibiotics inhibits bacterial growth.
Биохимические признаки штамма приведены в таб. 1.  Biochemical characteristics of the strain are given in table. one.
Таблица 1.  Table 1.
Показатели Характеристика  Indicators Characteristic
Окраска по Граму + Gram stain +
Пигментация - Pigmentation -
Форма Э Form E
Расположение ц  Location c
Спора:  Spore:
Раздутость  Bloated
- спорангия  - sporangia
Подвижность +  Mobility +
Рост в анаэробных условиях +  Growth under anaerobic conditions +
Катал азная активность +  Katalny activity +
Оксидазная активность +сл  Oxidase activity + cl
Тест Фогес-Проскауэра - Voges-Proskauer Test -
Использование цитрата -The use of citrate -
Редукция нитратов +газ Nitrate reduction + gas
Казеина - Casein -
Гидролиз Желатины + Hydrolysis of Gelatins +
Крахмала - Starch -
Образование Глюкозы - кислоты из: Маннита -Glucose Education - acids from: Mannitol -
Арабинозы -Arabinose -
Ксилозы -Xylose -
Лактозы -Lactose -
Маннозы + Mannose +
Сорбита - Sorbitol -
+2°С -+ 2 ° С -
Рост при Growth at
+8°С +  + 8 ° С +
температуре:  temperature:
+43°С +  + 43 ° C +
6,5% NaCl +СЛ  6.5% NaCl + SL
10% NaCl - 10% NaCl -
15% NaCl - рН 4 - рН 5 +СЛ 15% NaCl - pH 4 - pH 5 + SL
Реакция на  Reaction to
рН 5,5 +  pH 5.5 +
среду  Wednesday
рН 8,5 +  pH 8.5 +
рН 9 +  pH 9 +
рН 10 +  pH 10 +
рН 10,5 +  pH 10.5 +
рН 11 +СЛ  pH 11 + SL
Аммиака - Ammonia -
Образование на Education on
Индола - МПБ:  Indola - BCH:
H2S + H 2 S +
Фиксация N2 + Fixation N 2 +
Э - эллипсовидная спора; Ц - центральное расположение споры, - - отрицательная реакция; + - положительная реакция; +сл - слабоположительная реакция. 3. ТЕСТИРОВАНИЕ КУЛЬТУРЫ Bacillus sp. НА ВЫСШИХ ОРГАНИЗМАХE - ellipsoidal spore; C - the central location of the spores, - - a negative reaction; + - positive reaction; + SL - weakly positive reaction. 3. CULTURE TESTING Bacillus sp. AT HIGHER ORGANISMS
ПЕРЕЧЕНЬ ЧЕРТЕЖЕЙ LIST OF DRAWINGS
На фиг. 1 показано влияние культуры Bacillus sp. на продолжительность жизни Drosophila melanogaster In FIG. 1 shows the effect of a culture of Bacillus sp. on the life span of Drosophila melanogaster
На фиг. 2 - влияние культуры Bacillus sp. при парентеральном введении 5000 клеток на продолжительность жизни лабораторных мышей 17-ти месячного возраста.  In FIG. 2 - the influence of the culture of Bacillus sp. with parenteral administration of 5000 cells for the life expectancy of laboratory mice of 17 months of age.
На фиг. 3 - уровни IFN-γ через 6 дней (А) и TNF-α через 90 минут (В) в сыворотке крови мышей после интраперитонеальной инъекции Bacillus sp.  In FIG. 3 - levels of IFN-γ after 6 days (A) and TNF-α after 90 minutes (B) in the blood serum of mice after intraperitoneal injection of Bacillus sp.
ПРИМЕРЫ РЕАЛИЗАЦИИ ИЗОБРЕТЕНИЯ  MODES FOR CARRYING OUT THE INVENTION
3.1. Тестирование на мушках Drosophila melanogaster  3.1. Flies Testing Drosophila melanogaster
Для эксперимента отбирались особи Drosophila melanogaster одного возраста (1 сутки). Их помещали в пробирки с питательной средой (5-7 мл) по 5 пар. Объем выборки для каждого варианта составил 100 мух. Отбор особей для эксперимента проводили путем эфиризации, погибших и выживших мух учитывали каждые третьи сутки. Эксперимент проводили с суточной культурой Bacillus sp. МЗ, выращенной на мясопептонном бульоне. В опытную пробирку вносили культуру в объёме 20 мкл. Эксперимент включал контрольный вариант, в котором мух содержали на среде с добавлением дрожжей; и опытный вариант, когда первые 5 суток мух содержали на среде с добавлением дрожжей, затем 1 сутки на среде с бациллой (чередование весь период наблюдения). Для определения плодовитости отбирались виргинные мухи в день вылета. Самок и самцов помещали отдельно и выдерживали 5 суток по достижении ими максимальной плодовитости. Затем помещали попарно в пробирки со съемными крышками. Дно крышек заливали агар-агаром кондитерским с добавлением сахара. Учет плодовитости проводили ежесуточно на протяжении 6 дней. Учитывали количество отложенных яиц, а через сутки определяли количество неразвившихся яиц. For the experiment, individuals of Drosophila melanogaster of the same age (1 day) were selected. They were placed in test tubes with a nutrient medium (5-7 ml) of 5 pairs. The sample size for each variant was 100 flies. Individuals for the experiment were selected by etherification; dead and surviving flies were counted every third day. The experiment was conducted with a daily culture of Bacillus sp. MOH grown on meat and peptone broth. A culture of 20 μl was added to the test tube. The experiment included a control variant in which the flies were kept on medium supplemented with yeast; and the experimental version, when the first 5 days of flies were kept on the medium with the addition of yeast, then 1 day on the medium with the bacillus (alternating the entire observation period). Virgin flies were selected on the day of departure to determine fecundity. Females and males were placed separately and kept for 5 days after they reached maximum fecundity. Then placed in pairs in test tubes with removable caps. The bottom of the lids was poured with sugar agar confectionery agar. Fertility accounting was carried out daily for 6 days. The number of eggs laid was taken into account, and the number of undeveloped eggs was determined after 24 hours.
При добавлении на поверхность питательной среды 75 мкл штамма Bacillus sp. в физрастворе (1млрд. кл/мл) наблюдалось снижение плодовитости по отношению к контролю в 5 раз. Средняя плодовитость самки составила в контроле 58,1±8,61 шт., в опытном варианте эта величина 10,2±3,44 шт. При добавлении бактериальной культуры после выдерживания мушек на дрожжях, несмотря на гибель мушек в опыте и контроле после 50 дней, отмечалось превышение доли выживших мух с 24-х по 42-е сутки эксперимента по сравнению с контролем (фиг. 1).  When 75 μl of the strain Bacillus sp. in saline solution (1 billion cells / ml), a decrease in fertility by a factor of 5 was observed in relation to the control. The average female fecundity in the control was 58.1 ± 8.61 pcs., In the experimental version this value was 10.2 ± 3.44 pcs. When the bacterial culture was added after the flies were kept on yeast, despite the death of the flies in the experiment and control after 50 days, the proportion of surviving flies from the 24th to the 42nd day of the experiment exceeded the control (Fig. 1).
3.2.Тестирование на лабораторных мышах  3.2 Testing in laboratory mice
Подготовка бактериальной культуры проводилась аналогично методике тестирования на дрозофилах; использовалась суточная культура Bacillus sp., однако перед введением проводили ее замораживание и оттаивание. Эксперименты проводились на мышах Fl СВА/В1аск-6, в среднем по 15 особей в каждой группе. В первой серии экспериментов изучалось влияние доз культуры на параметры иммунной системы молодых животных (возраст 3-4 месяца). В контроле использовали две группы животных, одна из которых была интактной, а второй группе вводили физиологический раствор. Бактериальную культуру Bacillus sp. вводили животным однократно внутрибрюшинно 5 000; 50 000; 500 000; 5 000 000 и 50 000 000 микробных тел (м.т.) на животное. Во второй серии экспериментов оценивалось влияние бактериальной культуры на физиологические и поведенческие реакции «пожилых» мышей (возраст 17 месяцев), при этом культуру вводили однократно внутрибрюшинно в дозе 5000 м.т. Контрольная группа была представлена животными того же возраста. Эвтаназия животных проводилась методом дислокации шейных позвонков. По стандартным методикам оценивались морфофизиологическая активность тимуса и селезенки по индексу органа (отношение веса органа к весу тела, %), активность факторов неспецифической иммунорезистентности по уровню фагоцитарной (ФП, %) и метаболической (НСТ-тест, %) активности селезеночных макрофагов, клеточного иммунитета в реакции ГЗТ in vivo, активность гуморального иммунитета по числу антителообразующих клеток (АОК) в 1 миллионе ядросодержащих клеток в селезенке, мышечная сила животных в тесте поднятия груза, поведенческие реакции в тесте «Открытое поле», а также продолжительность жизни. The preparation of the bacterial culture was carried out similarly to the testing method on Drosophila; The diurnal culture of Bacillus sp. was used, however, before administration, it was frozen and thawed. The experiments were conducted on Fl CBA / B1ask-6 mice, on average 15 individuals in each group. In the first series of experiments, the effect of culture doses on the parameters of the immune system of young animals (age 3-4 months) was studied. Two groups of animals were used in the control, one of which was intact, and physiological saline was administered to the second group. Bacterial culture of Bacillus sp. 5,000 animals were administered once intraperitoneally; 50,000; 500,000; 5,000,000 and 50,000,000 microbial bodies (bw) per animal. In the second series of experiments, the effect of bacterial culture to the physiological and behavioral reactions of "elderly" mice (age 17 months), while the culture was injected once intraperitoneally at a dose of 5000 MT The control group was represented by animals of the same age. Euthanasia of animals was carried out by the method of cervical vertebra dislocation. The standard methods were used to evaluate the morphophysiological activity of the thymus and spleen by the organ index (ratio of organ weight to body weight,%), the activity of non-specific immunoresistance factors by the level of phagocytic (AF,%) and metabolic (HCT test,%) activity of splenic macrophages, cellular immunity in HRT in vivo, the activity of humoral immunity by the number of antibody-forming cells (AOK) in 1 million nucleated cells in the spleen, the muscle strength of animals in the load-lifting test, behavioral reactions in the test Open field ", as well as life expectancy.
Оказалось, что Bacillus sp. в дозе 5 000 микробных тел способствует увеличению индексов тимуса и селезенки. Культура бацилл в малой дозе (5000 м.т.) стимулируют, а в средних дозах (500000 и 5000000 м.т.) - подавляют фагоцитарную активность селезеночных макрофагов. Культура бацилл Bacillus sp. почти во всех дозах повышает активность гуморального иммунитета, а доза в 5000 м.т. способствует увеличению функциональной активности и клеточного и гуморального иммунитета.  It turned out that Bacillus sp. at a dose of 5,000 microbial bodies, it increases the thymus and spleen indices. The culture of bacilli in a small dose (5000 mt) is stimulated, and in medium doses (500,000 and 5,000,000 mt) they inhibit the phagocytic activity of splenic macrophages. Bacillus culture Bacillus sp. in almost all doses, increases the activity of humoral immunity, and a dose of 5000 mt contributes to an increase in functional activity and cellular and humoral immunity.
В этой связи для исследований влияния культуры на продолжительность жизни была выбрана доза в 5 000 м.т. Минимальный срок жизни мышей из контрольной группы составил 589 дней, а максимальный - 833 дня. Минимальный срок жизни мышей из опытной группы составил 836 дней, а максимальные - 897 (фиг. 2). Вес тела у животных опытной группы был выше, чем у животных контрольной группы через 2 месяца после введения культуры. Мышечная сила у опытных животных увеличилась (около 60%) относительно сверстников из контрольной группы. О повышении у животных способности к ориентации в пространстве и исследовательской активности свидетельствовало более частое посещение ими внутренних секторов открытого поля, увеличение числа вертикальных стоек и посещения норок. По-видимому, бактериальная культура при парентеральном введении стимулирует иммунную систему и улучшает эмоциональную устойчивость лабораторных животных. Увеличение продолжительности жизни мышей свидельствует о возможном присутствии в культуре Bacillus sp. геропротекторов. In this regard, for studies of the effect of culture on life expectancy, a dose of 5,000 mt was chosen. The minimum lifespan of mice from the control group was 589 days, and the maximum was 833 days. Minimum life mice from the experimental group amounted to 836 days, and the maximum - 897 (Fig. 2). Body weight in animals of the experimental group was higher than in animals of the control group 2 months after the introduction of the culture. Muscle strength in experimental animals increased (about 60%) relative to peers from the control group. An increase in the ability of animals to orient in space and research activity was evidenced by their more frequent visits to the internal sectors of the open field, an increase in the number of uprights and visits to minks. Apparently, a parenteral bacterial culture stimulates the immune system and improves the emotional stability of laboratory animals. An increase in the life span of mice indicates the possible presence of Bacillus sp. geroprotectors.
Кроме того, штамм исследован в качестве иммуномодулирующих биокорректоров, способных запускать врожденный иммунный ответ на животных моделях. Активация иммунитета была исследована в экспериментах по изменению метаболической активности макрофагов, уровней интерферона- гамма (IFN-γ), фактора некроза опухолей (TNF-α) и неспецифического цитотоксического действия Т-лимфоцитов под действием клеток Bacillus sp., введенных мышам линии С57В1/6. Установлено, что внутрибрюшинные инъекции штамма в диапазоне доз 5 000 - 5 000 000 м. т. на животное приводят к дозозависимой активации метаболизма интраперитонеальных макрофагов, оцениваемой по уровню химически активных промежуточных соединений аэробного метаболизма (ROIs) более чем в 2 раза. Однократные внутрибрюшинные инъекции в дозе 5 000 м.т. приводят к увеличению уровней IFN-γ в 2,6 раза, а также способствуют значительному снижению TNF-α (фиг. 3). Цитотоксическое действие Т-лимфоцитов, полученных из селезенки животных после инъекций Bacillus sp., было исследовано in vitro против клеток лимфосаркомы мыши RLS. Установлено, что достоверная активация неспецифического клона Т-лимфоцитов происходит при дозе 5 ООО ООО м.т., при этом цитотоксическое действие таких Т-лимфоцитов против клеток лимфосаркомы было в 1,8 раз выше, чем их действие без активации штаммом. Таким образом, усиление метаболизма макрофагов, повышение уровня интерферона-гамма, снижение фактора некроза опухолей и активация цитотоксического действия Т-лимфоцитов под действием Bacillus sp. свидетельствует об активации врожденного иммунитета у лабораторных животных и о ярко выраженных иммуномодулирующих свойствах штамма Bacillus sp. In addition, the strain was investigated as immunomodulating biocorrectors capable of triggering an innate immune response in animal models. The activation of immunity was studied in experiments on changes in the metabolic activity of macrophages, interferon-gamma levels (IFN-γ), tumor necrosis factor (TNF-α) and the non-specific cytotoxic effect of T-lymphocytes under the influence of Bacillus sp. Cells introduced into C57B1 / 6 mice . It was found that intraperitoneal injections of the strain in the dose range of 5,000 - 5,000,000 mt per animal lead to a dose-dependent activation of the metabolism of intraperitoneal macrophages, estimated by the level of chemically active aerobic metabolism intermediates (ROIs) more than 2 times. Single intraperitoneal injections at a dose of 5,000 mt lead to an increase in levels of IFN-γ by 2.6 times, and also contribute to a significant decrease in TNF-α (Fig. 3). The cytotoxic effect of T lymphocytes obtained from animal spleen after injection of Bacillus sp. Was studied in vitro against RLS mouse lymphosarcoma cells. It was established that reliable activation of a non-specific clone of T-lymphocytes occurs at a dose of 5 LLC LLC bw, while the cytotoxic effect of such T-lymphocytes against lymphosarcoma cells was 1.8 times higher than their effect without activation by strain. Thus, increased metabolism of macrophages, increased levels of interferon-gamma, decreased tumor necrosis factor and activation of the cytotoxic effect of T-lymphocytes under the action of Bacillus sp. indicates the activation of innate immunity in laboratory animals and the pronounced immunomodulating properties of the strain Bacillus sp.

Claims

Формула изобретения Claim
Штамм бактерий Bacillus sp. ВКПМ В- 10130, обладающий иммуномодулирующей и геропротекторной активностью.  The bacterial strain Bacillus sp. VKPM B-10130 with immunomodulating and geroprotective activity.
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