CN109486707B - Bacillus subtilis strain and application thereof - Google Patents
Bacillus subtilis strain and application thereof Download PDFInfo
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- CN109486707B CN109486707B CN201811409452.XA CN201811409452A CN109486707B CN 109486707 B CN109486707 B CN 109486707B CN 201811409452 A CN201811409452 A CN 201811409452A CN 109486707 B CN109486707 B CN 109486707B
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- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 70
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Classifications
-
- 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
-
- 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
-
- 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
Abstract
The embodiment of the invention discloses a bacillus subtilis strain and application thereof, wherein the bacillus subtilis strain is preserved in China general microbiological culture Collection center (CGMCC), and the preservation numbers are as follows: CGMCC NO. 16569; the preservation time is as follows: 10 and 10 months and 10 days in 2018. The embodiment of the invention also provides a bacillus subtilis high-density fermentation broth containing the metabolite of the bacillus subtilis strain. The bacillus subtilis strain provided by the embodiment of the invention can effectively inhibit aspergillus carbonarius, aspergillus niger and penicillium chrysogenum which are moulds easy to grow on grapes, so that the bacillus subtilis high-density fermentation liquid containing the metabolite of the bacillus subtilis strain can effectively prolong the storage period of the grapes, and has the characteristics of high efficiency, safety, environmental protection and the like, and has a wide application prospect.
Description
Technical Field
The embodiment of the invention relates to the technical field of microorganism application, and particularly relates to a bacillus subtilis strain and application thereof.
Background
Bacillus subtilis is a kind of Bacillus, and has effects in preventing biological oxidation, resisting pathogenic microorganism, improving in vivo and in vitro ecological environment, enhancing animal immunity, and producing various digestive enzymes and nutrients. During the growth of the bacillus subtilis, active substances such as subtilin, polymyxin, nystatin, short bacitracin and the like which have obvious inhibition effect on pathogenic bacteria or pathogenic bacteria with endogenous infection. The bacillus subtilis in the intestinal tract can rapidly consume free oxygen, so that the oxygen in the intestinal tract can be reduced, beneficial anaerobic bacteria can better survive, and partial pathogenic bacteria can be inhibited. Researches prove that the average level of salmonella in chicken ceca can be obviously reduced by 58% by adding bacillus subtilis into chicken feed, so that the risk of infection of living bodies is reduced, and the food safety is improved. Therefore, the bacillus subtilis with high antifungal activity plays an important role in improving the health condition of human bodies and guaranteeing the food safety. Bacillus subtilis, a gram-positive bacterium, has long been used to ferment soybean foods, widely used in the production of industrial enzymes and chemicals.
However, rarely found are strains which have good effects in inhibiting carbon aspergillus niger, aspergillus niger and penicillium chrysogenum.
Disclosure of Invention
Therefore, the embodiment of the invention provides a bacillus subtilis strain and application thereof, and aims to solve the problem that strains which have better effects in inhibiting aspergillus niger, aspergillus niger and penicillium chrysogenum are rarely found in the prior art.
In order to achieve the above object, an embodiment of the present invention provides the following:
in a first aspect of an embodiment of the present invention, there is provided a bacillus subtilis strain, which is deposited in the china general microbiological culture collection center of the culture collection management committee under the following collection number: CGMCC NO. 16569; the preservation time is as follows: 10 and 10 months and 10 days in 2018.
In a second aspect of embodiments of the present invention, there is provided a high density fermentation broth of Bacillus subtilis comprising a metabolite of the Bacillus subtilis strain.
In a third aspect of the embodiments of the present invention, there is provided a method for preparing a high-density fermentation broth of bacillus subtilis, comprising:
preparing a strain seed solution: activating the bacillus subtilis, streaking the activated bacillus subtilis single colony on a culture medium, sealing the culture medium with glue, culturing the culture medium in an incubator to obtain an activated strain, and transferring the activated strain into the culture medium for culturing to obtain the strain seed solution;
preparing fermentation liquor: inoculating the strain seed liquid into a container filled with a high-density culture medium for culturing to obtain a bacterial suspension, centrifuging the bacterial suspension, collecting a supernatant, filtering the supernatant, and sterilizing to obtain the bacillus subtilis high-density fermentation liquid.
In one embodiment of the present invention, the preparation method of the strain seed solution comprises: activating bacillus subtilis on an NA plate culture medium, taking a single colony of the activated bacillus subtilis, streaking the single colony on the NA culture medium, sealing the mixture, inverting the mixture in an incubator at the temperature of 33 ℃ for re-culture for 24 hours to obtain an activated strain, transferring the activated strain to an LB culture medium by using a bacterium transfer ring, and culturing for 24-50 hours under the conditions that the temperature is 33-50 ℃ and the rotating speed is 180-300 r/min to obtain a strain seed solution;
in one embodiment of the invention, the strain seed solution is stored at a temperature of-1 to 4 ℃ for later use.
In one embodiment of the present invention, the raw materials of the NA plate medium or the NA medium include: distilled water, beef extract, peptone, glucose and agar; wherein, each 1000mL of distilled water corresponds to 3.0g of beef extract, 7.0g of peptone, 10.0g of glucose and 15.0g of agar;
and/or the raw materials of the LB culture medium comprise: distilled water, sodium chloride, yeast extract and tryptone; wherein each 1000mL of distilled water corresponds to 10.0g of sodium chloride, 5.0g of yeast extract and 10.0g of tryptone.
In one embodiment of the present invention, the method for preparing the fermentation broth comprises: inoculating the strain seed liquid into a triangular flask filled with a high-density culture medium according to the volume ratio of 3-6% of the inoculation amount, culturing for 24-48 h under the conditions that the temperature is 33-50 ℃ and the rotating speed is 180-300 r/min to obtain a strain suspension, centrifuging the strain suspension in a centrifuge with the rotating speed of 10000r/min, taking a supernatant, filtering the supernatant by using a microporous filter membrane and sterilizing to obtain the bacillus subtilis high-density fermentation liquid.
In one embodiment of the present invention, the bacillus subtilis high-density fermentation broth is stored at 4 ℃ for further use.
In one embodiment of the present invention, the raw materials of the high-density medium include: distilled water, soybean meal, corn meal, yeast extract, NaCl and CaCl2And MnSO4·H2O; wherein, each 1000mL of distilled water corresponds to 20.03g of soybean meal, 24.41g of corn meal, 12.69g of yeast extract, 3g of NaCl and 2g of CaCl2、2g MnSO4·H2O。
In a fourth aspect of the embodiments of the present invention, there is provided the use of the bacillus subtilis high-density fermentation broth for inhibiting aspergillus carbonarius, aspergillus niger and penicillium chrysogenum.
In a fourth aspect of embodiments of the present invention, there is provided a grape preservative comprising the bacillus subtilis high-density fermentation broth.
According to the embodiment of the invention, the following advantages are provided:
1. the bacillus subtilis strain provided by the embodiment of the invention can effectively inhibit aspergillus carbonarius, aspergillus niger and penicillium chrysogenum which are moulds easy to grow on grapes, so that the bacillus subtilis high-density fermentation liquid containing the metabolite of the bacillus subtilis strain can effectively prolong the storage period of the grapes, and has the characteristics of high efficiency, safety, environmental protection and the like, and has a wide application prospect.
2. The bacillus subtilis strain provided by the embodiment of the invention can effectively reduce the number of microorganisms on the surface of grapes, the hardness of fruit grains is high, the color of grape stalks is more fresh green, the loss rate of grape nutrients is small, the appearance freshness and the taste are well maintained, and the preservation period of the whole grapes is prolonged by 7-10 days.
The Bacillus subtilis strain (Bacillus subtilis) provided by the application is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation addresses are as follows: the collection number of the microbial research institute of the Chinese academy of sciences, No. 3 Xilu-Beijing province, Chaoyang, and the collection number is: CGMCC NO. 16569; the preservation time is as follows: 10 and 10 months and 10 days in 2018.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a preparation method of a bacillus subtilis high-density fermentation broth, which comprises the following steps:
1) preparing a strain seed solution: activating bacillus subtilis on an NA plate culture medium, taking a single colony to streak on the NA culture medium, pouring a sealing compound into a 33 ℃ incubator to culture for 24 hours to obtain an activated strain, transferring the activated strain into an LB culture medium by using a transfer ring, culturing for 37 hours under the conditions of 41.5 ℃ and 240r/min, and storing the prepared strain seed liquid at 1.5 ℃ for later use;
2) preparing high-density fermentation liquor: inoculating the strain seed liquid prepared in the step 1) into a triangular flask of a high-density culture medium with the inoculation amount of 4.5 percent (V/V), culturing for 36 hours at the temperature of 41.5 ℃ and the speed of 240r/min, and enabling the viable count of the bacillus subtilis CGMCC NO.16569 to reach 3.27 multiplied by 1011CFU/mL. Obtaining bacterial suspension, centrifuging the bacterial suspension at 10000r/min, taking supernatant, filtering and sterilizing by using a microporous filter membrane to obtain high-density fermentation liquor, and storing the high-density fermentation liquor at 4 ℃ for later use.
Example 2
The embodiment provides a preparation method of a bacillus subtilis high-density fermentation broth, which comprises the following steps:
1) preparing a strain seed solution: activating bacillus subtilis on an NA plate culture medium, taking a single colony to mark on the NA culture medium, pouring a sealing compound into a 33 ℃ incubator to culture for 24 hours to obtain an activated strain, transferring the activated strain into an LB culture medium by using a transfer ring, culturing for 24 hours under the conditions of 33 ℃ and 180r/min, and storing the prepared strain seed liquid at-1 ℃ for later use;
2) preparing high-density fermentation liquor: inoculating the strain seed liquid prepared in the step 1) into a triangular flask of a high-density culture medium in an inoculation amount of 3% (V/V), and culturing for 24h at the temperature of 33 ℃ and 180r/min until the viable count of the bacillus subtilis CGMCC NO.16569 reaches 3.41 multiplied by 1011CFU/mL. Obtaining bacterial suspension, centrifuging the bacterial suspension at 10000r/min, taking supernatant, filtering and sterilizing by using a microporous filter membrane to obtain high-density fermentation liquor, and storing the high-density fermentation liquor at 4 ℃ for later use.
Example 3
The embodiment provides a preparation method of a bacillus subtilis high-density fermentation broth, which comprises the following steps:
1) preparing a strain seed solution: activating bacillus subtilis on an NA plate culture medium, taking a single colony to streak on the NA culture medium, pouring a sealing compound into a 33 ℃ incubator to be cultured for 24 hours to obtain an activated strain, transferring the activated strain into an LB culture medium by using a transfer ring, culturing for 50 hours under the conditions of 50 ℃ and 300r/min, and storing the prepared strain seed liquid at 4 ℃ for later use;
2) preparing high-density fermentation liquor: inoculating the strain seed liquid prepared in the step 1) into a triangular flask of a high-density culture medium in an inoculation amount of 6% (V/V), and culturing for 48h at the temperature of 50 ℃ and the speed of 300r/min until the viable count of the bacillus subtilis CGMCC NO.16569 reaches 3.60 multiplied by 1011CFU/mL. Obtaining bacterial suspension, centrifuging the bacterial suspension at 10000r/min, taking supernatant, filtering and sterilizing by using a microporous filter membrane to obtain high-density fermentation liquor, and storing the high-density fermentation liquor at 4 ℃ for later use.
Example 4
In the case of adopting any of the embodiments 1-3The composition of the NA plate medium or NA medium is as follows: every 1000mL of distilled water corresponds to 3.0g of beef extract, 7.0g of peptone, 10.0g of glucose and 15.0g of agar; the composition of the LB medium was: 10.0g of sodium chloride, 5.0g of yeast extract and 10.0g of tryptone per 1000mL of distilled water; the composition of the high-density medium was: each 1000mL of distilled water, 20.03g of soybean meal, 24.41g of corn meal, 12.69g of yeast extract, 3g of NaCl and 2g of CaCl2，2g MnSO4·H2O。
Application example 1 antibacterial action of Bacillus subtilis on Aspergillus carbonarius, Aspergillus niger and Penicillium chrysogenum
To further illustrate the resistance effect of Bacillus subtilis numbers on fungi, the present inventors investigated the antibacterial effect of this strain on Aspergillus carbonarius, Aspergillus niger and Penicillium chrysogenum, which are commonly present on grape surfaces, and examined the following:
firstly activating strains and preparing bacterial suspensions of three moulds, continuously subculturing aspergillus carbonarius, aspergillus niger, penicillium chrysogenum and the like twice on a PDA culture medium plate, and respectively growing cultures of the aspergillus carbonarius, the aspergillus niger and the penicillium chrysogenum on a PDA culture medium and a czapek culture medium for 5 days at 25 ℃. A colony with the diameter of more than or equal to 2mm is selected and inoculated into 5ml of sterile normal saline, and the colony is smashed by using an inoculating loop and is shaken for 5min, so that the colony is uniformly distributed in the sterile normal saline to form bacterial suspension. Transferring a little bacterial suspension onto a blood cell counting plate, observing and counting under a microscope, and adjusting the concentration to be 1-5 multiplied by 106CFU/mL。
The prepared three spore suspensions (100 μ L) were plated on PDA medium. mu.L of the high-density supernatant of the liquid culture of Bacillus subtilis and sterile distilled water were respectively added dropwise to the same PDA medium containing Aspergillus carbonarius, Aspergillus niger and Penicillium chrysogenum, respectively, using an Oxford cup, and incubated at 25 ℃. The diameter of the zone of inhibition was measured after 5 days using the cross-striped method.
As shown in the experimental results in Table 1, the culture solution can effectively inhibit the growth of fungi. The inhibition zone of the black aspergillus is 24.3mm, the inhibition zone of the aspergillus niger is 20.3mm, and the inhibition zone of the penicillium chrysogenum is 27.5 mm.
TABLE 1 inhibition of microorganisms by high-Density fermentation broths
Application example 2 action of Bacillus subtilis in grape antistaling agent
The culture generated in the culture process of the strain related in the embodiment can be applied to berries such as film-coated fresh-keeping grapes, and the specific method is as follows:
immersing grape bunch with uniform color and size and no damage into high-density fermentation liquid of Bacillus subtilis for 1 min, drying the surface under sterile airflow, placing into a cardboard box, and storing in a refrigerator at-1-0 deg.C.
In order to detect whether the culture of the strain is used on food such as grapes to cause adverse effects on the nutritional quality and the sensory quality, the invention carries out nutritional and sensory evaluation on the grapes treated by the high-density fermentation liquid.
The grapes treated with the strain were used as experimental group, and the untreated grapes were used as control group. Through experimental inspection, the weight loss rate, the rotting rate and the falling rate of the grapes in the experimental group are obviously lower than those in the control group.
Grape firmness is related to water content, which affects the expansion pressure and brittleness of the tissue. In both the control and treated samples, the grape firmness decreased significantly with increasing storage time. Water loss increases the activity of pectinases, pectinesterases and polygalacturonases, leading to degradation of pectin and polygalacturonic acid, resulting in fruit softening. From day 5 to day 30, the high-density broth treated grapes were harder than the control grapes.
The titratable acid content is an important index of the quality of fruits and vegetables, and experiments prove that the grapes in the experimental group have no significant difference from the grapes in the control group. It is stated that the titratable acid content of the grapes is not affected by treatment with this strain.
The content of vitamin C is an important index of the nutritive value of fruits and vegetables and is also one of key indexes for evaluating the storage effect of the fruits and the vegetables. Experiments prove that the experimental group and the control group show a rising trend and a falling trend in the first 15 days, and the Vc content is on the same level. This indicates that the use of high density fermentation broth of this strain will maintain the vitamin C content of the grapes.
The respiration intensity and the storage relation of the fruits are inseparable, and the consumption of nutrient substances of the fruits and the vegetables and the aging state of the fruits and the vegetables can be judged according to the change of the respiration intensity. By inhibiting the respiration rate of the fruits and vegetables, the senescence of the fruits and vegetables can be delayed. The respiration rate of the control group was 74.3 mg/kg-h on the first day of storage, but decreased with increasing storage time, from 54.0 mg/kg-h on the tenth day to 31.0 mg/kg-h on the thirty th day. The respiratory rates of the experimental group were 46.7 mg/kg-h and 21.3 mg/kg-h on the tenth and thirty days, respectively. The results show that the respiratory frequency is effectively reduced by the high-density fermentation liquor treatment of the strain, the weight loss and the aging of the grapes can be effectively prevented, and the quality of the grapes is maintained.
The strain is applied to the fresh keeping of the grapes, and the loss of the grapes due to rotting can be reduced by 50% in one month. 300 plants are planted per mu, 5 strings are planted per plant, two jin of each string is taken as a standard, the yield per mu is about 3000 jin, and the cost is about 1500 yuan. Calculated by 15 yuan per jin, the loss of the farmers can be reduced by 11250 yuan per mu per month. The harvest season is 7 months to 10 months and 4 months, and the loss is reduced by 45000 yuan a year.
In conclusion, the grape preservative containing the bacillus subtilis high-density fermentation liquid provided by the application can effectively preserve grapes and has great economic benefits.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) A strain characterized by: it is preserved in China general microbiological culture Collection center, and the preservation numbers are: CGMCC NO. 16569; the preservation time is as follows: 10 months and 10 days in 2018;
the bacillus subtilis high-density fermentation liquid containing the bacillus subtilis strain metabolite can inhibit aspergillus carbonarius, aspergillus niger and penicillium chrysogenum.
2. A method for preparing a high-density fermentation broth of Bacillus subtilis according to claim 1, comprising:
preparing a strain seed solution: activating the bacillus subtilis, streaking the activated bacillus subtilis single colony on a culture medium, sealing the culture medium with glue, culturing the culture medium in an incubator to obtain an activated strain, and transferring the activated strain into the culture medium for culturing to obtain the strain seed solution;
preparing fermentation liquor: inoculating the strain seed liquid into a container filled with a high-density culture medium for culturing to obtain a bacterial suspension, centrifuging the bacterial suspension, collecting a supernatant, filtering the supernatant, and sterilizing to obtain the bacillus subtilis high-density fermentation liquid.
3. The method for preparing a high-density fermentation broth of Bacillus subtilis as claimed in claim 2, wherein the seed liquid of the strain is prepared by: activating bacillus subtilis on an NA plate culture medium, taking a single colony of the activated bacillus subtilis, streaking the single colony on the NA culture medium, sealing the mixture, inverting the mixture in an incubator at the temperature of 33 ℃ for re-culture for 24 hours to obtain an activated strain, transferring the activated strain to an LB culture medium by using a bacterium transfer ring, and culturing for 24-50 hours under the conditions that the temperature is 33-50 ℃ and the rotating speed is 180-300 r/min to obtain a strain seed solution; and the strain seed liquid is stored for later use in an environment with the temperature of-1-4 ℃.
4. The method for preparing a high-density fermentation broth of Bacillus subtilis according to claim 3, wherein the raw materials of the NA plate medium or the NA medium comprise: distilled water, beef extract, peptone, glucose and agar; wherein, each 1000mL of distilled water corresponds to 3.0g of beef extract, 7.0g of peptone, 10.0g of glucose and 15.0g of agar;
the raw materials of the LB culture medium comprise: distilled water, sodium chloride, yeast extract and tryptone; wherein each 1000mL of distilled water corresponds to 10.0g of sodium chloride, 5.0g of yeast extract and 10.0g of tryptone.
5. The method of preparing a high-density fermentation broth of Bacillus subtilis according to claim 2, wherein the fermentation broth is prepared by: inoculating the strain seed liquid into a triangular flask filled with a high-density culture medium according to the volume ratio of 3-6% of the inoculation amount, culturing for 24-48 h under the conditions that the temperature is 33-50 ℃ and the rotating speed is 180-300 r/min to obtain a strain suspension, centrifuging the strain suspension in a centrifuge with the rotating speed of 10000r/min, taking a supernatant, filtering the supernatant by using a microporous filter membrane and sterilizing to obtain the bacillus subtilis high-density fermentation liquid.
6. The method for preparing a high-density fermentation broth of Bacillus subtilis according to claim 5, wherein: the bacillus subtilis high-density fermentation liquid is stored for later use in an environment with the temperature of 4 ℃.
7. The method for preparing a high-density fermentation broth of Bacillus subtilis according to claim 5, wherein the raw materials of the high-density culture medium comprise: distilled water, soybean meal, corn meal, yeast extract, NaCl and CaCl2And MnSO4·H2O; wherein, each 1000mL of distilled water corresponds to 20.03g of soybean meal, 24.41g of corn meal, 12.69g of yeast extract, 3g of NaCl and 2g of CaCl2、2g MnSO4·H2O。
8. A grape preservative is characterized in that: a high-density fermentation broth comprising the bacillus subtilis of claim 1.
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| CN111172085B (en) * | 2020-03-15 | 2021-04-02 | 青岛宝创生物科技有限公司 | Application of bacillus subtilis in production of substances inhibiting fungi |
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