CN107603912B

CN107603912B - Soybean endophyte and application thereof

Info

Publication number: CN107603912B
Application number: CN201710989377.8A
Authority: CN
Inventors: 王秀伶; 张红蕾; 郝庆红; 周博; 贾扬
Original assignee: Heibei Agricultural University
Current assignee: Heibei Agricultural University
Priority date: 2015-04-08
Filing date: 2015-04-08
Publication date: 2020-02-14
Anticipated expiration: 2035-04-08
Also published as: CN107603912A; CN108676737A; CN107653205B; CN108676737B; CN107653205A; CN105018370B; CN107629983A; CN107574135B; CN107629983B; CN105018370A; CN107574135A

Abstract

The present invention is a divisional application of the invention patent application No. 201510162612.5. The invention discloses soybean endophyte and application thereof, belonging to the technical field of microorganisms. A soybean endophyte is a black bean sprout endophyte, which is Bacillus amyloliquefaciens EnB-bsys5(Bacillus amyloliquefaciens) belonging to the genus Bacillus with the preservation number of CGMCC No. 10525. The soybean endophyte can obviously prolong the average life of caenorhabditis elegans, improve the heat resistance of the caenorhabditis elegans, can be planted in the body of the caenorhabditis elegans, and has wide prospect of being developed into health-care food or medicine.

Description

Soybean endophyte and application thereof

The application is a divisional application of an invention patent application with application number 201510162612.5, application date 2015, 04 and 08, and invention name of soybean endophyte and application thereof.

Technical Field

The invention relates to the technical field of microorganisms.

Background

Soybean is an annual herbaceous legume whose seeds, also known as soybeans, are the most important legumes in the world. The soybean is not only soybean, but also black soybean and green soybean. Soybeans are important oil, food and feed crops, and protein in the soybeans can provide a plurality of amino acids such as lysine, leucine, threonine and the like which are necessary for human bodies. In addition, the soybeans also contain a large amount of unsaturated fatty acids, phospholipids, cellulose, vitamins, rich inorganic salts such as calcium, phosphorus, magnesium, potassium and the like, and trace elements such as copper, iron, zinc, iodine, molybdenum and the like. The soybean has multiple functions of resisting oxidation, resisting aging, resisting cancer, preventing angiosclerosis, promoting bone development, reducing blood sugar and the like. The black soybean is a black seed of soybean, can be used as both medicine and food, and is different from soybean in that the black skin contains a large amount of flavonoid compound anthocyanidin pigment. In addition, black beans contain abundant proteins, fats, vitamins, nicotinic acid, carotene, trace elements and crude fibers, wherein the protein content is more than 48%, the black beans are higher than meat, eggs and milk, and the vegetable has the reputation of 'the king of vegetable protein'. The black bean has the effects of softening blood vessels, moistening skin, delaying aging and the like.

Soybeans contain various endophytes, like other plants, in addition to the various "non-biological" nutrient-like substances mentioned above. Endophytes are microorganisms, mainly fungi and bacteria, that live inside the tissues or organs of healthy plants at some or all stages of their life history without causing the host plant to exhibit significant symptoms of infection. At present, the separation of endophytes from different tissues and organs of leguminous plants such as soybeans, peas, kidney beans, dwarf green beans, alfalfa and the like has been reported, but the separation of endophytes from black beans has not been reported. The main purpose of previous isolation of endophytes from leguminous plants was to use the isolated beneficial strains in the agricultural field. For example, the endophyte can increase the plant height and dry weight, enhance the growth vigor of the plant, improve the germination rate of seeds and the survival rate of seedlings, increase the tillering growth of the plant and the formation of inflorescence, and the like. The plant endophyte can directly promote the growth of plants by fixing nitrogen and generating auxin, gibberellin, cytokinin and the like for promoting the growth of plants; endophytes can also promote plant growth by inducing host plants to produce phytohormones, and by enhancing the absorption of nutrient elements such as N, P and other minerals by plants (Zhouyi et al, Soy science, 2009, 28(3): 502-506). In addition, the previously reported endophytes also have the capability of enhancing the stress resistance and the pest resistance of the host. The enhancement of the stress resistance of the plants is mainly shown in two aspects of biotic stress (such as pest and disease attack resistance, insect and herbivore feeding inhibition and the like) and abiotic stress (such as high temperature resistance, drought resistance, cold damage resistance, saline alkali resistance and the like), and the main mechanism is that endophytes change the physiological characteristics of the plants by influencing the metabolism of host plant substances and generating physiologically active substances, so that the stress resistance of the plants is improved, and the growth of the plants is stimulated; the endophyte can compete with the pathogenic bacteria for nutrition and space or directly generate antagonistic substances to inhibit the growth of the pathogenic bacteria; or by inducing the plants to produce phenols and quinones which are accumulated in the intercellular space, thereby forming a chemical barrier for the pathogenic bacteria to enter or move in the plants.

According to the international passing judgment standard, the mark for judging whether a city or a country enters an aging society is whether the proportion of the population over 60 years old reaches or exceeds 10 percent of the total population. Recent data shows that the existing aged population in china has exceeded 1.6 billion and is increasing at a rate of nearly 800 million per year. At present, China enters an aging society, the population of the elderly is rapidly increased year by year, and the elderly diseases such as coronary heart disease, diabetes, osteoporosis, neurodegenerative diseases and the like are obviously increased, so that great pressure and challenge are brought to the economy, culture, medical treatment and the like of China. Although these senile diseases are caused by multiple factors and involve multiple genes, aging of the body is a common factor. Aging is an inevitable physiological process of the body and is a major problem which has been studied and failed to solve by human beings, so that the purpose of searching for an aging delaying method to effectively reduce the senile diseases caused by aging of the body is a major problem to be solved urgently at present.

Caenorhabditis elegans belongs to the phylum lineata, and in a natural state, Caenorhabditis elegans is a transparent organism living in soil, feeding on bacteria and humus and capable of sensing smell, light and temperature, and is also a unique organism in which all cells in a body can be sequentially checked and classified. The caenorhabditis elegans has a short life history, the average life history at 20 ℃ is about 3.5 days, and the whole life cycle is about 3 weeks. Caenorhabditis elegans is a multi-cellular eukaryote that also undergoes complex cell division and proliferation processes to eventually form more complex tissue and organ systems similar to higher organisms, such as skin, muscle, digestion, nerves, reproduction, etc., which makes caenorhabditis elegans have some cellular, molecular structure and life-span control pathways similar to those of higher organisms. Thus, the biological knowledge gained by the model organism C.elegans can be directly applied to higher organisms including even humans (Barbieri et al. American Journal of Physiology and Endocrinology Metabolism, 2003, 285: E1064-1071). Shtonda and Avery, when studying the behavior of caenorhabditis elegans on a diet, found that nematodes have the ability to self-judge whether bacteria in the surrounding environment are toxic and to select for the bacteria that are consumed (Journal of Experimental biology, 2006, 209: 89-102). Furthermore, it has been shown that nematodes are either infected or killed when fed to C.elegans with the human pathogens enterococcus faecium (Sifri et al., Infectious Immunology, 2002, 70: 5647-; in contrast, when C.elegans is fed on probiotic lactic acid bacteria, the mean life span of C.elegans is significantly extended, while the ability of the nematode to fight against pathogenic Salmonella bacteria is also enhanced (Applied and environmental microbiology, 2007, 73: 6404-. Furthermore, Gusarov et al, when fed to C.elegans with B.subtilis, found that the mean life span of C.elegans was significantly extended by the insulin signal transduction pathway, and that the extended life span of C.elegans was due to the ability of B.subtilis to produce nitric oxide gas by nitric oxide synthase (Cell, 2013, 152: 818-. Nitric oxide is the "most attractive molecule" known to humans. In the 90 s of the 20 th century, the research on nitric oxide became a wind tide, scientists all over the world had more and more interest in nitric oxide, and the research also reached a new height. In 1992, nitric oxide was selected as an "annual star molecule" by the well-known journal of science as a new finding in academia. In 1998, researchers in the United states were awarded the Nobel biomedical prize by Robert Frigold, Lewis eagerlo and Friedel-crafts. They found that nitric oxide is a signal molecule of cardiovascular system, confirmed the role of nitric oxide as a signal molecule in cardiovascular system, and found and demonstrated how nitroglycerin and related drugs make the body healthy by releasing nitric oxide and have positive effects on the body. Nitric oxide is an important signaling molecule that controls blood pressure, regulates blood flow, allows blood to reach tissues, supplies oxygen and nutrients to the body, and virtually all tissues are not isolated from nitric oxide. More and more research has shown that nitric oxide has an important role in the treatment of cardiovascular disease and many other major chronic diseases. The main physiological functions of nitric oxide include actions on the cardiovascular system, immune system, circulatory system, central nervous system and urogenital system (nitric oxide keeps you away from cardiovascular and cerebrovascular diseases, Louis j. lgnaro, kindergarten: wu shou ling, yangming; published by beijing university medical press, 2007).

Endophytes currently isolated from different parts of soybeans are mainly classified into Bacillus (Bacillus), Pseudomonas (Pseudomonas), Enterobacter (Enterobacter), Burkholderia (Burkholderia), Klebsiella (Klebsiella), Xanthomonas (Xanthomonas), etc. (Jiitendra Dalal1and Nikhilesh Kulkarni1, British Microbiology Research Journal, 2013, 3 (1): 96-105), and the isolation of endophytes from soybeans is only for agricultural production purposes. In addition to the "non-biological" nutrients contained in soybeans, no report has been made to select soybean endophytes from which endophytes have beneficial effects on humans or other animals.

Disclosure of Invention

The invention aims to solve the technical problem of providing soybean endophytes and application thereof, wherein the six soybean endophytes are respectively from black bean sprouts, soybean seeds and soybean sprouts and all belong to the genus Bacillus; the six soybean endophytes can obviously prolong the average life of the caenorhabditis elegans, improve the heat resistance of the caenorhabditis elegans and can be planted in the body of the caenorhabditis elegans; has certain effects on resisting aging and prolonging life, and can be used in health food or medicine.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a soybean endophyte is a black bean sprout endophyte, which belongs to the Bacillus and has a preservation number of CGMCC No.10524, and is Bacillus licheniformis EnB-bsys4(Bacillus licheniformis).

A soybean endophyte is a black bean sprout endophyte, which is Bacillus amyloliquefaciens EnB-bsys5(Bacillus amyloliquefaciens) belonging to the genus Bacillus with the preservation number of CGMCC No. 10525.

A soybean endophyte is soybean seed endophyte, which is Bacillus cereus EnB-sy3(Bacillus cereus) and belongs to the genus Bacillus with the preservation number of CGMCC No. 10687.

A soybean endophyte is soybean seed endophyte, which belongs to the genus Bacillus and has a preservation number of CGMCC No.10688, and is Bacillus licheniformis EnB-sy8(Bacillus licheniformis).

A soybean endophyte is a soybean sprout endophyte, which is Bacillus pumilus EnB-sys1(Bacillus pumilus) belonging to the genus Bacillus with the preservation number of CGMCC No. 10689.

A soybean endophyte is a soybean sprout endophyte, which is Bacillus cereus EnB-sys2(Bacillus cereus) belonging to the genus Bacillus with the preservation number of CGMCC No. 10690.

The invention relates to six soybean endophytes: bacillus licheniformis EnB-bsys4(Bacillus licheniformis 4) and Bacillus amyloliquefaciens EnB-bsys5(Bacillus amyloliquefaciens) separated from black bean sprouts are preserved in the China general microbiological culture Collection center (CGMCC for short) at 2-5 days 2015, and the preservation numbers are CGMCC No.10524 and CGMCC No.10525 respectively; bacillus cereus EnB-sy3(Bacillus cereus), Bacillus licheniformis EnB-sy8(Bacillus licheniformis) and Bacillus pumilus EnB-sys1(Bacillus pumilus) and Bacillus cereus EnB-sys2(Bacillus cereus) which are separated from soybean sprouts and have been preserved in the China general microbiological culture Collection center (CGMCC for short) on 4-7 th month 2015, wherein the preservation numbers are CGMCC No.10687, CGMCC No.10688, CGMCC No.10689 and CGMCC No.10690 respectively. China academy of sciences microbiological research institute Strain Collection center Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

The taxonomic characteristics of the six soybean endophytes are respectively as follows:

(1) bacillus licheniformis EnB-bsys 4: the diameter of a bacterial colony is 5-10 mm, the middle pink edge is pale and irregular and round, the middle pink color is dry, the edge is milky and wet, and when the bacterial colony is cultured in an LB liquid culture medium, the thallus is a small short straight rod under an optical microscope;

the strain can not grow at 50 ℃ or 10 ℃ but can grow under anaerobic conditions, the starch hydrolase activity, the nitrate reduction and the lactose fermentation produce acid which are all positive, and the hemolytic reaction and the indole reaction are negative, when detected by using the API50CH kit, the strain EnB-bsys4 can use glycerol, L-arabinose, ribose, D-xylose, glucose, fructose, mannose, inositol, mannitol, sorbitol, α -methyl-D-glucoside, amygdalin, arbutin, esculin, saligenin, cellobiose, maltose, melibiose, sucrose, trehalose, inulin, raffinose, starch, glycogen, D-turanose, D-tagatose and gluconate, but can not use erythrol, D-arabinose, L-xylose, adonitol, β -methyl-D-xyloside, galactose, sorbose, rhamnose, citronellol, α -methyl-D-mannose, N-acetyl-glucosamine, gentisic-D-xyloside, xylitol, D-xyloside, D-D.

(2) Bacillus amyloliquefaciens EnB-bsys 5: the diameter of the bacterial colony is about 5 mm, the bacterial colony is white, the edge of the central protruding blister sample is flat and divergent, the bacterial colony is not easy to pick, and the bacterial colony in an LB liquid culture medium is a short straight rod and is slightly longer;

the strain has positive starch hydrolase activity, positive nitrate reduction, positive hemolytic reaction, acid production by fermented lactose and growth in 12% sodium chloride, when the test is carried out by using an API50CH kit, the strain EnB-bsys5 can utilize glycerol, L-arabinose, ribose, D-xylose, L-xylose, adonitol, glucose, fructose, mannose, mannitol, sorbitol, α -methyl-D-glucoside, amygdalin, arbutin, esculin, saligenin, cellobiose, maltose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, D-turanose, D-lyxose and gluconate, and the rest 20 saccharides in the API50CH kit cannot be utilized.

(3) Bacillus cereus EnB-sy 3: the colony diameter is about 5 millimeters, white colony, and is opaque, dull polish form, and the centre has a little protrusion, and the periphery is flat, and the edge is more neat, drier. The thalli in the LB liquid culture medium is short and straight, and some of the thalli are slightly long;

the strain grows at 50 ℃, and is negative in nitrate reduction, positive in citrate, positive in hydrogen peroxide and positive in arginine double hydrolysis. When the test is carried out by using the API50CH kit, the strain EnB-sy3 is found to be capable of utilizing glycerol, L-arabinose, glucose, fructose, mannose, mannitol, inositol, N-acetyl-glucosamine, arbutin, esculin, saligenin, cellobiose, maltose, sucrose, trehalose and D-tagatose, and the rest 33 saccharides in the API50CH kit are not capable of being utilized.

(4) Bacillus licheniformis EnB-sy 8: the diameter of the colony is about 6 mm, the periphery of the middle light pink is light whitish, and the edge of the colony is irregular and is relatively wet. The thallus is made into a short straight rod in an LB liquid culture medium;

when the strain is detected by using an API50CH kit, the strain EnB-sy8 can utilize L-arabinose, ribose, D-xylose, glucose, fructose, mannose, inositol, mannitol, sorbitol, α -methyl-D-glucoside, N-acetyl-glucosamine, esculetin, cellobiose, maltose, sucrose, trehalose, inulin, starch, D-turanose and D-tagatose, and the rest 29 saccharides in the API50CH kit cannot be utilized.

(5) Bacillus pumilus EnB-sys 1: the colony diameter is about 2 ~ 4 millimeters, and the circular colony that inclines, white, drier, central fold-shaped protruding edge is even neat. The thallus is made into a short straight rod in an LB liquid culture medium;

the strain is positive in citrate utilization, positive in arginine double hydrolysis, negative in nitrate reduction, negative in hemolysis and negative in starch acid production, can grow at the pH value of 5.5. when the test is carried out by using an API50CH kit, the strain EnB-sys1 can utilize L-arabinose, ribose, glucose, fructose, mannose, inositol, mannitol, sorbitol, α -methyl-D-glucoside, N-acetyl-glucosamine, esculetin, saligenin, cellobiose, maltose, sucrose, trehalose and D-tagatose, and the rest 32 saccharides in the API50CH kit can not be utilized.

(6) Bacillus cereus EnB-sys 2: the colony diameter is about 5 millimeters, white colony, and is opaque, dull polish form, and the centre has a little protrusion, and the periphery is flat, and the edge is more neat, drier. The thalli in an LB liquid culture medium forms a medium-long rod, and some of the thalli are divided into sections;

the strain is positive in nitrate reduction, positive in citrate utilization, positive in hemolysis, capable of growing at the pH of 8, negative in arginine double hydrolysis and negative in 10% sodium chloride. When the API50CH kit is used for detection, the strain EnB-sys2 can only utilize ribose, glucose, fructose, N-acetyl-glucosamine, esculin, maltose, sucrose and trehalose, and the other 41 saccharides in the API50CH kit cannot be utilized.

The invention discloses an application of soybean endophyte in preparing health food and medicine with the functions of prolonging life and resisting aging.

The invention also discloses the application of the soybean endophyte in preparing medicines and health-care foods for improving cardiovascular diseases, immune system diseases, circulatory system diseases, central nervous system diseases and urogenital system diseases.

The specific separation and screening method of the six soybean endophytes comprises the following steps:

10 grains of black soya bean and soybean seeds which are cleaned by running water are soaked in 70 percent alcohol for 3 minutes, then are washed by sterile water and are soaked in 3 percent sodium hypochlorite for 5 minutes, and finally are washed by sterile water and are soaked for 4 hours. A control of 100. mu.l of LB-coated plates was taken from the sterile water of the last immersion to check whether the surface of the seeds was thoroughly sterilized. Then, 100. mu.l of the ground seeds were inoculated into LB liquid medium and pre-cultured in a common biochemical incubator at 37 ℃ for 24 hours. And (3) performing gradient dilution on the culture solution, coating an LB flat plate, and culturing at 37 ℃ for 1-2 days. Colonies with different forms are picked respectively and are subjected to pure culture, the picked colonies are subjected to plate streaking, and the test is repeated at least three times. Meanwhile, the seeds soaked with sterile water are placed in the dark for culturing, after the bean sprouts grow out, the bean sprouts are cut into small sections with the length of 1 cm by a cutter sterilized in advance, and the endophytes are separated by adopting the same method. The resulting pure culture was frozen in 50% glycerol at-80 ℃. And carrying out thallus morphology observation, physiological and biochemical analysis and 16S rDNA sequence analysis on the separated single colony. Endophytes in the separated black beans, soybean seeds and bean sprouts are respectively fed with wild type caenorhabditis elegans, and the influence of different foods on the life span and the heat resistance of the wild type caenorhabditis elegans is researched. Finally, two bacilli in soybean seeds and two bacilli in soybean sprouts can be found from the black bean sprout endophytes, the average life of wild type caenorhabditis elegans can be prolonged remarkably, and the heat resistance of the wild type caenorhabditis elegans can be improved remarkably. The wild type caenorhabditis elegans used in the present invention is hereinafter referred to as caenorhabditis elegans.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: the six soybean endophytes can obviously prolong the average life of wild caenorhabditis elegans and improve the heat resistance of the wild caenorhabditis elegans, and can be planted in the wild caenorhabditis elegans, wherein four soybean endophytes EnB-bsys4, EnB-bsys5, EnB-sy 8and EnB-sys1 can generate NO. The six soybean endophytes have certain effects on human body aging resistance and life prolonging, and can be applied to health-care food or medicines, wherein the four soybean endophytes capable of generating NO can also be applied to preparation of medicines and health-care food for improving cardiovascular diseases, immune system diseases, circulatory system diseases, central nervous system diseases and urogenital system diseases.

Because NO is a signal molecule, it can play an important role in regulating the cardiovascular system, immune system and nervous system. Therefore, once the soybean endophyte is planted in a human body, the incidence rate of aging diseases can be reduced by generating NO, and the immunity and the life quality of the organism can be improved; for patients with hypertension, the live bacteria preparation can lower blood pressure and reduce the incidence of cardiovascular and cerebrovascular diseases. As the two strains of the bacillus cereus which do not produce NO are fed with wild type caenorhabditis elegans, the average life span of the caenorhabditis elegans can be prolonged, and the heat resistance of the caenorhabditis elegans is improved, the six strains of the soybean endophytes can also play a beneficial regulation role on the caenorhabditis elegans through other ways after the planting. In addition, when the bacillus strain is in the form of a vegetative mass, most of the bacillus nutrients are "bitten" by caenorhabditis elegans and die when fed to it, so that the caenorhabditis elegans obtains nutrients from the "bitten" bacteria, which nutrients also serve to prolong the life of the caenorhabditis elegans. In 2013, Komura of Japanese scholars discovered that the life of caenorhabditis elegans can be prolonged when crushed bifidobacteria are used for feeding wild caenorhabditis elegans, and further experiments show that a certain teichoic acid component possibly in the middle of thallus cell walls and cytoplasm plays a role in prolonging the life of caenorhabditis elegans. Therefore, the six bacteria with the life-prolonging effect on the caenorhabditis elegans can play a role in improving the immunity of the human body after being made into high-protein bacteria powder.

Drawings

FIG. 1 shows the effect of endophytes from black bean sprouts (A), soybean seeds and soybean sprouts (B) on the longevity of caenorhabditis elegans;

FIG. 2 is a phylogenetic tree of soybean endophytes;

FIG. 3 shows the effect of soybean endophytes on caenorhabditis elegans heat resistance.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The nematodes used in the examples were all wild type C.elegans.

Example 1

1. Isolation of soybean endophytes

Black beans and soybean seeds were purchased from baoding. 10 grains of black soya bean and soybean seeds which are cleaned by running water are soaked in 70 percent alcohol for 3 minutes, then are washed by sterile water and are soaked in 3 percent sodium hypochlorite for 5 minutes, and finally are washed by sterile water and are soaked for 4 hours. A control of 100. mu.l of LB-coated plates was taken from the sterile water of the last immersion to check whether the surface of the seeds was thoroughly sterilized. Then, 100. mu.l of the ground seeds were inoculated into LB liquid medium and pre-cultured in a common biochemical incubator at 37 ℃ for 24 hours. And (3) performing gradient dilution on the culture solution, coating an LB flat plate, and culturing at 37 ℃ for 1-2 days. Colonies with different forms are picked respectively and are subjected to pure culture, the picked colonies are subjected to plate streaking, and the test is repeated at least three times. Meanwhile, the seeds soaked with sterile water are placed in the dark for culturing, after the bean sprouts grow out, the bean sprouts are cut into small sections with the length of 1 cm by a cutter sterilized in advance, and the endophytes are separated by adopting the same method. The pure culture obtained above was frozen in 50% glycerol at-80 ℃. And carrying out thallus morphology observation and physiological and biochemical analysis on the separated single colony, and carrying out 16S rDNA sequence analysis on the colony, the thallus or the bacterial strain with obvious difference in physiological and biochemical characteristics.

2. Screening of Soybean endophytes

(1) Activating strains: all soybean endophytes preserved at low temperature are thawed and inoculated into LB liquid culture medium according to the inoculation amount of 10 percent, and activated and cultured for 24 hours at 37 ℃. The next day, the activated strain was re-transferred to fresh LB liquid medium for subculture for 12 hours.

(2) Activation of wild-type caenorhabditis elegans: taking out the wild type caenorhabditis elegans frozen at-80 ℃, quickly thawing at 37 ℃, centrifuging, discarding the supernatant, adding the rest polypide to an NGM culture plate containing E.coli OP50, and culturing at constant temperature of 20 ℃.

(3) Preparation of caenorhabditis elegans standard food e.coli OP 50: inoculating E.coli OP50 single colony into autoclaved fresh 2 ml LB liquid medium, shake culturing at 37 deg.C 180 r/min for 8-12 hr to OD₆₀₀When the concentration is about 1.0, 100. mu.l of the bacterial suspension is aspirated and uniformly spread on the center of an NGM plate prepared in advance (about 0.5 mm from the edge of the plate), and the plate is cultured in an inverted manner at 37 ℃ overnight and stored at 4 ℃ for later use.

The culture method of the strain E.coli OP50 was the same as that for the preparation of a standard food E.coli OP50 for liquid culture of caenorhabditis elegans, except that OD was obtained by culturing₆₀₀Centrifuging at about 1.0 deg.C and 4000 rpm for 10 min, collecting thallus, washing the obtained precipitate with S.Medium culture solution twice, discarding supernatant, adding S.Medium culture solution, and adjusting thallus concentration to OD₆₀₀About 1.0 deg.C, and storing at 4 deg.C for use.

(4) Culturing caenorhabditis elegans soybean endophyte food: the activated and passaged soybean endophytes were respectively pipetted 150. mu.l with a pipette gun and spread on an LB solid plate, and cultured at 37 ℃ for 12 hours. The next day, the cells on LB plates were washed with M9 buffer, collected in centrifuge tubes, the resulting pellet was washed twice with S.Medium broth, the supernatant was discarded, and S.Medium broth was added to adjust the cell concentration to OD₆₀₀About 1.0 deg.C, and storing at 4 deg.C for use.

(5) Synchronization of caenorhabditis elegans: selecting a caenorhabditis elegans culture plate which is not polluted by infectious microbes and has more adults in the egg-laying period, washing the worm bodies and eggs by using M9 buffer solution, and collecting the worm bodies and eggs into a centrifuge tube to ensure that the worm body quantity of each tube is as consistent as possible and the worm body density cannot be too high. Centrifuge tubes containing oviposition adults at 1500 rpm at 4 ℃ for 1 minute, and discard the supernatant. And adding 650 microliters of M9 buffer solution and 350 microliters of ready-prepared nematode lysate into each centrifugal tube, fully shaking and uniformly mixing until the worm bodies cannot be seen by naked eyes, immediately taking a small amount of liquid to observe under a microscope to determine whether only worm eggs are left, wherein the process cannot exceed 10 minutes, and otherwise, the worm eggs are influenced and even die. At the moment when only eggs remained in the centrifuge tube, the tube was immediately centrifuged at 3500 rpm at 4 ℃ for 30 seconds, and the supernatant was discarded. The remaining precipitate was washed by adding M9 buffer to the centrifuge tube, centrifuged at 3500 rpm at 4 ℃ for 30 seconds, and the supernatant was discarded. The above washing step was repeated four times. The finally obtained eggs of the caenorhabditis elegans were aspirated to the edge of the fresh NGM plate (step (3)) containing E.coli OP50, and cultured at a constant temperature of 20 ℃ for about 48 hours, and then the eggs developed into the synchronized L4 nematode for experimental study.

(6) Life analysis of caenorhabditis elegans: 30 synchronized C.elegans at L4 stage were individually transferred to a medium containing 500. mu.l OD₆₀₀Culturing in 24-well plate containing about 1.0 liquid food of soybean endophyte at 20 deg.C, and adding E.coli OP50 liquid food as control, wherein the number of nematodes is 30 per well. Nematodes were transferred daily to wells containing the corresponding fresh food and dead insects were recorded daily. Data from life-span experiments on caenorhabditis elegans were analyzed using the Kalpan-Meir survival Curve analysis program from SPSS 19.0.

The results show that: the average life of wild type caenorhabditis elegans can be remarkably prolonged by the two endophytes screened from the black bean sprouts, the two endophytes screened from the soybean seeds and the two endophytes screened from the soybean sprouts, as shown in figure 1. When caenorhabditis elegans is fed with the standard food E.coli OP50, the average survival time of the caenorhabditis elegans is 20.467 +/-1.657 days; when two endophytes EnB-bsys4 and EnB-bsys5 separated from black bean sprouts are used for feeding caenorhabditis elegans, the average survival time of the caenorhabditis elegans is 30.517 +/-1.608 (P <0.001) and 27.636 +/-1.540 (P <0.01), and the life of the caenorhabditis elegans is respectively prolonged by 49.10% and 35.03% compared with that of the caenorhabditis elegans fed with a standard food E.coli OP 50; when two endophytes EnB-sy3 and EnB-sy8 separated from soybean seeds are used for feeding caenorhabditis elegans, the average survival time of the caenorhabditis elegans is 32.385 +/-2.008 (P <0.001) and 36.353 +/-1.790 (P <0.001), and the life span of the caenorhabditis elegans is respectively prolonged by 58.23% and 77.61% compared with that of the caenorhabditis elegans fed with a standard food E.coli OP 50; when two endophytes EnB-sys 1and EnB-sys2 separated from soybean sprout are used for feeding caenorhabditis elegans, the average survival time of the caenorhabditis elegans is 26.714 +/-1.405 (P <0.05) and 28.429 +/-1.407 (P <0.01), and the life span of the caenorhabditis elegans is 30.52 percent and 38.90 percent respectively longer than that of the caenorhabditis elegans fed with a standard food E.coli OP 50.

A phylogenetic tree was constructed from the six endophytes that significantly extended the life of C.elegans according to the 16S rDNA sequence, as shown in FIG. 2. API detection was performed on the six endophytes that could prolong the life of C.elegans using the API50CH kit, and no indicators (e.g., amylolytic enzyme activity, etc.) listed in the API50CH kit were performed according to the bacterial identification Manual (6 th edition 2002, Star publishing Co., Ltd.) such as Jousimes-Somer. According to a strain 16S rDNA sequence, and by combining colony and thallus morphological characteristics and physiological and biochemical index detection results, two endophytes EnB-bsys4 and EnB-bsys5 which can remarkably prolong the life of the caenorhabditis elegans are respectively identified as Bacillus licheniformis and Bacillus amyloliquefaciens. In the same way, two endophytes EnB-sy3 and EnB-sy8 which can remarkably prolong the life span of caenorhabditis elegans and are separated from soybean seeds are respectively identified as Bacillus cereus and Bacillus licheniformis; two endophytes EnB-sys 1and EnB-sys2 which can obviously prolong the life of caenorhabditis elegans and are separated from soybean sprouts are respectively identified as Bacillus pumilus and Bacillus cereus.

3. Detection of soybean endophyte in caenorhabditis elegans intestinal tract colonization

The bacillus in black beans and soybeans can produce spores with extremely strong resistance, and can bear various adverse environments, so that the bacillus is likely to colonize the intestinal tract of caenorhabditis elegans. In order to detect whether six endophytes screened from black beans and soybeans can be planted in intestinal tracts of caenorhabditis elegans after being fed for a period of time, the following experiments are carried out:

feeding the synchronized caenorhabditis elegans at L4 stage for 3 days, one week and three weeks respectively with the above six soybean endophytes, and performing surface disinfection treatment and culture according to the following steps:

(1) c.elegans in a 24-well plate was pipetted into a 1.5-ml EP tube, M9 buffer was added, 1200g was centrifuged under centripetal force for 2 minutes, the supernatant was discarded, 100. mu.l of 70% ethanol was added to the centrifuge tube, the surface of the nematode was sterilized for 20 seconds, and then 1 ml of M9 buffer (diluted ethanol) was immediately added.

(2) After washing twice with M9 buffer, the supernatant was discarded, 100 μ l of 10% sodium hypochlorite was added to the centrifuge tube to disinfect the nematode surface for 40 seconds, then 1 ml of M9 buffer (diluted sodium hypochlorite) was immediately added, and the nematodes were washed twice with M9 buffer.

(3) Absorbing 100 microliters of buffer solution in the surface-sterilized nematode tube into sterilized LB liquid culture, culturing at 37 ℃ and using the buffer solution as a control to detect whether the surface sterilization of the nematodes is thorough.

(4) The remaining worm bodies and buffer were triturated with a grinding rod (breaking up the worm bodies, bacteria overflow) and then 100. mu.l of triturated worm body buffer was taken to be cultured in sterilized LB medium at 37 ℃.

(5) And (3) if the aseptic growth of the LB culture in the step (3) indicates that the surface of the nematode is thoroughly disinfected, coating the thallus growing in the LB culture medium in the step (4) to obtain a single colony, and verifying whether the thallus is the soybean endophyte feeding the caenorhabditis elegans or not according to the colony morphology and the 16SrDNA gene sequence.

Grinding and coating the nematodes fed with the soybean endophytes for 3 days, finding that the plate has two colony forms, one colony form is similar to that of the fed soybean endophytes, respectively purifying the two colony forms, then carrying out DNA extraction and 16S rDNA sequencing and carrying out BLAST comparison, and finding that the two bacteria are respectively escherichia coli and the fed soybean endophytes; grinding and plating the nematodes of soybean endophytes fed for one week and three weeks, finding that only one colony form exists on the plate, namely the colony of the fed soybean endophytes, and carrying out DNA extraction and 16S rDNA sequencing on the colony, and finding that the strain is the fed soybean endophyte. In conclusion, the black bean and soybean endophytes are considered to be related to the nutrient substances provided by the thallus and the beneficial regulation effect exerted by the thallus after the thallus is planted in the nematode body to prolong the service life of the caenorhabditis elegans.

4. Determination of soybean endophyte in vitro release of NO

Gusarov, 2013, found that the longevity of nematodes were significantly prolonged when wild-type C.elegans was fed with Bacillus subtilis, and further studies found that NO released from the fed Bacillus subtilis could be used as a signaling molecule to regulate nematode longevity via the ILS pathway (Cell 2013, 152: 818-. A Bacillus subtilis strain (CGMCC No.1.4255) was purchased from the strain collection center of institute of microbiology, China academy of sciences, and when the strain was fed to wild caenorhabditis elegans, it was found that there was no significant prolongation of nematode longevity compared to the control fed to E.coli OP 50. The purchased bacillus subtilis was found to produce little NO as determined by the nitrate/nitrite assay kit. Therefore, we speculate that the isolated six soybean endophytes with life-prolonging effect on wild C.elegans are likely to produce NO.

The six soybean endophyte bacterial suspensions cultured for 12 hours in advance are used for measuring NO generation by a nitric oxide kit (Nanjing Pistak) and the specific steps are as follows:

(1) 100 microliter of double distilled water is taken as a blank tube, a standard product is taken as a standard tube, and soybean endophyte bacterial suspension is taken as a determination tube.

(2) Adding 400 microliter of mixed reagent, mixing evenly, and carrying out accurate water bath at 37 ℃ for 60 minutes.

(3) Adding three 200 microliter of reagent and four 100 microliter of reagent, fully and uniformly mixing by vortex for 30 seconds, standing for 40 minutes at room temperature, and centrifuging for 10 minutes at 3500g under the centripetal force.

(4) And respectively taking 500 microliters of supernatant and 600 microliters of color developing agent, uniformly mixing, and standing for 10 minutes at room temperature for color development.

(5) Adjusting the light path to zero with double distilled water at 550 nm and 0.5 cm, and measuring the absorbance value of each tube.

(6) And calculating the NO content according to a formula.

As a result, it was found that both of the two black soybean endophytes EnB-bsys4 and EnB-bsys5 were capable of producing NO at an average content of 48.85. mu. mol/L and 44.04. mu. mol/L, respectively. Soybean endophytes EnB-sy 8and EnB-sys1 also produced NO at average levels of 28.36. mu. mol/L and 73.24. mu. mol/L, respectively, whereas soybean endophytes EnB-sy3 and EnB-sys2 did not produce NO. As the six soybean endophytes of the bacillus which have the life-prolonging effect on the wild type caenorhabditis elegans can be planted in intestinal tracts of the caenorhabditis elegans, the reason is that four soybean endophytes of the bacillus which can produce NO can regulate and control the related metabolism of the caenorhabditis elegans through producing NO signal molecules, and finally the caenorhabditis elegans are obviously prolonged.

5. Effect of Soybean endophyte on caenorhabditis elegans heat resistance

The synchronized caenorhabditis elegans of L4 stage is respectively added into 24-hole plates containing different soybean endophyte foods and standard foods E.coli OP50, each hole contains 500 mu L of E.coli OP50 bacterial suspension and soybean endophyte bacterial suspension, each hole contains 30 nematodes, adults are transferred into new 24 holes containing corresponding fresh foods every 24 hours, and the incubation is carried out at the constant temperature of 20 ℃. By day 5, it was placed at 37 ℃ for heat stress testing and nematode survival was observed every 2 hours. The nematode death criterion was the same as the above caenorhabditis elegans life test, and the test data was repeated at least three times.

The results show that: the two endophytes in the black bean sprouts, the two endophytes in the soybean seeds and the two endophytes in the soybean sprouts obviously improve the heat resistance of wild type caenorhabditis elegans, and the result is shown in figure 3.

When the caenorhabditis elegans fed by the black bean endophyte and the E.coliOP50 are respectively placed at 37 ℃ for 3-4 hours, the nematodes begin to die, when the caenorhabditis elegans are placed at 37 ℃ for 5, 11 and 13 hours, the nematodes fed by the E.coliOP50, the nematodes fed by the black bean endophytes EnB-bsys4 and EnB-bsys5 die in large quantities respectively, and the average survival time of the nematodes fed by different foods is respectively as follows: coliop50 is fed for 5.37 + -0.31 hours, while the endophytes EnB-bsys4 and EnB-bsys5 are fed for 9.97 + -0.48 and 10.23 + -0.68 hours. Anova showed that caenorhabditis elegans fed with the black bean endophytes EnB-bsys4 and EnB-bsys5 had significant resistance to acute heat stress (P <0.001) compared to e.coliopop 50 fed nematodes, as shown in fig. 3-a.

When soybean endophytes and elegans fed by E.coliOP50 are respectively placed at 37 ℃ for 2-4 hours, the nematodes begin to die, when the soybean endophytes and the elegans fed by E.coliOP50 are placed at 37 ℃ for 4, 6 and 5 hours, the nematodes fed by E.coliOP50, EnB-sy3, EnB-sy8, EnB-sys 1and EnB-sys2 respectively begin to die in large areas, and the average survival time of the nematodes fed with different foods is as follows: the nematode feeding time of E.coliOP50 is 4.73 +/-0.34 hours, and the nematode feeding time of soybean endophytes EnB-sy3, EnB-sy8, EnB-sys 1and EnB-sys2 is 6.60 +/-0.34, 6.74 +/-0.47, 5.84 +/-0.32 and 6.53 +/-0.36 hours respectively. Analysis of variance showed that C.elegans fed with soybean endophytes EnB-sy3(P <0.01), EnB-sy8(P <0.01), EnB-sys1(P <0.05) and EnB-sys2(P <0.01) had significant resistance to acute heat stress as compared to E.coliOP50 fed nematodes, as shown in FIG. 3-B.

Claims

1. A soybean endophyte is characterized by being a black bean sprout endophyte, wherein the black bean sprout endophyte is Bacillus amyloliquefaciens EnB-bsys5(Bacillus amyloliquefaciens) which belongs to the genus Bacillus and has the preservation number of CGMCCNo.10525.

2. Use of the soybean endophyte of claim 1 in the preparation of a health food product having an extended life span.