CN118308278A - High-density liquid fermentation medium of bacillus subtilis and fermentation method - Google Patents
High-density liquid fermentation medium of bacillus subtilis and fermentation method Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 130
- 230000004151 fermentation Effects 0.000 title claims abstract description 130
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 65
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 65
- 239000007788 liquid Substances 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 35
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims abstract description 48
- 235000010288 sodium nitrite Nutrition 0.000 claims abstract description 24
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims abstract description 20
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 16
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 16
- 235000010265 sodium sulphite Nutrition 0.000 claims abstract description 10
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 9
- 240000003183 Manihot esculenta Species 0.000 claims abstract description 8
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims abstract description 8
- 229920002472 Starch Polymers 0.000 claims abstract description 8
- 239000011790 ferrous sulphate Substances 0.000 claims abstract description 8
- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 8
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 8
- 229940099596 manganese sulfate Drugs 0.000 claims abstract description 8
- 239000011702 manganese sulphate Substances 0.000 claims abstract description 8
- 235000007079 manganese sulphate Nutrition 0.000 claims abstract description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 8
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 8
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims abstract description 8
- 229910000029 sodium carbonate Inorganic materials 0.000 claims abstract description 8
- 239000008107 starch Substances 0.000 claims abstract description 8
- 235000019698 starch Nutrition 0.000 claims abstract description 8
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 8
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 8
- 239000002609 medium Substances 0.000 claims description 26
- 239000001963 growth medium Substances 0.000 claims description 18
- 238000011218 seed culture Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 18
- 235000010633 broth Nutrition 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 230000008859 change Effects 0.000 description 4
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- 238000004659 sterilization and disinfection Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
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- 239000012138 yeast extract Substances 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 239000000306 component Substances 0.000 description 2
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- 238000011031 large-scale manufacturing process Methods 0.000 description 2
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- 125000001477 organic nitrogen group Chemical group 0.000 description 2
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
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- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
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- 229910001437 manganese ion Inorganic materials 0.000 description 1
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Abstract
The invention discloses a high-density liquid fermentation medium of bacillus subtilis and a fermentation method, belonging to the technical field of microbial fermentation. The components of the high-density liquid fermentation medium comprise: 20-60g/L of tapioca starch, 1-6g/L of sodium nitrite, 0.5-3g/L of magnesium sulfate, 0.5-3g/L of monopotassium phosphate, 0.5-3g/L of light calcium carbonate, 0.5-2g/L of sodium carbonate, 0.1-1g/L of manganese sulfate, 0.1-1g/L of zinc sulfate and 0.1-1g/L of ferrous sulfate. Inoculating bacillus subtilis into the high-density liquid fermentation medium for fermentation, and adding sodium sulfite at regular time in the fermentation process, so that the viable count of bacillus subtilis YXSY-01 can be remarkably improved to 82 multiplied by 10 8 cfu/ml, and the spore conversion rate is up to 98%. Provides a new idea for reducing cost and enhancing efficiency in the mass production process of bacillus subtilis YXSY-01.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a high-density liquid fermentation medium of bacillus subtilis and a fermentation method.
Background
Bacillus subtilis (Bacillus subtilis) is a kind of bacillus, and single cells (0.7-0.8) × (2-3) microns are uniformly colored. The bacterium has no capsule, and can move around flagellum, is gram positive bacterium, can form endogenous stress-resistant spore, and the spore is (0.6-0.9) x (1.0-1.5) micrometer, elliptic to columnar, is positioned in the center of the bacterium or slightly deviated, and does not expand after the spore is formed. The growth and propagation speed of the bacterial colony are high, the surface of the bacterial colony is rough and opaque, the bacterial colony is dirty white or yellowish, and when the bacterial colony grows in a liquid culture medium, the bacterial colony often forms wrinkling, so the bacterial colony is an aerobic bacterium. Is widely distributed in soil and putrefactive organic matters. The existing production modes of the bacillus subtilis comprise a solid fermentation method and a liquid fermentation method, the liquid fermentation is uniform due to the fermentation, and fermentation conditions (temperature, pH value and aseptic conditions) are easy to control, so that the method is a main mode adopted by industrial mass production of the bacillus subtilis.
However, in the current liquid fermentation production of bacillus subtilis, firstly, the liquid culture medium needs to be sterilized at high temperature and high pressure, and the steam energy consumption needed by sterilization is high, secondly, the pH of the fermentation liquid is reduced due to the acid production of the bacillus subtilis in the fermentation process, and alkaline substances (sodium hydroxide, ammonia water and the like) are required to be continuously added in the fermentation process to improve the pH. Thirdly, organic nitrogen sources (yeast extract, peptone and the like) are used for liquid fermentation culture base, so that the production cost of bacillus subtilis is high.
The inventor screens out a bacillus subtilis YXSY-01 for producing nitrite oxidoreductase from a small shed shrimp culture pond in Taishan city of Guangdong in the earlier stage and discloses the bacillus subtilis in a patent CN 117568235B. However, the fermentation production of Bacillus subtilis YXSY-01 by the fermentation production technique in the prior art cannot achieve the expected effect of high-density fermentation. Therefore, the liquid fermentation of the bacillus subtilis is improved, the purposes of saving cost, simplifying operation, improving fermentation yield, saving cost and improving efficiency are achieved, and the method is a key for improving the fermentation yield of the bacillus subtilis.
Disclosure of Invention
The invention aims to provide a high-density liquid fermentation medium of bacillus subtilis and a fermentation method thereof, so as to solve the problems in the prior art. The high-density liquid fermentation medium and the fermentation method provided by the invention can obviously improve the viable count of bacillus subtilis YXSY-01 in liquid fermentation to 82 multiplied by 10 8 cfu/ml, the spore conversion rate is up to 98%, the medium does not need sterilization treatment, the pH is stable, and a new idea is provided for cost reduction and synergy in the large-scale production process of bacillus subtilis YXSY-01.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides a high-density liquid fermentation medium of bacillus subtilis, which comprises the following components: 20-60g/L of tapioca starch, 1-6g/L of sodium nitrite, 0.5-3g/L of magnesium sulfate, 0.5-3g/L of monopotassium phosphate, 0.5-3g/L of light calcium carbonate, 0.5-2g/L of sodium carbonate, 0.1-1g/L of manganese sulfate, 0.1-1g/L of zinc sulfate and 0.1-1g/L of ferrous sulfate;
The bacillus subtilis is bacillus subtilis YXSY-01, and the preservation number is GDMCC No:64078.
The invention also provides a method for high-density liquid fermentation of bacillus subtilis, which comprises the step of inoculating seed liquid of the bacillus subtilis into the high-density liquid fermentation medium for fermentation;
The bacillus subtilis is bacillus subtilis YXSY-01, and the preservation number is GDMCC No:64078.
Further, sodium sulfite is required to be added during the fermentation process.
Further, the sodium sulfite is supplemented at 12-16 hours and 24-28 hours of fermentation.
Further, the adding amount of the sodium sulfite is 1g of sodium nitrite added to each liter of fermentation liquor.
Further, the fermentation temperature is 33 ℃ and the fermentation time is 36h.
Further, the seed liquid is obtained by culturing the bacillus subtilis on a seed culture medium;
The seed culture medium comprises 15g/L of tapioca starch, 1g/L of sodium nitrite, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of light calcium carbonate, 0.5g/L of sodium carbonate, 0.1g/L of manganese sulfate, 0.1g/L of zinc sulfate and 0.1g/L of ferrous sulfate.
Further, the temperature of the culture was 33℃and the culture time was 24 hours.
The invention discloses the following technical effects:
the high-density liquid fermentation medium and the fermentation method provided by the invention can obviously improve the viable count of bacillus subtilis YXSY-01 in liquid fermentation to 82 multiplied by 10 8 cfu/ml, and the spore conversion rate is up to 98%. The culture medium does not need sterilization treatment, the pH is generally 7.2-8.0, the pH does not need to be adjusted in the fermentation process, and the cost caused by the sterilization of the culture medium and the pH adjustment is saved. As the culture medium does not contain an organic nitrogen source, the culture medium is not suitable for the survival of mixed bacteria, and thus the mixed bacteria pollution can not be generated. The bacillus subtilis high-density liquid fermentation medium and the fermentation method provided by the invention provide a new thought for cost reduction and synergy in the bacillus subtilis YXSY-01 large-scale production process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the change in sodium nitrite content in a fermentation broth of Bacillus subtilis YXSY-01 subjected to fermentation culture in the medium and fermentation method of scheme (1) in example 1;
FIG. 2 is a graph showing changes in pH of fermentation broth of Bacillus subtilis YXSY-01 in the medium and fermentation method of scheme (1) of example 1;
FIG. 3 is a graph showing the total number of viable bacteria in a fermentation broth obtained by fermenting Bacillus subtilis YXSY-01 in the medium and the fermentation method of the scheme (1) in example 1.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Bacillus subtilis YXSY-01 used in the examples of the present invention was deposited in the microorganism strain collection, guangdong province, accession number: GDMCC No:64078, disclosed in patent CN 117568235B. Commercial bacillus subtilis for control was purchased from Jiangsu green biotechnology limited.
EXAMPLE 1 high Density fermentation culture of Bacillus subtilis YXSY-01
1 Preparation of culture Medium
YXSY seed medium: 15g/L of tapioca starch, 1g/L of sodium nitrite, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of light calcium carbonate, 0.5g/L of sodium carbonate, 0.1g/L of manganese sulfate, 0.1g/L of zinc sulfate and 0.1g/L of ferrous sulfate.
YXSY fermentation medium: 60g/L of tapioca starch, 1g/L of sodium nitrite, 1g/L of magnesium sulfate, 2g/L of monopotassium phosphate, 1g/L of light calcium carbonate, 1g/L of sodium carbonate, 0.2g/L of manganese sulfate, 0.2g/L of zinc sulfate and 0.2g/L of ferrous sulfate. Sodium nitrite 1g/L was added to the feed stream.
LB liquid seed culture medium: 10g/L tryptone, 3.0g/L yeast extract, and initial pH value of 7.0.
LB liquid fermentation medium: tryptone 30g/L, yeast extract 9.0g/L, and initial pH 7.0.
Preparation of seed solution and fermentation culture
Seed solutions were prepared and fermentation cultured according to the protocols shown in Table 1, respectively. The seed solution and fermentation mode prepared by commercial bacillus subtilis under the same condition are used as a control.
TABLE 1 different schemes for seed liquid preparation and fermentation culture
According to different schemes of Table 1, single colonies of bacillus subtilis strain YXSY-01 and commercial bacillus subtilis are selected and respectively inoculated in corresponding seed culture media, and shake cultivation is carried out at 33 ℃ and 150rpm for 24 hours to obtain seed liquid for later use.
The seed solutions prepared above were inoculated into the corresponding fermentation media at an inoculum size of 5%, respectively, and shake-cultured at 33℃and 150rpm for 36 hours. When fermentation is carried out in YXSY fermentation medium, the feeding is carried out according to the amount of 1g of sodium nitrite added to each liter of fermentation liquid at 16 hours and 28 hours of fermentation culture.
3. Viable count and pH determination
The total number of viable bacteria and the pH in the obtained fermentation broth were detected according to GB/T34224-2017 and GB/T9724-2007, respectively.
The measurement results are shown in Table 2.
TABLE 2 total number of viable bacteria and pH in fermentation broth
As is clear from Table 1, the seed solution was prepared and fermented by the method of the present embodiment (1), and the total number of viable bacteria in the fermentation broth of Bacillus subtilis YXSY-01 was 81X 10 8 cfu/ml. After the commercial bacillus subtilis is fermented under the same fermentation conditions and fermentation method, the total number of viable bacteria in the fermentation broth is 2X 10 6 cfu/ml. The pH values before and after fermentation of the two bacteria are not obviously changed. The culture medium and the fermentation method of the embodiment (1) can realize high-density fermentation culture of bacillus subtilis YXSY-01, and the sodium nitrite content, the pH change and the total viable bacteria change in the fermentation broth are respectively shown in figures 1-3 in the fermentation process. As is clear from the results of the above-mentioned schemes (1) to (4), the fermentation medium has a greater influence on the total number of viable bacteria in the fermentation broth of Bacillus subtilis YXSY-01 than the seed medium.
EXAMPLE 2 Effect of Medium components on fermentation culture of Bacillus subtilis YXSY-01
To search for an optimal medium for high-density fermentation culture of Bacillus subtilis YXSY-01, the medium composition was adjusted according to Table 3 on the basis of YXSY fermentation medium, and Bacillus subtilis YXSY-01 was subjected to fermentation culture according to the inoculum size and fermentation method in example 1.
TABLE 3 different Medium Components
The seed solution of Bacillus subtilis YXSY-01 prepared in the scheme (1) in example 1 was subjected to fermentation culture on different fermentation media designed in Table 3, and after the fermentation was completed, the total number of viable bacteria, the spore conversion rate and the pH of the obtained fermentation broth were measured. The results are shown in Table 4.
TABLE 4 total number of viable bacteria, spore conversion and pH in different groups of fermentation broths
| Group of | Total number of viable bacteria | Conversion of spores | PH before fermentation | Fermentation end pH |
| 1 | 80×108cfu/ml | 96% | 7.52 | 7.35 |
| 2 | 81×108cfu/ml | 98% | 7.51 | 7.43 |
| 3 | 43×108cfu/ml | 74% | 7.52 | 7.34 |
| 4 | 55×108cfu/ml | 75% | 7.52 | 7.33 |
| 5 | 54×108cfu/ml | 30% | 7.52 | 5.23 |
| 6 | 56×108cfu/ml | 63% | 7.52 | 7.18 |
As is clear from Table 4, the fermentation medium of group 1 and group 2 was used to ferment the seed solution of Bacillus subtilis YXSY-01, the total viable count and the spore conversion rate in the fermentation solution were significantly higher than those in the other groups, and there was no significant change in pH before and after fermentation. The light calcium carbonate in the culture medium is beneficial to the formation of spores, and meanwhile, acidic substances generated in the fermentation process of bacillus can be neutralized, so that the acid-base level in the fermentation liquid is kept balanced, zinc ions and manganese ions are auxiliary factors of enzyme activity, the activity of fermenting and producing nitrite oxidoreductase by bacillus subtilis YXSY-01 can be improved, the fermentation process can be promoted, and the fermentation efficiency can be improved.
As can be seen from the above, the most suitable culture medium for high-density fermentation culture of bacillus subtilis YXSY-01 comprises 20-60g/L of tapioca starch, 1-6g/L of sodium nitrite, 0.5-3g/L of magnesium sulfate, 0.5-3g/L of monopotassium phosphate, 0.5-3g/L of light calcium carbonate, 0.5-2g/L of sodium carbonate, 0.1-1g/L of manganese sulfate, 0.1-1g/L of zinc sulfate and 0.1-1g/L of ferrous sulfate.
Example 3 Effect of fed-batch Nitrogen Source on fermentation results during fermentation
From previous studies (patent CN 117568235B), it is known that bacillus subtilis YXSY-01 can use sodium nitrite as the sole nitrogen source, and the capability of converting sodium nitrite into nitrate is outstanding. However, sodium nitrite can exert an inhibitory effect on microorganisms, even induce mutation, within a certain dosage range. Therefore, in order to ensure the high density effect of fermentation and smooth fermentation, the operation of feeding sodium nitrite was optimally adjusted during the fermentation by the fermentation method of the scheme (1) in example 1, and the influence of feeding sodium nitrite during the fermentation on the fermentation result was explored. The optimization results are shown in table 5.
TABLE 5 influence of fed-batch sodium nitrite on fermentation results during fermentation
As is clear from Table 5, the time for adding sodium sulfite to the medium during fermentation greatly affects the total number of viable bacteria in the fermentation broth. Too early addition, too little nitrogen source consumption in the original culture medium, too much sodium nitrite content accumulation after feeding, and influence the activity of bacillus subtilis YXSY-01; after the feeding is too late, the nitrogen source in the original culture medium is consumed before the feeding, and the fermentation of the bacillus subtilis YXSY-01 is blocked due to the lack of the nitrogen source, so that the fermentation process is delayed on the whole level even if sodium sulfite is subsequently fed, and the total viable count in the fermentation liquor is reduced. In addition, since sodium nitrite is a common preservative component, excessive use of sodium nitrite can inhibit the activity of microorganisms and even induce mutation. Therefore, in the fermentation culture process of bacillus subtilis YXSY-01, the timing and the dosage of sodium sulfite are respectively the 12 th to 16 th hours and 24 th to 28 th hours of fermentation culture, and 1g of sodium nitrite is respectively added into each liter of YXSY fermentation culture medium for feeding.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A high-density liquid fermentation medium for bacillus subtilis, the components of the high-density liquid fermentation medium comprising: 20-60g/L of tapioca starch, 1-6g/L of sodium nitrite, 0.5-3g/L of magnesium sulfate, 0.5-3g/L of monopotassium phosphate, 0.5-3g/L of light calcium carbonate, 0.5-2g/L of sodium carbonate, 0.1-1g/L of manganese sulfate, 0.1-1g/L of zinc sulfate and 0.1-1g/L of ferrous sulfate;
The bacillus subtilis is bacillus subtilis YXSY-01, and the preservation number is GDMCC No:64078.
2. A method for high-density liquid fermentation of bacillus subtilis, which is characterized by comprising the step of inoculating a seed liquid of the bacillus subtilis into the high-density liquid fermentation medium according to claim 1 for fermentation;
The bacillus subtilis is bacillus subtilis YXSY-01, and the preservation number is GDMCC No:64078.
3. The method according to claim 2, characterized in that sodium sulfite is required to be added during the fermentation.
4. A method according to claim 3, characterized in that the sodium sulfite is fed in at 12-16 hours and 24-28 hours of fermentation.
5. A method according to claim 3, wherein the sodium sulfite is added in an amount of 1g sodium nitrite per liter of fermentation broth.
6. The method according to claim 2, wherein the fermentation temperature is 33 ℃ and the fermentation time is 36h.
7. The method according to claim 2, wherein the seed liquid is obtained by culturing the bacillus subtilis on a seed medium;
The seed culture medium comprises 15g/L of tapioca starch, 1g/L of sodium nitrite, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 0.5g/L of light calcium carbonate, 0.5g/L of sodium carbonate, 0.1g/L of manganese sulfate, 0.1g/L of zinc sulfate and 0.1g/L of ferrous sulfate.
8. The method of claim 7, wherein the temperature of the culturing is 33 ℃ and the culturing time is 24 hours.
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