CN109486707A - A kind of bacillus subtilis strain and its application - Google Patents
A kind of bacillus subtilis strain and its application Download PDFInfo
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- CN109486707A CN109486707A CN201811409452.XA CN201811409452A CN109486707A CN 109486707 A CN109486707 A CN 109486707A CN 201811409452 A CN201811409452 A CN 201811409452A CN 109486707 A CN109486707 A CN 109486707A
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- bacillus subtilis
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 69
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 68
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 238000000855 fermentation Methods 0.000 claims abstract description 42
- 230000004151 fermentation Effects 0.000 claims abstract description 42
- 235000009754 Vitis X bourquina Nutrition 0.000 claims abstract description 33
- 235000012333 Vitis X labruscana Nutrition 0.000 claims abstract description 33
- 235000014787 Vitis vinifera Nutrition 0.000 claims abstract description 33
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 15
- 241000228212 Aspergillus Species 0.000 claims abstract description 13
- 241000228150 Penicillium chrysogenum Species 0.000 claims abstract description 13
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 239000002207 metabolite Substances 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 32
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- 238000002360 preparation method Methods 0.000 claims description 22
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
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- UOQHWNPVNXSDDO-UHFFFAOYSA-N 3-bromoimidazo[1,2-a]pyridine-6-carbonitrile Chemical compound C1=CC(C#N)=CN2C(Br)=CN=C21 UOQHWNPVNXSDDO-UHFFFAOYSA-N 0.000 description 1
- 241000252983 Caecum Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
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- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
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- 230000001717 pathogenic effect Effects 0.000 description 1
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- 229920001277 pectin Polymers 0.000 description 1
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- 108020004410 pectinesterase Proteins 0.000 description 1
- 229940041153 polymyxins Drugs 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- General Health & Medical Sciences (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The embodiment of the invention discloses a kind of bacillus subtilis strain and its application, the bacillus subtilis strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC NO.16569；The preservation time are as follows: on October 10th, 2018.The embodiment of the invention also provides a kind of bacillus subtilis high density fermentation liquid containing the bacillus subtilis strain metabolite.A kind of bacillus subtilis strain provided in an embodiment of the present invention can effectively inhibit Aspergillus carbonerius, aspergillus niger, penicillium chrysogenum, and Aspergillus carbonerius, aspergillus niger, penicillium chrysogenum are the easily raw moulds of grape, therefore the bacillus subtilis high density fermentation liquid containing bacillus subtilis strain metabolite can effectively extend grape storage period, have the characteristics that efficient, safety and environmental protection, has broad application prospects.
Description
Technical field
The present embodiments relate to technical field of microbe application, and in particular to a kind of bacillus subtilis strain and its answers
With.
Background technique
Bacillus subtilis (Bacillus subtilis) is one kind of bacillus, and having prevents biological oxidation, a word used in person's names
Anti- pathogenic microorganisms improves inside and outside ecological environment, enhancing Immune Function In Animals, the work for generating a variety of digestive ferments and nutriment
With.Subtilin, polymyxins, nystatin, gramicidins etc. be can produce in bacillus subtilis growth course to pathogenic bacteria
Or the conditioned pathogen of autogenous infection has the active material of obvious inhibiting effect.Bacillus subtilis in enteron aisle can because of it
To rapidly deplete free oxygen, so as to reduce enteron aisle oxygen, so that beneficial anaerobic bacteria is preferably survived and part is inhibited to cause a disease
Bacterium.There are some researches prove add bacillus subtilis in chicken feed the average level of salmonella in chicken caecum can be made to substantially reduce
58%, this not only reduced by only the risk of life entity infection, also contribute to improve food safety.Therefore there is high antifungal
No matter active bacillus subtilis is helping improve human health status or is suffering from the aspect that ensures food safety important
Effect.Bacillus subtilis, as a kind of gram-positive bacterium, be used to always for a long time fermented soybean food, extensively
Production for industrial enzyme and chemicals.
But the rare discovery of existing bacillus subtilis inhibit Aspergillus carbonerius, aspergillus niger, obtain in penicillium chrysogenum compared with
The bacterial strain of good effect.
Summary of the invention
For this purpose, the embodiment of the present invention provides a kind of bacillus subtilis strain and its application, it is rare in the prior art to solve
It is found the problem of obtaining the bacterial strain of better effects in inhibition Aspergillus carbonerius, aspergillus niger, penicillium chrysogenum.
To achieve the goals above, embodiments of the present invention provide the following technical solutions:
In the first aspect of embodiments of the present invention, a kind of bacillus subtilis strain is provided, is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are as follows: CGMCC NO.16569；The preservation time
Are as follows: on October 10th, 2018.
In the second aspect of embodiments of the present invention, provide a kind of containing bacillus subtilis strain metabolism
The bacillus subtilis high density fermentation liquid of product.
In the third aspect of embodiments of the present invention, a kind of bacillus subtilis high density fermentation liquid is provided
Preparation method, comprising:
The preparation of strain seed liquor: the bacillus subtilis is activated, and the bacillus subtilis single colonie after taking activation is drawn
For line on culture medium, progress sealing later is simultaneously put into incubator the bacterial strain cultivated, activated, and the bacterial strain of activation is transferred to training
It supports and is cultivated in base, obtain the strain seed liquor；
The preparation of fermentation liquid: the strain seed liquor being seeded in the container equipped with high-density culture medium and is cultivated, system
Bacteria suspension is obtained, supernatant is collected after centrifugation in the bacteria suspension, supernatant liquid filtering and degerming obtain the bacillus subtilis
Bacterium high density fermentation liquid.
In one embodiment of the invention, the preparation method of the strain seed liquor includes: that bacillus subtilis exists
It is activated on NA plating medium, the bacillus subtilis single colonie after taking activation lines on NA culture medium, carries out sealing later
And being inverted in temperature is that 33 DEG C of incubators cultivate the bacterial strain activated for 24 hours again, and the bacterial strain of activation is transferred to LB with transfering loop
It is 33~50 DEG C in temperature in culture medium, revolving speed cultivates 24~50h under conditions of being 180~300r/min, and the bacterium is made
Kind seed liquor；
In one embodiment of the invention, the strain seed liquor saves standby in the environment of being -1~4 DEG C in temperature
With.
In one embodiment of the invention, the raw material of the NA plating medium or NA culture medium includes: distilled water, ox
Meat medicinal extract, peptone, glucose and agar；Wherein, every 1000mL distilled water correspond to 3.0g beef extract, 7.0g peptone,
10.0g glucose, 15.0g agar；
And/or the raw material of the LB culture medium includes: distilled water, sodium chloride, yeast extract and tryptone；Wherein,
Every 1000mL distilled water corresponds to 10.0g sodium chloride, 5.0g yeast extract, 10.0g tryptone.
In one embodiment of the invention, the preparation method of the fermentation liquid include: by the strain seed liquor with
The volume ratio that inoculum concentration is 3~6% is inoculated in the triangular flask equipped with high-density culture medium, is 33~50 DEG C, revolving speed in temperature
To cultivate 24~48h under conditions of 180~300r/min, bacteria suspension is obtained, the bacteria suspension is 10000r/min's in revolving speed
Supernatant is taken after being centrifuged in centrifuge, it is highly dense that supernatant filtering with microporous membrane and degerming obtain the bacillus subtilis
Spend fermentation liquid.
In one embodiment of the invention, the ring that the bacillus subtilis high density fermentation liquid is 4 DEG C in temperature
It is saved backup under border.
In one embodiment of the invention, the raw material of the high-density culture medium includes: distilled water, soybean powder, corn
Powder, yeast extract, NaCl, CaCl2And MnSO4·H2O；Wherein, it is beautiful to correspond to 20.03g soybean powder, 24.41g for every 1000mL distilled water
Rice flour, 12.69g yeast extract, 3g NaCl, 2g CaCl2、2g MnSO4·H2O。
In the fourth aspect of embodiments of the present invention, above-mentioned bacillus subtilis high density fermentation liquid is provided in suppression
Charcoal processing aspergillus niger, aspergillus niger, the application in penicillium chrysogenum.
In the fourth aspect of embodiments of the present invention, a kind of antistaling agent for grape is provided, it includes the withered grass buds
Spore bacillus high density fermentation liquid.
Embodiment according to the present invention has the advantages that
1, a kind of bacillus subtilis strain provided in an embodiment of the present invention can be yellow green to Aspergillus carbonerius, aspergillus niger, production
It is mould effectively to be inhibited, and Aspergillus carbonerius, aspergillus niger, penicillium chrysogenum are the easily raw moulds of grape, therefore contain bacillus subtilis
The bacillus subtilis high density fermentation liquid of bacteria strain metabolite can effectively extend grape storage period, have efficient, safety
And the features such as environmental protection, have broad application prospects.
2, bacillus subtilis strain provided in an embodiment of the present invention can be effectively reduced grape surface microorganism quantity, fruit
Grain hardness is high, and the color of grape pedicel is more bud green, and grape nutriment turnover rate is small, and appearance freshness and mouthfeel obtain well
It keeps, the freshness date of whole grape will extend 7~10 days.
Bacillus subtilis strain (Bacillus subtilis) provided by the present application is preserved in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology, deposit number are as follows: CGMCC NO.16569；The preservation time are as follows: on October 10th, 2018.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily, it is clear that described embodiment is the present invention one
Section Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not doing
Every other embodiment obtained under the premise of creative work out, shall fall within the protection scope of the present invention.
Embodiment 1
The preparation method for present embodiments providing a kind of bacillus subtilis high density fermentation liquid includes the following steps:
1) prepared by strain seed liquor: bacillus subtilis being activated on NA plating medium, single colonie is taken to line NA
On culture medium, sealing is inverted in 33 DEG C of incubator cultures for 24 hours, the bacterial strain activated, is shifted the bacterial strain of activation with transfering loop
Into LB culture medium, at 41.5 DEG C, under conditions of 240r/min, 37h is cultivated, strain seed liquor obtained is standby in 1.5 DEG C of preservations
With；
2) preparation of high density fermentation liquid: the strain seed liquor that step 1) is got ready is connect with the inoculum concentration of 4.5% (V/V)
Kind under the conditions of temperature is 41.5 DEG C, 240r/min, cultivates 36h, bacillus subtilis in the triangular flask of high-density culture medium
CGMCC NO.16569 viable count reaches 3.27 × 1011CFU/mL.Bacteria suspension is obtained, by bacteria suspension after 10000r/min is centrifuged
Supernatant is taken, with filtering with microporous membrane degerming, high density fermentation liquid is obtained and is saved backup in 4 DEG C.
Embodiment 2
The preparation method for present embodiments providing a kind of bacillus subtilis high density fermentation liquid includes the following steps:
1) prepared by strain seed liquor: bacillus subtilis being activated on NA plating medium, single colonie is taken to line NA
On culture medium, sealing is inverted in 33 DEG C of incubator cultures for 24 hours, the bacterial strain activated, is shifted the bacterial strain of activation with transfering loop
Into LB culture medium, at 33 DEG C, under conditions of 180r/min, for 24 hours, strain seed liquor obtained is saved backup in -1 DEG C for culture；
2) preparation of high density fermentation liquid: the strain seed liquor that step 1) is got ready is inoculated with the inoculum concentration of 3% (V/V)
In the triangular flask of high-density culture medium, under the conditions of temperature is 33 DEG C, 180r/min, cultivate for 24 hours, bacillus subtilis
CGMCC NO.16569 viable count reaches 3.41 × 1011CFU/mL.Bacteria suspension is obtained, by bacteria suspension after 10000r/min is centrifuged
Supernatant is taken, with filtering with microporous membrane degerming, high density fermentation liquid is obtained and is saved backup in 4 DEG C.
Embodiment 3
The preparation method for present embodiments providing a kind of bacillus subtilis high density fermentation liquid includes the following steps:
1) prepared by strain seed liquor: bacillus subtilis being activated on NA plating medium, single colonie is taken to line NA
On culture medium, sealing is inverted in 33 DEG C of incubator cultures for 24 hours, the bacterial strain activated, is shifted the bacterial strain of activation with transfering loop
Into LB culture medium, at 50 DEG C, under conditions of 300r/min, 50h is cultivated, strain seed liquor obtained is saved backup in 4 DEG C；
2) preparation of high density fermentation liquid: the strain seed liquor that step 1) is got ready is inoculated with the inoculum concentration of 6% (V/V)
In the triangular flask of high-density culture medium, under the conditions of temperature is 50 DEG C, 300r/min, 48h, bacillus subtilis are cultivated
CGMCC NO.16569 viable count reaches 3.60 × 1011CFU/mL.Bacteria suspension is obtained, by bacteria suspension after 10000r/min is centrifuged
Supernatant is taken, with filtering with microporous membrane degerming, high density fermentation liquid is obtained and is saved backup in 4 DEG C.
Embodiment 4
In taking embodiment 1-3 in the case where any technical solution, the composition of NA plating medium or NA culture medium
Are as follows: every 1000mL distilled water corresponds to 3.0g beef extract, 7.0g peptone, 10.0g glucose, 15.0g agar；LB culture medium
Composition are as follows: every 1000mL distilled water corresponds to 10.0g sodium chloride, 5.0g yeast extract, 10.0g tryptone；High Density Cultivation
The composition of base are as follows: every 1000mL distilled water, 20.03g soybean powder, 24.41g corn flour, 12.69g yeast extract, 3g NaCl, 2g
CaCl2, 2g MnSO4·H2O。
Antibacterial action of 1 bacillus subtilis of application examples to Aspergillus carbonerius, aspergillus niger and penicillium chrysogenum
It is acted in order to further illustrate resistance of the bacillus subtilis number to fungi, the present invention has studied the strain to normal
It is stored in the Aspergillus carbonerius on grape surface, the antibacterial action of aspergillus niger and penicillium chrysogenum and has done following inspection, the method is as follows:
The bacteria suspension of three kinds of moulds of activated strains and preparation first, Aspergillus carbonerius, aspergillus niger and penicillium chrysogenum etc. is continuous
Passage is inoculated in PDA culture medium plate twice, by Aspergillus carbonerius, aspergillus niger and penicillium chrysogenum culture respectively in PDA and
It is grown 5 days on czapek culture medium in 25 DEG C.One ring diameter of picking >=2mm bacterium colony accesses in 5ml sterile saline, with connecing
5min is smashed and shaken to kind ring, is uniformly distributed in it in sterile saline and is formed bacteria suspension.Pipette a little bacteria suspension
In on blood cell counting plate, observation is counted under the microscope, and adjustment concentration control is 1~5 × 106CFU/mL。
Prepare three kinds of spore suspensions (100 μ L) are coated in PDA culture medium.Using Oxford cup by 200 μ L's
The high density supernatant and sterile distilled water of bacillus subtilis liquid culture are added drop-wise to respectively identical contains carbon black respectively
In the PDA culture medium of aspergillus, aspergillus niger and penicillium chrysogenum, and it is incubated at 25 DEG C.It is measured after 5 days using crossbanding method
The diameter of inhibition zone.
Such as table 1, the experimental results showed that, culture solution effectively can inhibit fungi to grow.The inhibition zone of Aspergillus carbonerius is
24.3mm, the inhibition zone of aspergillus niger are 20.3mm, and the inhibition zone of penicillium chrysogenum is 27.5mm.
Inhibiting effect of the 1. high density fermentation liquid of table to microorganism
Effect of 2 bacillus subtilis of application examples in antistaling agent for grape
The culture that strain involved in the present embodiment generates during the cultivation process can be applied to coating-film fresh-keeping grape etc.
Berry, the specific method is as follows:
Color and uniform in size, undamaged grape cluster are immersed in the high density fermentation liquid of bacillus subtilis 1 minute,
It is dry in germ-free air flow lower surface, be put into carton, be placed in refrigerator and save, best storage temperature be -1 to
0℃。
In order to detect whether the culture of the strain can cause nutritional quality and sense organ product on the food such as grape to it
Adverse effect in matter, the present invention have carried out the nutrition and sensory evaluation to the grape by the processing of high density fermentation liquid.
The grape for using the strain to handle is cooked into experimental group, untreated grape is as a control group.It is examined through experiment
It tests, experimental group grape weight-loss ratio, rotting rate and shattering rate are significantly lower than control group.
Grape hardness is related with water content, this will affect the bulbs of pressure and brittleness of tissue.In the sample for compareing and handling
In, the significant reduction with the increase of storage time of grape hardness.Moisture loss increases pectase, pectinesterase and poly half
The activity of lactobionic acid enzyme causes pectin and polygalacturonase to be degraded, leads to fruit softening.It is high from the 5th day to the 30th day
The processed grape of density fermentation liquid is more stronger than control grape hardness.
Titratable acid content is fruit-vegetable quality important indicator, it is demonstrated experimentally that experimental group grape is not shown with control group grape
Write sex differernce.Illustrate, the titratable acid content for not interfering with grape is handled using the strain.
Vitamin C content be fruit vegetable nutrient value important indicator and fruit vegetables storing effect assessment key index it
One.It is demonstrated experimentally that experimental group as control group, is presented the trend risen in first 15 days, downward trend is then presented, and Vc contains
Amount is also in same level.This explanation, the use of the strain high density fermentation liquid can keep grape Vitamin C content.
Respiratory intensity is inseparable with the storage relationship of fruit, and the size variation of respiratory intensity may determine that fruit vegetable nutrient object
The consumption of matter and the aging state of fruits and vegetables.By inhibiting fruits and vegetables respiratory rate, the aging of fruits and vegetables can be delayed.Control group is exhaled
Suction rate was 74.3mg/kgh at first day of storage, but was declined with the increase of storage time, from the tenth day 54.0mg/
Kgh is down to the 30th day 31.0mg/kgh.And experimental group the tenth day was respectively with the 30th day respiratory rate
46.7mg/kgh and 21.3mg/kgh.These are the result shows that the high density fermentation liquid processing of the strain is effectively reduced and exhaled
Frequency is inhaled, the weightlessness and aging of grape can be effectively prevented, keep the quality of grape.
The strain be applied to grape it is fresh-keeping in, can reduce within one month grape because rot due to the 50% of loss amount.With every
Mu plantation 300 plants, every plant 5 string, it is every string two jin be standard, 3000 jin or so of every per mu yield, about 1500 yuan of cost.With 15 yuan per jin
It calculates, every month can reduce 11250 yuan of loss per acre for peasant.To be July to 4 months October, 1 year is reduced harvest season
45000 yuan of loss.
To sum up, the antistaling agent for grape provided by the present application comprising the bacillus subtilis high density fermentation liquid can be effective
It is fresh-keeping to grape progress, and there is huge economic benefit.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (10)
1. a kind of bacillus subtilis strain, it is characterised in that: it is general that it is preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, deposit number are as follows: CGMCC NO.16569；The preservation time are as follows: on October 10th, 2018.
2. a kind of bacillus subtilis high density fermentation containing bacillus subtilis strain metabolite as described in claim 1
Liquid.
3. the preparation method of bacillus subtilis high density fermentation liquid described in a kind of claim 2 characterized by comprising
The preparation of strain seed liquor: the bacillus subtilis is activated, and the bacillus subtilis single colonie after taking activation lines
On culture medium, sealing is carried out later and is put into incubator the bacterial strain cultivated, activated, the bacterial strain of activation is transferred to culture medium
Middle culture obtains the strain seed liquor；
The preparation of fermentation liquid: the strain seed liquor being seeded in the container equipped with high-density culture medium and is cultivated, and bacterium is made
Supernatant is collected after centrifugation in the bacteria suspension by suspension, and it is high that supernatant liquid filtering and degerming obtain the bacillus subtilis
Density fermentation liquid.
4. the preparation method of bacillus subtilis high density fermentation liquid according to claim 3, which is characterized in that the strain
The preparation method of seed liquor includes: to activate bacillus subtilis on NA plating medium, the bacillus subtilis after taking activation
Bacterium single colonie lines on NA culture medium, carries out sealing later and is inverted in temperature to be that 33 DEG C of incubators are cultivated for 24 hours again, be lived
The bacterial strain of activation is transferred in LB culture medium by the bacterial strain of change with transfering loop, temperature be 33~50 DEG C, revolving speed be 180~
24~50h is cultivated under conditions of 300r/min, and the strain seed liquor is made；The strain seed liquor in temperature be -1~
It is saved backup in the environment of 4 DEG C.
5. the preparation method of bacillus subtilis high density fermentation liquid according to claim 4, which is characterized in that the NA is flat
The raw material of plate culture medium or NA culture medium includes: distilled water, beef extract, peptone, glucose and agar；Wherein, often
1000mL distilled water corresponds to 3.0g beef extract, 7.0g peptone, 10.0g glucose, 15.0g agar；
And/or the raw material of the LB culture medium includes: distilled water, sodium chloride, yeast extract and tryptone；Wherein, often
1000mL distilled water corresponds to 10.0g sodium chloride, 5.0g yeast extract, 10.0g tryptone.
6. the preparation method of bacillus subtilis high density fermentation liquid according to claim 3, which is characterized in that the fermentation
The preparation method of liquid includes: to be inoculated in the strain seed liquor with the volume ratio that inoculum concentration is 3~6% to train equipped with high density
In the triangular flask for supporting base, 24~48h is cultivated under conditions of temperature is 33~50 DEG C, revolving speed is 180~300r/min, obtains bacterium
Suspension, the bacteria suspension take supernatant, supernatant miillpore filter mistake after being centrifuged in the centrifuge that revolving speed is 10000r/min
It filters and degerming obtains the bacillus subtilis high density fermentation liquid.
7. the preparation method of bacillus subtilis high density fermentation liquid according to claim 6, it is characterised in that: described is withered
Careless bacillus high density fermentation liquid saves backup in the environment of being 4 DEG C in temperature.
8. the preparation method of bacillus subtilis high density fermentation liquid according to claim 6, which is characterized in that described highly dense
The raw material for spending culture medium includes: distilled water, soybean powder, corn flour, yeast extract, NaCl, CaCl2And MnSO4·H2O；Wherein, often
1000mL distilled water corresponds to 20.03g soybean powder, 24.41g corn flour, 12.69g yeast extract, 3g NaCl, 2g CaCl2、2g
MnSO4·H2O。
9. bacillus subtilis high density fermentation liquid described in a kind of claim 2 is inhibiting Aspergillus carbonerius, aspergillus niger, penicillium chrysogenum
In application.
10. a kind of antistaling agent for grape, it is characterised in that: include bacillus subtilis high density fermentation liquid described in claim 2.
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