CN105018370B - Soybean endophyte and application thereof - Google Patents
Soybean endophyte and application thereof Download PDFInfo
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- CN105018370B CN105018370B CN201510162612.5A CN201510162612A CN105018370B CN 105018370 B CN105018370 B CN 105018370B CN 201510162612 A CN201510162612 A CN 201510162612A CN 105018370 B CN105018370 B CN 105018370B
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- bacillus
- bacterium
- caenorhabditis elegans
- soybean
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- 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
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- 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
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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Abstract
The invention discloses soybean endophyte and application thereof, belonging to the technical field of microorganisms. The six soybean endophytes are respectively Bacillus licheniformis EnB-bsys4, Bacillus amyloliquefaciens EnB-bsys5, Bacillus cereus EnB-sy3, Bacillus licheniformis EnB-sy8, Bacillus pumilus EnB-sys1 and Bacillus cereus EnB-sys2 with the preservation numbers of CGMCC No.10524, CGMCC No.10525, CGMCC No.10687, CGMCC No.10688, CGMCC No.10689 and CGMCC No. 10690. The six soybean endophytes can obviously prolong the average life of the caenorhabditis elegans, improve the heat resistance of the caenorhabditis elegans, can be planted in the body of the caenorhabditis elegans, and have wide prospect of being developed into health-care food or medicine.
Description
Technical field
The present invention relates to microbial technology field.
Background technology
Soybean is annual herb legume, and its seed is also referred to as soybean, is most important beans in the world.Soybean is not
Singly refer to soya bean, it also includes black soya bean and green soya bean.Soya bean is important oil plant, edible and forage crop, the protein energy in soya bean
Many essential amino acids such as lysine, leucine, threonine are provided.In addition, also contain a large amount of unsaturated fats in soya bean
The trace element such as inorganic salts and copper, iron, zinc, iodine, molybdenum such as acid, phosphatide, cellulose, vitamin and abundant calcium, phosphorus, magnesium, potassium.
Soya bean has anti-oxidant, anti-aging, anticancer, prevents vascular sclerosis, promotes the multiple functions such as skeleton development and hypoglycemic.Black soya bean is big
The black seeds of beans, can medicine-food two-purpose, with soya bean it is maximum be not both that black crust contains a large amount of flavonoids anthocyanidin
Class pigment.In addition, black soya bean contains abundant protein, fat, vitamin, niacin, carrotene, trace element and crude fibre,
Wherein protein content is occupied first of beans up to more than 48%, higher than meat, egg and milk, is known as the U.S. of " king of vegetable protein "
Reputation.Black soya bean has softening blood vessel, skin care, anti-aging and other effects.
In soybean in addition to containing above-mentioned various " abiotic " class nutriments, also contain as other plant
Various endophytes.Endophyte of plant refers to those groups that health plant is moved in the certain phase of its history of life or whole stages
Knit or organ inside, without making host plant show the microorganism of obvious infection symptoms, based on fungi and bacterium.At present from
Endophyte is separated in the legume different tissues organ such as soya bean, pea, Kidney bean, dwarf bean and clover it has been reported that but still
Have no the report that endophyte is separated from black soya bean.The main purpose that conventional people separate endophyte from legume is to divide
From beneficial bacterial strain be applied to agriculture field.As endophyte of plant can increase plant plant height and dry weight, enhancing plant strain growth gesture, carry
High percentage of seedgermination and survival rate of seedling, increase plant tillering growth and inflorescence formation etc..Endophyte of plant can pass through biology
Fixed nitrogen and generation promote auxin, gibberellin and basic element of cell division of plant growth etc., directly facilitate plant growth；Endophyte
Can also be by inducing host plant to produce plant hormone, by strengthening plant to the nutrients such as N, P and other mineral matters
Absorption promote plant growth（Week is happy etc., Soybean Science, 2009,28 (3):502-506）.In addition, previously reported plant
Endophyte also has enhancing host's resistance and diseases and insect pests resistance.The humidification of stress resistance of plant is mainly manifested in the biological side of body
Compel (as resistance pest and disease damage invasion and attack, prevent feeding of insect and herbivore etc.) and abiotic stress (such as high temperature resistance, it is drought-resistant,
Inappropriate ADH syndrome, salt resistance alkali etc.) two aspects, main mechanism is endophyte by influenceing host plant metabolism and producing physiologically active
Material changes the physiological property of plant, improves the resistance of plant, stimulates plant growth；Endophyte of plant can be with pathogen
Competition nutrition and space directly produce antagonistic substance and suppress growth of pathogenic bacteria；Or by inducing plant to produce phenols, quinones
Material accumulates in space between cells, enters the chemical barrier operated in plant or in plant so as to form pathogen.
According to the criterion of the current international practice, judge that the mark whether a city or country enters " aging society " is
Whether the population proportion of more than 60 years old has met or exceeded the 10% of total number of people.Latest data shows that China is existing
The aged is more than 1.6 hundred million and every year in the speed increase with nearly 8,000,000.Current China comes into aging society,
Elderly population are increasing sharply year by year, and the thing followed is that coronary heart disease, diabetes, osteoporosis, nerve degenerative diseases etc. are old
Year property disease showed increased, economy, culture and medical treatment to country etc. brings immense pressure and challenge.These geriatric diseases
Though by it is multifactor cause participated in polygenes, and body aging is then common factor.Aging is that a body is inevitably given birth to
Reason process, it is to study and have not been able to the significant problem solved always since the dawn of human civilization, it is therefore, purposeful to find anti-aging side
Method, it is current significant problem urgently to be resolved hurrily effectively to reduce the geriatric disease triggered by body aging.
Caenorhabditis elegans（Caenorhabditis elegans）Belong to Nemathelminthes, under nature, beautiful hidden bar
Nematode be it is a kind of live in soil using bacterium and humus as food, smell, light, the transparent life in the whole body of temperature can be perceived
All cells can successively be made an inventory and respectively return the biology of its class in thing, and only one body.The Caenorhabditis elegans history of life
Short, the average history of life under 20 °C is 3.5 days or so, and whole life cycle is about 3 weeks or so.Caenorhabditis elegans is a kind of
Eucaryote with many cells, it also to undergo the cell division of complexity and breeding can just ultimately form and higher organism
Similar more complicated tissue and tract, such as skin, muscle, digestion, nerve, reproduction, this causes Caenorhabditis elegans to have
There are some cells similar to higher organisms, molecular structure and life control path.Therefore, it is this by Caenorhabditis elegans
The Biological Knowledge that model organism is obtained may be directly applied to higher organism or even include the mankind（Barbieri et al.
American Journal of Physiology and Endocrinology Metabolism, 2003,285:E1064-
1071）.Shtonda and Avery has found that nematode, which has, voluntarily judges surrounding ring when studying Caenorhabditis elegans dietary behavior
Whether the bacterium in border is poisonous and the ability of selection is carried out to eaten bacterium（Journal of Experimental
Biology, 2006,209:89-102）.In addition, there is research to confirm, when with human pathogen VREF（Sifri et
Al., Infectious Immunology, 2002,70:5647-5650）And staphylococcus aureus（Sifri et al.,
Infectious Immunology, 2006,74:1091-1096）During feeding Caenorhabditis elegans, nematode or infected or killed
Extremely；On the contrary, when using probiotic lactic bacteria as food feeding Caenorhabditis elegans, Caenorhabditis elegans average life span substantially obtains
Extend, while nematode can also be made to resist pathogen salmonella ability to be strengthened（Applied and Environmental
Microbiology, 2007,73:6404-6409）.In addition, Gusarov etc. is with bacillus subtilis feeding Caenorhabditis elegans
When, it is found that Caenorhabditis elegans average life span is significantly extended by insulin signal transduction approach, and find beautiful hidden bar
Nematode life is due to that bacillus subtilis can produce nitric oxide gas by nitricoxide synthase（Cell, 2013,
152:818-830）.Nitric oxide is " most charming molecule " known to the mankind.In the 1990s, being ground to nitric oxide production
Studying carefully turns into a kind of agitation, and scientist all over the world is further dense to nitric oxide production interest, research also reached one it is new
Highly.1992, new discovery of the nitric oxide as academia, by famous《Science》Magazine is chosen as " annual star point
Son ".1998, researcher Robert Furchgott, Louis's Ignarro and the Freed Mo Lade quilts in the U.S.
Authorize Nobel Prize in Physiology or Medicine.They have found that nitric oxide is the signaling molecule of cardiovascular system, it was confirmed that nitric oxide is made
The effect for being signaling molecule in cardiovascular system, find and demonstrate nitroglycerin and how related drugs are by discharging an oxygen
Changing nitrogen makes health, and positive effect is produced to body.Nitric oxide is important signaling molecule, and it controls blood pressure, is adjusted
CBF is saved, blood is reached tissue, oxygen and nutrition are supplied for body, in fact almost all of tissue all be unable to do without an oxygen
Change nitrogen.Increasing research shows that nitric oxide has in treatment angiocardiopathy and many other great chronic diseases
Play the role of important.Nitric oxide production main Physiological Function is included to cardiovascular system, immune system, the circulatory system, maincenter god
Effect through system and urogenital system（《Nitric oxide allows you away from cardiovascular and cerebrovascular diseases》, original work Louis J. lgnarro
, it is main to translate：Wu Shouling, Yang Ganghong；Medical publishing society of Peking University publishes, 2007）.
At present from soya bean different parts separation endogenetic bacteria mainly adhere to separately bacillus (Bacillus), false unit cell
Pseudomonas（Pseudomonas）, Enterobacter（Enterobacter）, Burkholderia category（Burkholderia）, citric acid bar
Pseudomonas（Klebsiella）, xanthomonas（Xanthomonas）Deng（Jitendra Dalal1 and Nikhilesh
Kulkarni1, British Microbiology Research Journal, 2013,3 (1)：96-105）, people are from soybean
The purpose of middle separation screening endophyte is also intended merely to agricultural production needs.Except contained " abiotic " class nutriment in soybean
Outside, the report screened from soybean endogenetic bacterium to the endophyte of beneficial effect people or other animals is had not yet to see.
The content of the invention
The technical problem to be solved in the present invention is to provide soybean endogenetic bacterium and its application, six kinds of soybean endogenetic bacterium respectively from
In black bean sprout, soya bean seed and moyashi, and all belong to bacillus；This six kinds of soybean endogenetic bacterium can significantly extend show
The average life span of beautiful hidden rhabditida, and the heat resistance of Caenorhabditis elegans is improved, and can be in Caenorhabditis elegans body
Field planting；Also there is certain effect to flight against senium of human body and extension life-span, can be applied in health food or medicine.
In order to solve the above technical problems, the technical solution used in the present invention is：
A kind of soybean endogenetic bacterium, it is characterised in that be black bean sprout endogenetic bacteria, the black bean sprout endogenetic bacteria is lichens gemma
Bacillus EnB-bsys4 (Bacillus licheniformis), belong to bacillus, preserving number is CGMCC No.10524.
A kind of soybean endogenetic bacterium, it is characterised in that be black bean sprout endogenetic bacteria, the black bean sprout endogenetic bacteria is solution starch bud
Spore bacillus EnB-bsys5（Bacillus amyloliquefaciens）, belong to bacillus, preserving number CGMCC
No.10525。
A kind of soybean endogenetic bacterium, it is characterised in that be soya bean seed endogenetic bacteria, the soya bean seed endogenetic bacteria is waxy
Bacillus EnB-sy3 (Bacillus cereus), belong to bacillus, preserving number is CGMCC No.10687.
A kind of soybean endogenetic bacterium, it is characterised in that be soya bean seed endogenetic bacteria, the soya bean seed endogenetic bacteria is lichens
Bacillus EnB-sy8 (Bacillus licheniformis), belong to bacillus, preserving number CGMCC
No.10688。
A kind of soybean endogenetic bacterium, it is characterised in that be moyashi endogenetic bacteria, the moyashi endogenetic bacteria is short and small gemma
Bacillus EnB-sys1 (Bacillus pumilus), belong to bacillus, preserving number is CGMCC No.10689.
A kind of soybean endogenetic bacterium, it is characterised in that be moyashi endogenetic bacteria, the moyashi endogenetic bacteria is waxy gemma
Bacillus EnB-sys2 (Bacillus cereus), belong to bacillus, preserving number is CGMCC No.10690.
Six kinds of soybean endogenetic bacterium in the present invention：From the bacillus licheniformis EnB-bsys4 of black bean sprout separation
(Baclicus licheniformis), bacillus amyloliquefaciens EnB-bsys5（Bacillus amyloliquefaciens）
In on 2 5th, 2015 in China Committee for Culture Collection of Microorganisms's common micro-organisms center（Abbreviation CGMCC）Preservation,
Preserving number is respectively CGMCC No.10524, CGMCC No.10525；From the bacillus cereus EnB-sy3 of soya bean seed separation
（Bacillus cereus）, bacillus licheniformis EnB-sy8 (Bacillus licheniformis) and from moyashi point
From bacillus pumilus EnB-sys1（Bacillus pumilus）, bacillus cereus EnB-sys2 (Bacillus cereus), on April 7th, 2015 in China Committee for Culture Collection of Microorganisms's common micro-organisms center（Referred to as
CGMCC）Preservation, preserving number are respectively CGMCC No.10687, CGMCC No.10688, CGMCC No.10689 and CGMCC
No.10690.DSMZ of Institute of Microorganism, Academia Sinica address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The taxology feature of six kinds of soybean endogenetic bacterium of the present invention is respectively：
（1）Bacillus licheniformis EnB-bsys4：5 ~ 10 millimeters of colony diameter, the central shallow white irregular roundness in pink colour edge
Shape, central pink colour is dry, and edge milky moistening, when being cultivated in LB fluid nutrient mediums, thalline is under an optical microscope in small
Short straight rod；
The bacterial strain can not grow at 50 DEG C, can not be grown at 10 DEG C, but can grow under anaerobic.Starch
Hydrolytic enzyme activities, nitrate reduction and lactose fermentation production acid are positive, and hemolytic reaction and indole reaction are negative.Utilize
When API50 CH kits detect, bacterial strain EnB-bsys4 can utilize glycerine, L-arabinose, ribose, D- xyloses, glucose, fruit
Sugar, mannose, inositol, mannitol, sorbierite, Alpha-Methyl-D-Glucose glucoside, amygdalin, Arbutin, aesculin, salicin, fibre
Tie up disaccharides, maltose, melibiose, sucrose, trehalose, synanthrin, raffinose, starch, glycogen, D- turanoses, D-Tag and Portugal
Grape sugar lime；Red tinea alcohol, D-R, L- xyloses, adonite, Beta-methyl-D- xylosides, galactolipin, sorb can not be utilized
Sugar, rhamnose, melampyrin, Alpha-Methyl-D-MANNOSE glucoside, N- acetyl-aminoglucose, melezitose, xylitol, gentiobiose, lactose,
D- lyxoses, D- rocks sugar, L- rocks sugar, D-R alcohol, L-arabinose alcohol, 2- keto-D-gluconates salt and 5- ketone groups-Portugal
Grape sugar lime.
（2）Bacillus amyloliquefaciens EnB-bsys5：About 5 millimeters of colony diameter, white, central protrusion bubble sample edge
Flat diverging, is not easy picking, and thalline is slightly long into short straight rod in LB fluid nutrient mediums；
The bacterial strain Starch Hydrolysis enzymatic activity is positive, and nitrate reduction is positive, and hemolytic reaction is positive, lactose fermenters production acid, and 12%
Grown in sodium chloride.When being detected using API50 CH kits, bacterial strain EnB-bsys5 can utilize glycerine, L-arabinose, core
Sugar, D- xyloses, L- xyloses, adonite, glucose, fructose, mannose, mannitol, sorbierite, Alpha-Methyl-D-Glucose glucoside, hardship
Almond glucoside, Arbutin, aesculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, synanthrin, melezitose, cottonseed
Sugar, starch, glycogen, D- turanoses, D- lyxoses and gluconate, remaining 20 kinds of carbohydrate in API50 CH kits are then not
It can utilize.
（3）Bacillus cereus EnB-sy3：About 5 millimeters of colony diameter, white colony is opaque, frosted shape, and centre is slightly
There is projection, peripheral flat, edge is more neat, relatively dries.Thalline is slightly long into short straight rod, some in LB fluid nutrient mediums；
The bacterial strain grows under 50 °, nitrate reduction negative, and citrate is positive, and hydrogen peroxide is positive, the double water of arginine
Solution is positive.When being detected using API50 CH kits, find bacterial strain EnB-sy3 can utilize glycerine, L-arabinose, glucose,
Fructose, mannose, mannitol, inositol, N- acetyl-aminoglucose, Arbutin, aesculin, salicin, cellobiose, maltose, sugarcane
Sugar, trehalose and D-Tag, remaining 33 kinds of carbohydrate in API50 CH kits can not then utilize.
（4）Bacillus licheniformis EnB-sy8：About 6 millimeters of colony diameter, middle lightpink periphery is shallower to turn white, and edge is not
Regular bacterium colony, relatively moisten.Thalline is into short and small straight-bar in LB fluid nutrient mediums；
The bacterial strain grows under 50 °, Starch Hydrolysis enzymatic activity is positive, nitrate is positive, citrate is positive, lactose fermentation
Production acid is positive, and the double hydrolysis of arginine are negative, haemolysis is negative, indole reaction is negative.When being detected using API50 CH kits, find
Bacterial strain EnB-Sy8 can utilize L-arabinose, ribose, D- xyloses, glucose, fructose, mannose, inositol, mannitol, sorb
Alcohol, Alpha-Methyl-D-Glucose glucoside, N- acetyl-aminoglucose, aesculin, cellobiose, maltose, sucrose, trehalose, synanthrin, shallow lake
Powder, D- turanoses and D-Tag, remaining 29 kinds of carbohydrate in API50 CH kits can not then utilize.
（5）Bacillus pumilus EnB-sys1：About 2 ~ 4 millimeters of colony diameter, inclined circular colonies, white, relatively dry, in
Entreat pleated raised brim uniformly neat.Thalline is into short and small straight-bar in LB fluid nutrient mediums；
The bacterial strain citrate is positive using the double hydrolysis of positive, arginine, and nitrate reduction negative, haemolysis are negative, starch
Production acid is negative, can be grown in pH5.5.When being detected using API50 CH kits, find bacterial strain EnB-sys1 can utilize L- Ah
Draw uncle's sugar, ribose, glucose, fructose, mannose, inositol, mannitol, sorbierite, Alpha-Methyl-D-Glucose glucoside, N- acetyl-Portugal
Osamine, aesculin, salicin, cellobiose, maltose, sucrose, trehalose and D-Tag, remaining in API50 CH kits
32 kinds of carbohydrates can not then utilize.
（6）Bacillus cereus EnB-sys2：About 5 millimeters of colony diameter, white colony is opaque, frosted shape, middle
Slightly projection, peripheral flat, edge is more neat, relatively dries.Thalline has merogenesis into middle stock, some in LB fluid nutrient mediums；
The bacterial strain nitrate reduction is positive, citrate can be grown using positive, the haemolysis positive, pH8, the double hydrolysis of arginine
Negative, 10% sodium chloride feminine gender.When being detected using API50 CH kits, it is found that bacterial strain EnB-sys2 is only capable of utilizing ribose, grape
Sugar, fructose, N- acetyl-aminoglucose, aesculin, maltose, sucrose and trehalose, remaining 41 kinds of sugar in API50 CH kits
Class can not then utilize.
The invention discloses soybean endogenetic bacterium prepare with extend the life-span, anti-aging effects health food and medicine in
Application.
The invention also discloses soybean endogenetic bacterium to prepare improvement angiocardiopathy, disease of immune system, circulatory system disease
Application in the medicine and health food of disease, central nervous system disease and disease of the genitourinary system.
Wherein, the specific separation of six kinds of soybean endogenetic bacterium of the invention and screening technique are：
The black soya bean and soya bean seed that 10 are cleaned up with running water flowing water soak 3 minutes in 70% alcohol, then
With being dipped in 3% sodium hypochlorite 5 minutes after aseptic water washing, finally with after aseptic water washing and soaking 4 hours.From last
Take 100 microlitres to apply LB plates as control in the sterilized water of secondary immersion, examine the surface of the seed sterilization whether thorough with this.Afterwards will
Seed takes 100 microlitres to be connected in LB fluid nutrient mediums after being ground in mortar, preculture at 37 DEG C in common biochemical cultivation case
24 hours.LB flat boards are applied after nutrient solution is carried out into gradient dilution, 37 DEG C are cultivated 1 ~ 2 day.The different bacterium colony of picking form respectively
Pure culture is carried out, chooses bacterium colony and carries out plate streaking, experiment is at least in triplicate.Meanwhile by finally with sterilized water immersion
Seed is placed at dark and cultivated, and after bean sprouts is grown, bean sprouts is cut into 1 centimeter length segment with the knife to sterilize in advance, use is above-mentioned
Same method carries out the separation of endophyte.The pure culture of gained freezes for -80 DEG C in 50% glycerine.To the single bacterium colony of separation
Carry out thalli morphology observation, Physiology and biochemistry and 16S rDNA sequence analyses.By in isolated black soya bean, soya bean seed and bean sprouts
Endophyte difference feeding Wild-type C. elegans, study different foods to Wild-type C. elegans life-span and heat resistanceheat resistant
The influence of property.Two bacillus, the moyashi in two bacillus, soya bean seed are finally found from black bean sprout endophyte
In two bacillus can significantly extend the average life span of Wild-type C. elegans, and can significantly improve wild
Type Caenorhabditis elegans heat resistance.Wild-type C. elegans hereinafter referred to as Caenorhabditis elegans used in the present invention.
It is using beneficial effect caused by above-mentioned technical proposal：Six kinds of soybean endogenetic bacterium in the present invention can be significantly
Extend the average life span of wild Caenorhabditis elegans, improve the heat resistance of Wild-type C. elegans, and can be out of office
It is colonized in raw type Caenorhabditis elegans body, wherein four plants of soybean endogenetic bacterium EnB-bsys4, EnB-bsys5, EnB-sy8 and EnB-
Sys1 can produce NO.Six kinds of soybean endogenetic bacterium in the present invention also have certain effect to flight against senium of human body and extension life-span, can
To be applied in health food or medicine, wherein four kinds of soybean endogenetic bacterium that can produce NO can also improve angiocarpy preparing
Medicine and the health care of disease, disease of immune system, circulation system disease, central nervous system disease and disease of the genitourinary system
It is applied in food.
Because NO is signaling molecule, important regulative can be played in cardiovascular system, immune system, nervous system.
So above-mentioned soybean endogenetic bacterium then can reduce the senile disease incidence of disease by producing NO, improve machine once being colonized in human body
Body immunity and quality of life；To hypertensive patient, it can then be played by taking viable bacteria microbial inoculum and reduced blood pressure, reduce cardiovascular and cerebrovascular
The incidence of disease.During due to the two plants of bacillus cereus for not producing NO feeding Wild-type C. elegans, it can equally extend beautiful hidden
Rhabditida average life span and raising Caenorhabditis elegans heat resistance, it is thus regarded that above-mentioned six plants of soybean endogenetic bacterium are also possible to be colonized
Caenorhabditis elegans is played by other approach afterwards beneficial to adjustment effect.In addition, in the presence of gemma bacterial strain is with trophosome, feed
When eating Caenorhabditis elegans, most bacillus trophosomes can be broken and dead by Caenorhabditis elegans " stinging ", so beautiful
Hidden rhabditida just obtains nutriment from the thalline of these " being bitten into pieces ", and these nutrients can also play Caenorhabditis elegans
Lengthen the life effect.Japanese scholars Komura in 2013, during Caenorhabditis elegans wild using Bifidobacterium feeding after broken, find elegant
The beautiful hidden rhabditida life-span can also be extended, by further testing, thus it is speculated that be probably among somatic cells wall and cytoplasm
Certain LTA component serves effect of lengthening the life to Caenorhabditis elegans.So above-mentioned six kinds have to Caenorhabditis elegans and lengthen the life
After the bacterium of effect makes high protein bacterium powder, it may be played a role in terms of body immunity is improved.
Brief description of the drawings
Fig. 1 is black bean sprout（A）, soya bean seed and moyashi（B）Influence of the endophyte to the Caenorhabditis elegans life-span；
Fig. 2 is the phylogenetic tree of soybean endogenetic bacterium；
Fig. 3 is that soybean endogenetic bacterium influences on Caenorhabditis elegans heat resistance.
Embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.Line used in embodiment
Worm is Wild-type C. elegans.
Embodiment 1
1st, the separation of soybean endogenetic bacterium
Black soya bean and soya bean seed are purchased from Baoding market.The black soya bean and soya bean seed that 10 are cleaned up with running water flowing water
Soak in 70% alcohol 3 minutes, then with being dipped in after aseptic water washing in 3% sodium hypochlorite 5 minutes, finally rushed with sterilized water
After washing and soak 4 hours.Take 100 microlitres to apply LB plates as control from the sterilized water of last time immersion, kind is examined with this
Whether sub- surface sterilization is thorough.100 microlitres are taken to be connected in LB fluid nutrient mediums after seed is ground in mortar afterwards, commonly
Preculture 24 hours at 37 DEG C in biochemical cultivation case.LB flat boards, 37 DEG C of cultures 1 ~ 2 are applied after nutrient solution is carried out into gradient dilution
My god.The different bacterium colony of picking form carries out pure culture respectively, chooses bacterium colony and carries out plate streaking, experiment is at least in triplicate.
Meanwhile the seed that finally will be soaked with sterilized water is placed at dark culture, after bean sprouts is grown, by bean sprouts with the knife to sterilize in advance
1 centimeter length segment is cut into, the separation of endophyte is carried out using above-mentioned same method.By pure culture obtained as above in 50%
- 80 DEG C freeze in glycerine.And thalli morphology observation, physiological and biochemical analysis are carried out to the single bacterium colony of separation, and to bacterium colony, thalline
Or the bacterial strain that physiological and biochemical property has notable difference carries out 16S rDNA sequence analyses.
2nd, the screening of soybean endogenetic bacterium
（1）Actication of culture：All soybean endogenetic bacterium that cryogenic freezing is preserved are melted and are inoculated into LB liquid by 10% inoculum concentration
In body culture medium, 37 DEG C of activation cultures 24 hours.Next day, the strain of activation is transferred to fresh LB fluid nutrient mediums again
In passed on, cultivate 12 hours.
（2）Wild-type C. elegans activate：- 80 DEG C of Wild-type C. elegans frozen are taken out, 37 DEG C fast
Speed melt, centrifugation, discard supernatant, by remaining polypide be added to containingE.coliOn OP50 NGM culture plates, 20 DEG C incubated.
（3）Caenorhabditis elegans standard chowE.coliOP50 preparation：PickingE.coliOP50 single bacterium colonies access warp
Cross in autoclaved 2 milliliters of fresh LB fluid nutrient mediums, 180 revs/min of concussion and cultivates 8 ~ 12 hours at 37 DEG C,
To OD600For 1.0 or so when, draw 100 microlitres of bacterium solutions and even spread be to the middle position of the NGM flat boards prepared in advance
（Apart from about 0.5 millimeter of plate edge）, overnight incubation is inverted in 37 DEG C, 4 DEG C is put and saves backup.
Prepare Caenorhabditis elegans Liquid Culture standard chowE.coliDuring OP50 foods, bacterial strainE.coliOP50's
Cultural method is same as above, and simply arrives OD in culture600For 1.0 or so when in 4 DEG C 4000 revs/min centrifuge 10 minutes, collect bacterium
Body, obtained precipitation is washed twice with S. Medium nutrient solutions, supernatant discarding, add S. Medium nutrient solutions adjustment thalline
Concentration is to OD600For 1.0 or so, put 4 DEG C and save backup.
（4）The culture of Caenorhabditis elegans soybean endogenetic bacterium food：Respectively by above-mentioned activation and the soybean endogenetic that has passed on
Bacterium draws 150 microlitres of coating LB solid plates with liquid-transfering gun, and 37 DEG C are cultivated 12 hours.Next day, with M9 buffer solutions by LB plates
Thalline rinse, be collected into centrifuge tube, obtained precipitation washed twice with S. Medium nutrient solutions, supernatant discarding,
Add S. Medium nutrient solutions and adjust cell concentration to OD600For 1.0 or so, put 4 DEG C and save backup.
（5）The synchronization of Caenorhabditis elegans：Picking not by living contaminants, in more beautiful hidden of egg-laying season adult
Rhabditida culture plate, polypide and ovum are rinsed with M9 buffer solutions, are collected into centrifuge tube, the polypide amount of every pipe is tried one's best one
Cause, polypide density can not be excessive.Centrifuge tube containing egg-laying season adult is centrifuged 1 minute in 4 DEG C 1500 revs/min, discarded
Supernatant.Into above-mentioned centrifuge tube, often pipe adds 650 microlitres of M9 buffer solutions and 350 microlitres of nematode lysates now matched somebody with somebody again, fully
Concussion mixes, and untill visually seeing less than polypide, takes a small amount of liquid immediately in micro- Microscopic observation, it is determined whether only remaining
Worm's ovum, this process was no more than 10 minutes, and otherwise worm's ovum can also be affected, or even dead.It is only surplus in centrifuge tube is observed
The moment of lower worm's ovum, centrifuged 30 seconds in 4 DEG C 3500 revs/min immediately, discard supernatant.M9 is added into above-mentioned centrifuge tube to delay
The remaining precipitation of fliud flushing washing, 4 DEG C 3500 revs/min centrifuge 30 seconds, discard supernatant.The above-mentioned washing step of repetition four times.Will most
The beautiful hidden bar worm's ovum obtained afterwards be drawn to it is above-mentioned containingE.coliOP50 fresh NGM flat boards（Step（3））Edge be free of
Bacterium part, 20 DEG C incubated, and after about 48 hours, these ovum will bud into the L4 phase nematodes of synchronization, is ground for testing
Study carefully.
（6）The durability analysis of Caenorhabditis elegans：The Caenorhabditis elegans in the L4 phases turns respectively after 30 are synchronized
Contain 500 microlitres of OD to addition600In 24 orifice plates for 1.0 or so different soybean endogenetic bacteria liquid foods, 20 DEG C of constant temperature trainings
Support, with additionE.coliOP50 liquid foods are per 30, hole for control, nematode bar number.Nematode is transferred to containing corresponding daily
Fresh food hole in, the insect to die is recorded daily.The life experimental data of Caenorhabditis elegans uses SPSS
19.0 Kalpan-Meir survival curve parser analysis.
As a result show：Two plants of endophytes being screened from black bean sprout, the two plants of endophytes screened from soya bean seed, from Huang
The two plants of endophytes screened in bean sprouts can significantly extend the average life span of Wild-type C. elegans, as shown in Figure 1.With mark
Quasi- foodE.coliDuring OP50 feeding Caenorhabditis elegans, the mean survival time of nematode is 20.467 ± 1.657 days；With black
During two plants of endophyte EnB-bsys4 and EnB-bsys5 the feeding Caenorhabditis elegans separated in bean sprouts, the nematode mean survival time
Respectively 30.517 ± 1.608 (P<And 27.636 ± 1.540 (P 0.001)<0.01), the life-span of Caenorhabditis elegans is than feeding
Eat standard chowE.coliOP50 extends 49.10% and 35.03% respectively；With the two plants of endophyte EnB- separated in soya bean seed
During sy3 and EnB-sy8 feeding Caenorhabditis elegans, the nematode mean survival time is respectively 32.385 ± 2.008 (P<0.001)
With 36.353 ± 1.790 (P<0.001), the life-span of Caenorhabditis elegans is than feeding standard chowE.coliOP50 extends respectively
58.23% and 77.61%；With two plants of endophyte EnB-sys1 and EnB-sys2 the feeding Caenorhabditis elegans separated in moyashi
When, the nematode mean survival time is respectively 26.714 ± 1.405 (P<And 28.429 ± 1.407 (P 0.05)<0.01) it is, beautiful
The life-span of hidden rhabditida is than feeding standard chowE.coliOP50 extends 30.52% and 38.90% respectively.
According to above-mentioned six plants endophyte constructing systems that can significantly extend the Caenorhabditis elegans life-span of 16S rDNA sequence pairs
Development tree, as shown in Figure 2.Above-mentioned six plants of endophytes that can extend the Caenorhabditis elegans life-span are entered using API50 CH kits
Row API is detected, the index not being listed in API50 CH kits（Such as Starch Hydrolysis enzymatic activity）According to Jousimies-
The Bacteria Identification handbook such as Somer（2002 the 6th edition, Star publishing company）Carry out.According to bacterial strain 16S rDNA sequences, with reference to bacterium
Fall with morphological features and physiological and biochemical index testing result, can significantly extend beautiful hidden bar by separated in black bean sprout two plants
The endophyte EnB-bsys4 and EnB-bsys5 in nematode life-span are identified respectively as bacillus licheniformisBacillus licheniformisAnd bacillus amyloliquefaciensBacillus amyloliquefaciens., will be by soya bean with same method
The endophyte EnB-sy3 and EnB-sy8 that two plants separated in seed can significantly extend the Caenorhabditis elegans life-span are identified respectively as
Bacillus cereusBacillus cereusAnd bacillus licheniformisBacillus licheniformis；By in moyashi points
From two plants of endophyte EnB-sys1 and EnB-sys2 that can significantly extend the Caenorhabditis elegans life-span be identified respectively as short and small bud
Spore bacillusBacillus pumilusAnd bacillus cereusBacillus cereus。。
3rd, detection of the soybean endogenetic bacterium in Caenorhabditis elegans intestinal colonisation
Bacillus in black soya bean and soya bean can produce the extremely strong gemma of resistance, and it can bear a variety of poor environments,
Therefore, bacillus is possible to that Caenorhabditis elegans enteron aisle can be colonized in.Screened to detect from black soya bean and soya bean
Six plants of endophytes feeding Caenorhabditis elegans can be colonized in its enteron aisle after for a period of time, carried out following experiment：
The Caenorhabditis elegans that the L4 phases are in after synchronization is distinguished into feeding 3 days, one week with above-mentioned six plants of soybean endogenetic bacterium
And surface sterilization processing and culture are carried out after three weeks as follows：
(1) draw 24 orifice plates in Caenorhabditis elegans in 1.5 milliliters of EP pipes, add M9 buffer solutions, 1200g to
Centrifuged 2 minutes under mental and physical efforts, abandon supernatant, 100 microlitre of 70% alcohol is added in centrifuge tube, to nematode surface sterilization 20 seconds, then
1 milliliter of M9 buffer solution is added immediately（Dilute alcohol）.
(2) after being washed twice with M9 buffer solutions, supernatant is abandoned, 100 microlitre of 10% sodium hypochlorite is added in centrifuge tube, it is right
Nematode surface sterilization 40 seconds, 1 milliliter of M9 buffer solution is added immediately after（Dilute sodium hypochlorite）, and washed again with M9 buffer solutions
Nematode is twice.
(3) 100 microlitres of buffer solution in the nematode pipe after adsorption surface sterilization is in the LB Liquid Cultures of sterilizing, 37 °C
Cultivate and be used as control, whether detection nematode surface sterilization is thorough.
(4) remaining polypide and buffer solution are smashed to pieces with grinding rod（Rupture polypide, bacterium is overflowed）, then take 100 microlitres
The polypide buffer solution smashed to pieces 37 °C of cultures in the LB culture mediums of sterilizing.
(5) if step（3）Middle LB cultures asepsis growth illustrates that nematode surface sterilization is thorough, then step（4）Middle LB trainings
It is the thalline in nematode body to support the thalline grown in base, carries out coated plate to the bacterium, single bacterium colony is obtained, from colonial morphology, 16S
RDNA gene orders verify the bacterium whether be feeding Caenorhabditis elegans soybean endogenetic bacterium.
Coated plate after the nematode of 3 days soybean endophytes of feeding is ground, find there are two kinds of colonial morphologies on plate, one kind is similar to
The soybean endogenetic bacterium bacterium colony of feeding, by this two kinds of bacterium colonies difference after purification, carry out DNA extractions and 16S rDNA are sequenced and carried out
BLAST is compared, and as a result finds that two kinds of bacterium are respectively Escherichia coli and the soybean endogenetic bacterium of feeding；To the big of feeding one week and three weeks
Coated plate after the nematode of beans endophyte grinds, find there was only the soybean endogenetic bacterium bacterium colony of a kind of colonial morphology, i.e. feeding on flat board, together
Sample carries out DNA extractions and 16S rDNA sequencings to it, and it is exactly the soybean endogenetic bacterium of feeding as a result to find a kind of this bacterium.To sum up institute
State, it is believed that, the battalion that black soya bean and soya bean endophyte may be provided both Caenorhabditis elegans life-time dilatation in itself with thalline
Foster material is relevant, and the beneficial adjustment effect played after being also colonized with thalline in line polypide is relevant.
4th, soybean endogenetic bacterium release in vitro NO is determined
Gusarov, which is equal to 2013, to be found, during with bacillus subtilis feeding Wild-type C. elegans, the nematode life-span
Significantly extended, further study show that, the NO of the bacillus subtilis release of institute's feeding can be used as signaling molecule, pass through
ILS approach regulates and controls the life-span of nematode（Cell 2013,152：818-830）.We are from Institute of Microorganism, Academia Sinica bacterium
Kind collection have purchased a bacillus subtilis（Bacterial strain preserving number: CGMCC No. 1.4255）, when with the bacterial strain feeding
During wild Caenorhabditis elegans, discovery and feedingE.coliOP50 control is compared, not obvious to the nematode life-span to extend
Effect.Determined by nitrate/nitrite assay kit, it is found that bought bacillus subtilis is nearly free from NO.
Therefore, it is presumed that six plants of soybean endogenetic bacterium for having effect of lengthening the life to wild Caenorhabditis elegans of above-mentioned separation are likely to produce
Raw NO.
Above-mentioned six plants of soybean endogenetics bacterium bacteria suspension of 12 hours will be cultivated in advance, with nitric oxide kit（Nanjing is built
Into）NO generations are determined, are comprised the following steps that：
(1) 100 microlitres of distilled waters are taken to make as blank tube, standard items as standard pipe, soybean endogenetic bacterium bacteria suspension respectively
Managed for measure.
(2) 400 microlitres of mix reagent is added, is mixed, 37 °C of accurate water-baths 60 minutes.
(3) 3 200 microlitres of reagent, 4 100 microlitres of reagent are added, abundant vortex mixes 30 seconds, is stored at room temperature 40 points
Clock, centrifuge 10 minutes under 3500g centripetal force.
(4) 500 microlitres of supernatant plus 600 microlitres of developers are taken respectively, are stored at room temperature 10 minutes and are developed the color after mixing.
(5) distilled water returns to zero, and 550 nanometers, 0.5 centimetre of optical path, determines each pipe absorbance.
(6) NO contents are calculated according to formula.
As a result find, two plants of black soya bean endophytes EnB-bsys4 and EnB-bsys5 can produce NO, average content point
Wei not 48.85 μm of ol/L and 44.04 μm of ol/L.Soya bean endophyte EnB-sy8 and EnB-sys1 also produce NO, averagely contain
Amount is respectively 28.36 μm of ol/L and 73.24 μm of ol/L, and soya bean endophyte EnB-sy3 and EnB-sys2 do not produce NO then.
There is the bacillus soybean endogenetic bacterium for effect of lengthening the life can be in beautiful hidden bar to Wild-type C. elegans due to this six plants
Be colonized in nematode enteron aisle, we it is reasonable that, wherein four plants of bacillus soybean endogenetic bacterium that can produce NO may pass through production
NO signaling molecules regulate and control Caenorhabditis elegans associated metabolic, finally make it that nematode is significantly extended.
5th, soybean endogenetic bacterium influences on Caenorhabditis elegans heat resistance
Above-mentioned synchronized L4 phases Caenorhabditis elegans is respectively added to eat containing different soybean endogenetic bacterium foods and standard
ThingE.coliIn OP50 24 orifice plates, contain per holeE.coliOP50 bacteria suspensions and each 500 μ L of soybean endogenetic bacterium bacteria suspension,
30/hole of nematode, adult is transferred to inside the 24 new holes containing corresponding fresh food every 24 hours, 20 DEG C of constant temperature trainings
Support.During by the 5th day, heat stress experiment is carried out under the conditions of being put in 37 DEG C, a nematode survival condition was observed every 2 hours.
Nematode death criterion is with above-mentioned Caenorhabditis elegans life test, and test data is at least in triplicate.
As a result show：Two plants of endophytes are equal in two plants of endophytes, moyashi in two plants of endophytes, soya bean seed in black bean sprout
The heat resistance of Wild-type C. elegans is significantly improved, as a result as shown in Figure 3.
When black soya bean endophyte andE.coliThe Caenorhabditis elegans of OP50 feedings is put at 37 DEG C 3 ~ 4 hours respectively, nematode
Start it is dead, when be placed in 5 at 37 DEG C, 11 and 13 it is small when, feedingE.coliOP50 nematode, feeding black soya bean endophyte EnB-
Bsys4 and EnB-bsys5 nematode difference mortality, the nematode mean survival time of feeding difference food is respectively：FeedingE.coliOP50 nematode is 5.37 ± 0.31 hours, and feeding black soya bean endophyte EnB-bsys4 and EnB-bsys5 are respectively
9.97 ± 0.48 and 10.23 ± 0.68 hours.Variance analysis shows, feeding black soya bean endophyte EnB-bsys4 and
EnB-bsys5 Caenorhabditis elegans is compared with feedingE.coliOP50 nematode has significant anti-acute heat stress ability (P<
0.001), as shown in Fig. 3-A.
When soya bean endophyte andE.coliWhen the Caenorhabditis elegans of OP50 feedings is put in 2 ~ 4 hours at 37 DEG C respectively, line
Worm starts death, when be placed in 4 at 37 DEG C, 6,6 and 5 hours when, feedingE.coliOP50 nematode, feeding soya bean endophyte
EnB-sy3, EnB-sy8, EnB-sys1 and EnB-sys2 nematode start Large Scale Death, the nematode of feeding difference food respectively
Mean survival time is respectively：FeedingE.coliOP50 nematode is 4.73 ± 0.34 hours, feeding soya bean endophyte EnB-
Sy3, EnB-sy8, EnB-sys1 and EnB-sys2 are respectively 6.60 ± 0.34,6.74 ± 0.47,5.84 ± 0.32 and 6.53
± 0.36 hour.Variance analysis shows, feeding soya bean endophyte EnB-sy3 (P< 0.01)、EnB-sy8(P < 0.01)、
EnB-sys1 (P <And EnB-sys2 (P 0.05)<0.01) Caenorhabditis elegans is compared with feedingE.coliOP50 line
Worm has significant anti-acute heat stress ability, as shown in Fig. 3-B.
Claims (2)
1. a kind of soybean endogenetic bacterium, it is characterised in that be black bean sprout endogenetic bacteria, the black bean sprout endogenetic bacteria is lichens gemma bar
Bacterium EnB-bsys4 (Bacillus licheniformis), belong to bacillus, preserving number is CGMCC No.10524.
2. soybean endogenetic bacterium as claimed in claim 1 prepare with extend the life-span, anti-aging effects health products in should
With.
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| Bacterial Nitric Oxide Extends the lifespan of C. elegans;Ivan Gusarov,et al;《Cell》;20130214;第152卷(第4期);818-830 * |
| 地衣芽孢杆菌在食品工业中的应用;王聪等;《食品工业》;20141231(第12期);254-257 * |
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