CN114437984A - Preparation method of lactic acid-producing bacillus coagulans microbial preparation - Google Patents
Preparation method of lactic acid-producing bacillus coagulans microbial preparation Download PDFInfo
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 140
- 238000002360 preparation method Methods 0.000 title claims abstract description 103
- 241000193749 Bacillus coagulans Species 0.000 title claims abstract description 100
- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 100
- 230000000813 microbial effect Effects 0.000 title claims abstract description 87
- 239000004310 lactic acid Substances 0.000 title claims abstract description 70
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 70
- 238000000855 fermentation Methods 0.000 claims abstract description 132
- 230000004151 fermentation Effects 0.000 claims abstract description 132
- 239000001963 growth medium Substances 0.000 claims abstract description 99
- 239000007788 liquid Substances 0.000 claims abstract description 74
- 239000007787 solid Substances 0.000 claims abstract description 70
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 49
- 238000002156 mixing Methods 0.000 claims abstract description 37
- 238000009630 liquid culture Methods 0.000 claims abstract description 35
- 238000012258 culturing Methods 0.000 claims abstract description 30
- 238000011218 seed culture Methods 0.000 claims abstract description 30
- 239000008223 sterile water Substances 0.000 claims abstract description 15
- 238000011081 inoculation Methods 0.000 claims description 66
- 239000001888 Peptone Substances 0.000 claims description 60
- 108010080698 Peptones Proteins 0.000 claims description 60
- 235000015278 beef Nutrition 0.000 claims description 60
- 235000019319 peptone Nutrition 0.000 claims description 60
- 229920001817 Agar Polymers 0.000 claims description 48
- 239000008272 agar Substances 0.000 claims description 48
- 238000009423 ventilation Methods 0.000 claims description 37
- 238000003756 stirring Methods 0.000 claims description 36
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000002994 raw material Substances 0.000 claims description 25
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 24
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 24
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 24
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 24
- 229940099596 manganese sulfate Drugs 0.000 claims description 24
- 239000011702 manganese sulphate Substances 0.000 claims description 24
- 235000007079 manganese sulphate Nutrition 0.000 claims description 24
- 239000001301 oxygen Substances 0.000 claims description 24
- 229910052760 oxygen Inorganic materials 0.000 claims description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 23
- 244000061458 Solanum melongena Species 0.000 claims description 22
- 235000002597 Solanum melongena Nutrition 0.000 claims description 22
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 13
- 235000019733 Fish meal Nutrition 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 12
- 239000004467 fishmeal Substances 0.000 claims description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 12
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 12
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 12
- 238000001291 vacuum drying Methods 0.000 claims description 12
- -1 5g/L Substances 0.000 claims description 2
- ZRVNHXQGRJLLIT-UHFFFAOYSA-L dipotassium hydrogen sulfate Chemical compound [K+].[K+].OS([O-])(=O)=O.OS([O-])(=O)=O ZRVNHXQGRJLLIT-UHFFFAOYSA-L 0.000 claims description 2
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 abstract description 68
- 241001052560 Thallis Species 0.000 abstract description 50
- 241000894006 Bacteria Species 0.000 abstract description 28
- 230000004763 spore germination Effects 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract 1
- 230000002458 infectious effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 34
- 238000011068 loading method Methods 0.000 description 20
- 230000001276 controlling effect Effects 0.000 description 12
- 101710088194 Dehydrogenase Proteins 0.000 description 10
- 239000007836 KH2PO4 Substances 0.000 description 10
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 238000007790 scraping Methods 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 230000035784 germination Effects 0.000 description 9
- 241000209094 Oryza Species 0.000 description 8
- 235000007164 Oryza sativa Nutrition 0.000 description 8
- 235000009566 rice Nutrition 0.000 description 8
- 238000010411 cooking Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010042566 Superinfection Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000017525 heat dissipation Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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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
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Abstract
The invention relates to a preparation method of a lactic acid producing bacillus coagulans microbial preparation, which comprises the following steps: 1) transferring more than 80% of bacillus coagulans with thalli forming spores into sterile water to form a seed culture solution; 2) transferring the seed culture solution into a primary liquid culture medium for primary fermentation to prepare primary fermentation liquid; 3) transferring the primary fermentation liquid to a secondary liquid culture medium for secondary fermentation to prepare secondary fermentation liquid; 4) and (3) inoculating the secondary fermentation liquid into a bran solid culture medium, uniformly mixing, and culturing for 60-72 h to prepare the lactic acid producing bacillus coagulans microbial preparation. The invention provides a liquid culture medium suitable for bacillus coagulans and a liquid culture spore-producing condition, and the prepared lactic acid bacillus coagulans microbial preparation contains 83-132 billion live bacteria per gram, the spore germination rate is not lower than 92%, the infectious microbe rate is less than 0.2%, and the L-lactic acid content is not lower than 36.5 g/L.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method of a lactic acid producing bacillus coagulans microbial preparation.
Background
The microbial preparation has been widely accepted in the field of animal husbandry because of its functions of regulating intestinal dysfunction, maintaining the balance of flora in the intestinal tract, improving the health level and production performance of organisms, and avoiding drug resistance and superinfection caused by taking antibiotics. However, at present, the common microbial preparations such as lactic acid bacteria and bacillus in China often have the dual advantages of good production performance and strong stress resistance, so that the application of the additives in practice is limited.
The Bacillus coagulans (Bacillus coagulans) has the advantages that spores with high heat resistance and acid resistance can be formed, the spores are used as active factors to resist gastric acid and successfully reach intestines and stomach, the planting survival capability is high, and L-lactic acid can be generated under the anaerobic condition. These characteristics play an important role in promoting the digestion and absorption of animal nutrition, improving the feed conversion rate of animals, preventing diseases and promoting growth. Besides good production performance, the feed additive has unique biological characteristics of strong stress resistance, high temperature and high pressure resistance, easy storage and the like, and is becoming a new favorite in the feed additive industry at present, so that the addition of bacillus coagulans capable of producing L-lactic acid in the feed is a very green and healthy choice.
The existing bacillus coagulans microbial preparation is generally prepared by adopting an open bran solid traditional fermentation process and adopting natural wind, natural temperature and humidity, and the prepared bacillus coagulans microbial preparation has high mixed bacteria rate and large living bacteria number fluctuation. Although Bacillus coagulans forms spores well on various solid media, spores cannot be formed in ordinary liquid media, and the limitations of solid culture will certainly prevent the scale-up of production and practical application thereof.
Disclosure of Invention
The invention provides a preparation method of a lactic acid producing bacillus coagulans microbial preparation aiming at the technical problems in the prior art.
The technical scheme for solving the technical problems is as follows:
a method for preparing a lactic acid producing bacillus coagulans microbial preparation comprises the following steps:
1) transferring activated Bacillus coagulans with more than 80% of thallus to form spore into sterile water to make its concentration 5.0 × 108cfu/ml～2.0×109cfu/ml, forming a seed culture solution;
2) transferring the seed culture solution into a primary liquid culture medium by an inoculation amount of 3-5% for primary fermentation, wherein the fermentation temperature is 37 +/-1 ℃, the stirring speed is 200-350 rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, and the fermentation time is 8-16 h to prepare a primary fermentation solution;
3) transferring the primary fermentation liquid into a secondary liquid culture medium by 10 percent of inoculation amount for secondary fermentation, wherein the fermentation temperature is 37 +/-1 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80 percent, and the fermentation time is 12-30 hours, so that the bacillus coagulans grows to the late logarithmic growth stage to prepare the secondary fermentation liquid;
4) in an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 2-4%, uniformly mixing, and controlling the ventilation quantity as follows: ventilating for 3-10 times/hour, culturing for 60-72 hours at the temperature of 30-40 ℃ and the humidity of 80-90%, and turning over a bran solid culture medium during the culturing period to prepare the lactic acid producing bacillus coagulans microbial preparation;
wherein the first and second liquid culture media both contain MnSO4 0.5～2.0g/L、MgSO4 0.4～1.0g/L、KH2P04 0.5～3g/L、K2HP040.5-3 g/L, NaCl 3-10 g/L, 5-20 g/L peptone and 3-10 g/L beef extract, and the pH is 7.0 +/-1.0; the bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 40-90 parts of bran, 0.1-2 parts of fish meal, 0.1-0.6 part of ammonium sulfate, 0.1-0.6 part of dipotassium hydrogen phosphate, 0.1-0.5 part of manganese sulfate and 10-60 parts of water.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in the step 1), the specific operations for preparing the seed culture solution are as follows: inoculating Bacillus coagulans to test tube slant of beef extract peptone agar culture medium, culturing at 37 deg.C for 24 hr, inoculating to eggplant bottle slant of beef extract peptone agar culture medium, culturing at 37 deg.C until more than 80% thallus forms spore, transferring into sterile water to form 5.0 × 108cfu/ml～2.0×109cfu/ml seed culture.
Further, the beef extract peptone agar medium contains 10g/L of peptone, 5g/L, NaCl 5g/L of beef extract and 15-20 g/L of agar, and the pH value is 6.8-7.0.
Further, in the step 4), turning over the bran solid culture medium every 3-5 hours during the culture.
Further, in the step 4), the prepared lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃ and then packaged into a finished product, and the finished product can be used as a microbial preparation product after being detected to be qualified.
Further, in steps 2) and 3), the primary and secondary liquid media were sterilized at 121 ℃ for 20min before inoculation.
Further, in the step 4), the preparation method of the bran solid culture medium comprises the following steps: dissolving ammonium sulfate, dipotassium hydrogen sulfate and manganese sulfate in water, mixing with the rest solid raw materials, stirring uniformly, and respectively controlling the pressure and the temperature to be 1.05-1.3 Kg/cm3And sterilizing for 30-40 min at the temperature of 121-125 ℃, and cooling to 40-50 ℃ to obtain a bran solid culture medium for later use.
The invention has the beneficial effects that:
(1) the invention optimizes the fermentation culture medium, inoculates bacillus coagulans of which more than 80 percent of thalli form spores into a liquid culture medium as a seed culture solution, performs primary and secondary fermentation under proper conditions, inoculates the secondary fermentation broth into a bran solid culture medium, and performs culture under proper conditions, so that the bacillus coagulans is always in aerobic conditions and performs high-density fermentation to form a large amount of spores, and simultaneously avoids the influence of mixed bacteria.
(2) The invention effectively solves the problems of excessive mixed bacteria, uneven ventilation and heat dissipation, non-constant temperature and the like in the bran fermentation process in the prior art, greatly improves the viable count of bacillus coagulans, and tests prove that the lactic acid producing bacillus coagulans microbial preparation prepared by the method contains 83-132 hundred million viable bacteria per gram of microbial preparation, the spore germination rate is not less than 92%, the mixed bacteria rate is less than 0.2%, and the L-lactic acid content is not less than 36.5 g/L.
(3) The invention provides a liquid culture medium suitable for bacillus coagulans and spore production conditions of liquid culture, has certain practical significance for laying industrial production of bacillus coagulans microbial preparations, and is expected to solve the problem that bacillus coagulans cannot form spores in a common liquid culture medium, and the limitation of solid culture hinders the expanded production and practical application of bacillus coagulans.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Unless otherwise indicated, the raw materials and equipment used in the present invention are conventional in the art (conventional commercial products) and are commercially available. For example, the lyophilized species of Bacillus coagulans in examples 1-10 were purchased and are the existing biological material, with the collection number GDMCC 1.645, and the collection unit is the Guangdong province collection of microorganisms.
Example 1
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 2.0 × 109cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15g/L beef extract and 15g/L agar, has a pH of 6.8, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at an inoculation amount of 5%, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 200rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 8 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 250rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 30 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 2.0g/L、MgSO4 0.8g/L、KH2PO41.0g/L、K2HPO41.0g/L, NaCl 5g/L, peptone 10g/L and beef extract 5g/L, and pH 7.0, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 3% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 10 times/hour, carrying out anaerobic culture for 60 hours under the conditions that the temperature is 30-40 ℃ and the humidity is 80-90%, and turning over the bran solid culture base material once every 3 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 700 parts of bran, 2 parts of fish meal, 4 parts of ammonium sulfate, 2 parts of dipotassium hydrogen phosphate, 2 parts of manganese sulfate and 290 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then the pressure and the temperature are respectively 1.05 to 1.3Kg/cm in a rotary cooking pot or a rice cooker3And sterilizing for 30-40 min at 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 120 hundred million live bacteria, the germination rate of spores is 99 percent, and the rate of mixed bacteria is 0.08 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 57.4 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 2
From freeze-dried species of Bacillus coagulansSelecting a ring of thalli to inoculate into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 5.0 × 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 9g/L peptone, 6g/L, NaC14g/L beef extract and 22g/L agar, and has a pH of 7.0, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at 3% inoculation amount, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 350rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 16 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 150rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 12 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 0.5g/L、MgSO4 0.4g/L、KH2PO40.5g/L、K2HPO40.5g/L, NaCl 10g/L, peptone 5g/L and beef extract 10g/L, and pH 7.0, and autoclaving at 121 deg.C for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in the aseptic environment in an inoculation amount of 2%, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 3 times/hr, culturing at 30-40 deg.C and humidity of 80-90% for 72 hrIn the culture period, the solid culture base material of bran is turned over once every 6 hours, so as to prepare the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 880 parts of bran, 5 parts of fish meal, 1 part of ammonium sulfate, 1 part of dipotassium hydrogen phosphate, 3 parts of manganese sulfate and 100 parts of water. The preparation method comprises the steps of dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring uniformly. Then sterilizing at 123 deg.C for 25min, cooling to 38 deg.C, and packaging into shallow pots, 5 kg per pot.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 83 hundred million live bacteria, the germination rate of spores is 92 percent, and the rate of mixed bacteria is 0.15 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 38.1 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 3
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 1.0 × 109cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15g/L beef extract and 15g/L agar, and has a pH of 6.9, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at an inoculation amount of 4%, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 250rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 10 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 200rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 24 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 1.0g/L、MgSO4 1.0g/L、KH2PO40.5g/L、K2HPO43g/L, NaCl 3g/L, peptone 20g/L and beef extract 3g/L, and pH 6.8, autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, the ozone generator and the ultraviolet lamp switch of the culture room are firstly opened, and the culture room area is as follows: 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 4% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 6 times/hour, carrying out anaerobic culture at 30-40 ℃ and humidity of 80-90% for 66 hours, and turning over the bran solid culture base material once every 4 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 400 parts of bran, 1 part of fish meal, 6 parts of ammonium sulfate, 6 parts of dipotassium hydrogen phosphate, 1 part of manganese sulfate and 586 parts of water. The preparation method comprises the steps of dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring uniformly. Then in the rotary cooking pot or the rice cooker, the pressure and the temperature are respectively as follows: 1.05 to 1.3Kg/cm3And sterilizing for 30-40 min at 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 100 hundred million live bacteria, the germination rate of spores is 95 percent, and the rate of mixed bacteria is 0.08 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 50.2 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 4
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 23 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 8.0 × 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 16g/L agar, has a pH of 6.8, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at an inoculation amount of 5%, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 350rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 8 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 180rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 16 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 0.7g/L、MgSO4 0.5g/L、KH2PO43g/L、K2HPO41.8g/L, NaCl 3g/L, peptone 6g/L and beef extract 9g/L, and pH 7.0, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in the aseptic environment in an inoculation amount of 2%, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 4 times/hour, carrying out anaerobic culture at 30-40 ℃ and 80-90% humidity for 62 hours, and turning over the bran solid culture base material once every 4 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 500 parts of bran, 20 parts of fish meal, 2 parts of ammonium sulfate, 1 part of dipotassium hydrogen phosphate, 5 parts of manganese sulfate and 458 parts of water. The preparation method comprises the steps of dissolving the ammonium sulfate, the dipotassium phosphate and the manganese sulfate in water, mixing with the rest solid raw materials, and stirring uniformly. Then the pressure and the temperature are respectively 1.05 to 1.3Kg/cm in a rotary cooking pot or a rice cooker3And sterilizing for 30-40 min at the temperature of 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid-producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid-producing bacillus coagulans microbial preparation contains 91 hundred million live bacteria, the germination rate of spores is 93 percent, and the rate of mixed bacteria is 0.12 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 36.8 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 5
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 12 × 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 18g/L agar, has a pH of 6.9, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at an inoculation amount of 5%, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 320rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 8 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 220rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 18 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 0.8g/L、MgSO4 1.0g/L、KH2PO41.5g/L、K2HPO41.2g/L, NaCl 4g/L, peptone 9g/L and beef extract 8g/L, and pH 8.0, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2Sterilizing for 30min, preparing sterile environment, inoculating the secondary fermentation liquid into bran solid culture medium at an inoculation amount of 2%, mixing, and controlling ventilation amount in bran solid culture mediumComprises the following steps: ventilating for 5 times/hour, carrying out anaerobic culture for 70 hours under the conditions that the temperature is 30-40 ℃ and the humidity is 80-90%, and turning over the bran solid culture base material once every 5 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 450 parts of bran, 18 parts of fish meal, 3 parts of ammonium sulfate, 2 parts of dipotassium hydrogen phosphate, 4 parts of manganese sulfate and 600 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then the pressure and the temperature are respectively 1.05 to 1.3Kg/cm in a rotary cooking pot or a rice cooker3And sterilizing for 30-40 min at the temperature of 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 97 hundred million live bacteria, the germination rate of spores is 93 percent, and the rate of mixed bacteria is 0.13 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 43.5 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 6
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 14 × 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 17g/L agar, has a pH of 6.8, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at an inoculation amount of 4%, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 230rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 12 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 250rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 20 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 1.5g/L、MgSO4 0.9g/L、KH2PO41.5g/L、K2HPO43g/L, NaCl 6g/L, peptone 18g/L and beef extract 6g/L, and pH 6.0, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 3% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 6 times/hour, carrying out anaerobic culture at 30-40 ℃ and humidity of 80-90% for 68 hours, and turning over the bran solid culture base material once every 4.5 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 600 parts of bran, 15 parts of fish meal, 4 parts of ammonium sulfate, 3 parts of dipotassium hydrogen phosphate, 3 parts of manganese sulfate and 415 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then the pressure is applied to a rotary cooker or a rice cooker,The temperature is 1.05 to 1.3Kg/cm3And sterilizing for 30-40 min at 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that the lactic acid producing bacillus coagulans microbial preparation contains 110 hundred million live bacteria per gram, the spore germination rate is 94 percent, and the mixed bacteria rate is 0.08 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 52.7 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 7
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 16 × 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 19g/L agar, and has a pH of 7.0, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seeding tank filled with a primary liquid culture medium by 4 percent of inoculum size for fermentation culture, wherein the liquid loading amount is 70 percent, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 270rpm, the ventilation amount is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90 percent, the fermentation time is 16 hours, and the thalli reach the logarithmic phase to prepare primary fermentation liquid. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 230rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 26 hours, and the thallus reaches the late stage of logarithmic growth, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 1.3g/L、MgSO4 0.6g/L、KH2PO42.0g/L、K2HPO42.5g/L, NaCl 7g/L, peptone 12g/L and beef extract 8g/L, and pH 7.3, autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 4% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 8 times/hour, carrying out anaerobic culture at 30-40 ℃ and 80-90% humidity for 64 hours, and turning over the bran solid culture base material once every 3.5 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 650 parts of bran, 10 parts of fish meal, 5 parts of ammonium sulfate, 4 parts of dipotassium hydrogen phosphate, 1 part of manganese sulfate and 330 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then the pressure and the temperature are respectively 1.05 to 1.3Kg/cm in a rotary cooking pot or a rice cooker3And sterilizing for 30-40 min at 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 116 hundred million live bacteria, the germination rate of spores is 97 percent, and the rate of mixed bacteria is 0.06 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 58.6 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 8
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 18 × 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 20g/L agar, and has a pH of 7.0, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at 3% inoculation amount, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 250rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 14 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 250rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 28 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 1.8g/L、MgSO4 0.8g/L、KH2PO42.5g/L、K2HPO42.0g/L, NaCl 5g/L, peptone 14g/L and beef extract 7g/L, and pH 7.5, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 4% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 10 times/hour, carrying out anaerobic culture for 66 hours under the conditions that the temperature is 30-40 ℃ and the humidity is 80-90%, and turning over the bran solid culture base material once every 3 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 750 parts of bran, 12 parts of fish meal, 5 parts of ammonium sulfate, 5 parts of dipotassium hydrogen phosphate, 2 parts of manganese sulfate and 226 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then the pressure and the temperature are respectively 1.05 to 1.3Kg/cm in a rotary cooking pot or a rice cooker3And sterilizing for 30-40 min at the temperature of 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 125 hundred million live bacteria, the germination rate of spores is 98 percent, and the rate of mixed bacteria is 0.08 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and measuring the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the measured content of the L-lactic acid is 66.1 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 9
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, and packagingShaking in a 250ml triangular flask to homogenize the cells to a cell concentration of 6.0X 108cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 13g/L agar, has a pH of 6.8, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seed tank filled with a primary liquid culture medium for fermentation culture at an inoculation amount of 5%, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 300rpm, the ventilation rate is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, the fermentation time is 10 hours, and the thalli reach the logarithmic phase to prepare a primary fermentation solution. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 200rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 14 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 0.9g/L、MgSO4 0.5g/L、KH2PO40.8g/L、K2HPO41.5g/L, NaCl 9g/L, peptone 8g/L and beef extract 7g/L, and pH 7.0, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 3% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 7 times/hour, carrying out anaerobic culture for 72 hours under the conditions that the temperature is 30-40 ℃ and the humidity is 80-90%, and turning over the bran solid culture base material once every 5 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 900 parts of bran, 8 parts of fish meal, 2 parts of ammonium sulfate, 2 parts of dipotassium hydrogen phosphate, 5 parts of manganese sulfate and 183 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then sterilizing at high temperature and high pressure for 45min, cooling to 55 ℃, and subpackaging in shallow pots, 5 kg per pot, for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 90 hundred million live bacteria, the germination rate of spores is 94 percent, and the rate of mixed bacteria is 0.15 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 36.5 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
Example 10
Selecting a ring of thalli from the freeze-dried strain of bacillus coagulans, inoculating the selected ring of thalli into a beef extract peptone agar test tube slant culture medium, and culturing for 24 hours at 37 ℃; then transferring the test tube slant seeds into a beef extract peptone agar eggplant bottle slant culture medium, culturing for 24 hours at 37 ℃, and detecting that more than 80% of thalli form spores. Scraping mature bacterial mud on eggplant bottle with sterile water, placing into 250ml triangular flask, shaking to make thallus uniform, and thallus concentration is 2.0 × 109cfu/ml, forming a seed culture solution.
The beef extract peptone agar medium described in this example contains 10g/L peptone, 5g/L, NaC15, 15g/L beef extract and 17g/L agar, has a pH of 6.8, and is autoclaved at 121 ℃ for 20min before inoculation.
Transferring the seed culture solution into a 30L seeding tank filled with a primary liquid culture medium by 3 percent of inoculum size for fermentation culture, wherein the liquid loading amount is 70 percent, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 200rpm, the ventilation amount is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90 percent, the fermentation time is 12 hours, and the thalli reach the logarithmic phase to prepare primary fermentation liquid. Transferring the primary fermentation liquid with the inoculation amount of 10% into a 300L fermentation tank filled with a secondary liquid culture medium for further expanded culture, wherein the liquid loading amount is 70%, the fermentation temperature is 37 +/-1 ℃, the stirring speed is 250rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, the fermentation time is 30 hours, and the thalli reach the late logarithmic growth stage, thereby obtaining the secondary fermentation liquid.
Both the primary and secondary liquid media described in this example contain MnSO4 2.0g/L、MgSO4 0.7g/L、KH2PO43g/L、K2HPO42.5g/L, NaCl 8g/L, peptone 16g/L and beef extract 4g/L, and pH 7.0, and was autoclaved at 121 ℃ for 20min before inoculation.
Before inoculation, an ozone generator and an ultraviolet lamp switch of a culture room are turned on, and the area of the culture room is 25m2And (3) sterilizing for 30min, preparing an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 4% in the aseptic environment, uniformly mixing, and controlling the ventilation rate in the bran solid culture medium as follows: ventilating for 9 times/hour, carrying out anaerobic culture at 30-40 ℃ and humidity of 80-90% for 66 hours, and turning over the bran solid culture base material once every 3 hours during the culture period, thereby preparing the lactic acid producing bacillus coagulans microbial preparation.
The bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 700 parts of bran, 16 parts of fish meal, 5 parts of ammonium sulfate, 5 parts of dipotassium hydrogen phosphate, 3 parts of manganese sulfate and 350 parts of water. The preparation method comprises dissolving ammonium sulfate, dipotassium hydrogen phosphate and manganese sulfate in water, mixing with the rest solid raw materials, and stirring. Then the pressure and the temperature are respectively 1.05 to 1.3Kg/cm in a rotary cooking pot or a rice cooker3And sterilizing for 30-40 min at 121-125 ℃, cooling to 40-50 ℃, and subpackaging in shallow pots with 5 kg per pot for later use.
The lactic acid producing bacillus coagulans microbial preparation of the embodiment is tested to find that each gram of the lactic acid producing bacillus coagulans microbial preparation contains 132 hundred million live bacteria, the germination rate of spores is 96 percent, and the rate of mixed bacteria is 0.06 percent.
The lactic acid producing Bacillus coagulans microbial preparation of this example was dissolved in water, heated to 80 ℃ and centrifuged in a centrifuge at 5000rpm for 15 min. Taking the supernatant, and determining the content of the L-lactic acid by using an L-lactic acid dehydrogenase method, wherein the determined content of the L-lactic acid is 72.0 g/L.
The lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, packaged into a finished product, and the finished product can be used as a microbial preparation product after the detection is qualified.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. A method for preparing a lactic acid producing bacillus coagulans microbial preparation is characterized by comprising the following steps:
1) transferring activated Bacillus coagulans with more than 80% of thallus to form spore into sterile water to make its concentration 5.0 × 108cfu/ml～2.0×109cfu/ml, forming a seed culture solution;
2) transferring the seed culture solution into a primary liquid culture medium by an inoculation amount of 3-5% for primary fermentation, wherein the fermentation temperature is 37 +/-1 ℃, the stirring speed is 200-350 rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 80-90%, and the fermentation time is 8-16 h to prepare a primary fermentation solution;
3) transferring the primary fermentation liquid into a secondary liquid culture medium by 10% of inoculation amount for secondary fermentation, wherein the fermentation temperature is 37 +/-1 ℃, the stirring speed is 150-250 rpm, the ventilation volume is 0.8-0.9V/V.min, the dissolved oxygen concentration is 70-80%, and the fermentation time is 12-30 h, so that the bacillus coagulans grows to the last logarithmic growth stage to prepare the secondary fermentation liquid;
4) in an aseptic environment, inoculating the secondary fermentation liquid into a bran solid culture medium in an inoculation amount of 2-4%, uniformly mixing, and controlling the ventilation quantity as follows: ventilating for 3-10 times/hour, culturing for 60-72 hours at the temperature of 30-40 ℃ and the humidity of 80-90%, and turning over a bran solid culture medium during the culturing period to prepare the lactic acid producing bacillus coagulans microbial preparation;
wherein the first and second liquid culture media both contain MnSO40.5～2.0g/L、MgSO40.4～1.0g/L、KH2P040.5～3g/L、K2HP040.5-3 g/L, NaCl 3-10 g/L, 5-20 g/L peptone and 3-10 g/L beef extract, and the pH is 7.0 +/-1.0; the bran solid culture medium is prepared by mixing the following raw materials in parts by weight: 40-90 parts of bran, 0.1-2 parts of fish meal, 0.1-0.6 part of ammonium sulfate, 0.1-0.6 part of dipotassium hydrogen phosphate, 0.1-0.5 part of manganese sulfate and 10-60 parts of water.
2. The method for preparing a lactic acid producing microbial preparation of Bacillus coagulans according to claim 1, wherein the step 1) comprises the following steps: inoculating Bacillus coagulans to test tube slant of beef extract peptone agar culture medium, culturing at 37 deg.C for 24 hr, inoculating to eggplant bottle slant of beef extract peptone agar culture medium, culturing at 37 deg.C until more than 80% thallus forms spore, transferring into sterile water to form 5.0 × 108cfu/ml～2.0×109cfu/ml seed culture.
3. The method for preparing a lactic acid producing Bacillus coagulans microbial preparation according to claim 2, wherein the beef extract peptone agar medium contains 10g/L peptone, 5g/L, NaCl 5g/L beef extract and 15-20 g/L agar, and the pH is 6.8-7.0.
4. The method for preparing a lactic acid producing Bacillus coagulans microbial preparation according to claim 1, wherein in step 4), the bran solid medium is turned over every 3-5 h during the culture period.
5. The method for preparing a lactic acid producing bacillus coagulans microbial preparation according to claim 1, wherein in the step 4), the prepared lactic acid producing bacillus coagulans microbial preparation is subjected to vacuum drying treatment at 55-60 ℃, and then packaged into a finished product, and the finished product can be used as a microbial preparation product after being detected to be qualified.
6. The method of claim 1, wherein the primary and secondary liquid culture media are sterilized at 121 ℃ for 20min before inoculation in steps 2) and 3).
7. The method for preparing a lactic acid producing bacillus coagulans microbial preparation according to claim 1, wherein in the step 4), the bran solid medium is prepared by the following steps: dissolving ammonium sulfate, dipotassium hydrogen sulfate and manganese sulfate in water, mixing with the rest solid raw materials, stirring uniformly, and respectively controlling the pressure and the temperature to be 1.05-1.3 Kg/cm3And sterilizing for 30-40 min at the temperature of 121-125 ℃, and cooling to 40-50 ℃ to obtain a bran solid culture medium for later use.
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