CN104371960B - Composite fungus agent and the continuous fermentation method of complex microorganism adopted - Google Patents
Composite fungus agent and the continuous fermentation method of complex microorganism adopted Download PDFInfo
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- 230000004151 fermentation Effects 0.000 title claims abstract description 55
- 244000005700 microbiome Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000002131 composite material Substances 0.000 title claims abstract description 16
- 241000233866 Fungi Species 0.000 title description 6
- 239000007788 liquid Substances 0.000 claims abstract description 83
- 241000894006 Bacteria Species 0.000 claims abstract description 53
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 44
- 241000193403 Clostridium Species 0.000 claims abstract description 39
- 238000002360 preparation method Methods 0.000 claims abstract description 30
- 239000006041 probiotic Substances 0.000 claims abstract description 21
- 235000018291 probiotics Nutrition 0.000 claims abstract description 21
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 238000003756 stirring Methods 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 12
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 238000013019 agitation Methods 0.000 claims description 8
- 238000009423 ventilation Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 235000015278 beef Nutrition 0.000 claims description 4
- NKWPZUCBCARRDP-UHFFFAOYSA-L calcium bicarbonate Chemical compound [Ca+2].OC([O-])=O.OC([O-])=O NKWPZUCBCARRDP-UHFFFAOYSA-L 0.000 claims description 4
- 229910000020 calcium bicarbonate Inorganic materials 0.000 claims description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 235000010216 calcium carbonate Nutrition 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- 210000000582 semen Anatomy 0.000 claims description 3
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- 238000004321 preservation Methods 0.000 description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 244000063299 Bacillus subtilis Species 0.000 description 10
- 235000014469 Bacillus subtilis Nutrition 0.000 description 9
- 239000001301 oxygen Substances 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 241000193830 Bacillus <bacterium> Species 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000011068 loading method Methods 0.000 description 7
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- 238000010899 nucleation Methods 0.000 description 7
- 230000000529 probiotic effect Effects 0.000 description 7
- 235000011187 glycerol Nutrition 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 239000002068 microbial inoculum Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 235000014590 basal diet Nutrition 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
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- 235000013305 food Nutrition 0.000 description 3
- 230000037406 food intake Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
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- 238000001816 cooling Methods 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
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- 239000004310 lactic acid Substances 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000193171 Clostridium butyricum Species 0.000 description 1
- 206010009657 Clostridium difficile colitis Diseases 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 208000003100 Pseudomembranous Enterocolitis Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 201000009840 acute diarrhea Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 208000019902 chronic diarrheal disease Diseases 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 238000012262 fermentative production Methods 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- -1 lactic acid Chemical class 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- YKQOSKADJPQZHB-RGYSVOEGSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(6r,9s,12r,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-hydr Chemical compound CCC(C)CCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)C([C@@H](C)O)NC(=O)[C@@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O YKQOSKADJPQZHB-RGYSVOEGSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000027272 reproductive process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
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- 108010046845 tryptones Proteins 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of continuous fermentation method of complex microorganism, comprise the following steps: prepare subtilis bacterium liquid, Bacillus licheniformis liquid and clostridium butylicum bacterium liquid 1), respectively; 2), basal fermentation liquid will be added in fermentor tank; 3), the temperature that controls basal fermentation liquid is 36 ~ 38 DEG C, controls tank pressure 0.01Mpa, first drops into subtilis liquid fermentation culture, then drops into Bacillus licheniformis liquid and continue fermentation culture, finally drops into clostridium butylicum bacterium liquid and leaves standstill anaerobically fermenting and cultivate; 4), step 3) gained filtering fermentation liquor after centrifugal concentrating to 28 ~ 32% of original volume; Then spray-dried, obtain composite probiotics preparations.To instant invention overcomes in the independent fermenting process of microorganism sterilizing often, charging shortcoming often, simplify fermenting procedure, simultaneously by continuously fermenting and the synergy of different microorganisms, improve viable count, reduce miscellaneous bacteria number, improve the action effect of microorganism.
Description
Technical field
The invention belongs to field of microbial fermentation, relate to a kind of zymotechnique of complex microorganism---the zonal cooling zymotechnique of clostridium butylicum, Bacillus licheniformis and subtilis.
Background technology
Clostridium butylicum is a kind of Gram-positive genus bacillus of anaerobism, due to its strictly anaerobic, exists in yeasting if any oxygen, and clostridium butylicum thalline can not grow even dead.Heat-resisting, acidproof, the resistance to bile of clostridium butylicum, and have stronger tolerance to multiple feeding antibiotic, can use by compatibility; Amylase can be produced, hydrolyzed starch and carbohydrate; Produce the meta-bolitess such as butyric acid, acetic acid and lactic acid.It has been widely used in treatment digestion clinically, the enteric flora disturbance that a variety of causes such as children's cause, acute and chronic diarrhea, irritable bowel syndrome, the various intestinal tract disease such as Antibiotic-associated Colitis, and in Japan, the ground such as Korea S are applied on livestock and poultry cultivation as fodder additives and also achieve good effect, for the abuse reducing antibiotic product in present feed, reduce medicine remaining in meat, reduce the resistance of animal bacteria and ensure that animal health aspect has great significance, a kind of desirable, there is the probiotics of wide DEVELOPMENT PROSPECT.
Bacillus licheniformis is a kind of amphimicrobian microorganism, it all can grow under aerobic oxygen free condition, oxygen in intestines can be consumed in growth and breeding process after entering animal gastrointestinal tract, maintain enteron aisle anaerobic environment, promote that intestinal beneficial bacterium is as the propagation of bifidus bacillus and milk-acid bacteria and growth, prevent pathogenic bacteria and the spoilage organism abnormality proliferation in enteron aisle, thus regulate animal intestinal micro-ecology balance.Bacillus licheniformis can form spore, and energy high temperature resistant (100 DEG C), acid and alkali-resistance, anti-extrusion and resistance to temperature variation etc., can meet process for processing characteristic, and in feed, application has good production performance.The application and development of China to Bacillus licheniformis is started late, though therefore cause there are some Bacillus licheniformis products in the market, but its living bacteria count is low, complex manufacturing and cost is higher, and quality product is uneven, have impact on its result of use.
Subtilis is aerobic microorganism, grow under aerobic conditions, the free oxygen in digestive tube can be consumed rapidly after entering animal gastrointestinal tract, form enteron aisle low-oxygen environment, promote that useful anerobe grows, and produce the organic acids such as lactic acid, reduce gut pH, indirectly suppress the growth of other pathogenic bacterium.Subtilis thalline self can synthesize digestibility enzyme, as proteolytic enzyme, amylase, lipase etc., jointly plays a role in digestive tube with endogenous enzyme, improves feed digestibility.Subtilis can synthesize multivitamin in reproductive process simultaneously, improves the activity of animal body internal interference element and scavenger cell.There is the advantages such as yield of enzyme is high, bacteriostasis is strong, security is good, environmental protection.In addition, subtilis has the characteristic of acidproof, salt tolerant, high temperature resistant (100 DEG C) and anti-extrusion, preserves good, be conducive to applying in feed manufacturing.Its microbial strains as great exploitation potential for its has been widely used in all fields of industry, agricultural, medical and health, food, livestock industry, aquatic products and scientific research.
The problems such as the fermentation costs that the independent zymophyte of above-mentioned probiotic bacterium faces is high, viable count is low, miscellaneous bacteria is many, and to three fermentation techniques, there is not been reported both at home and abroad.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of contact new process for fermenting of complex microorganism, to overcome in the independent fermenting process of microorganism sterilizing often, charging shortcoming often, simplify fermenting procedure, reduce the production cost of fermentable, simultaneously by continuously fermenting and the synergy of different microorganisms, improve viable count, reduce miscellaneous bacteria number, improve the action effect of microorganism.
In order to solve the problems of the technologies described above, the invention provides a kind of continuous fermentation method of complex microorganism, comprising the following steps:
1), subtilis bacterium liquid, Bacillus licheniformis liquid and clostridium butylicum bacterium liquid is prepared respectively;
2), fermented liquid based on 15 tons of substratum III will be added in fermentor tank;
3), the temperature that controls basal fermentation liquid is 36 ~ 38 DEG C, controls tank pressure 0.01Mpa, drops into the subtilis liquid of 140 ~ 160L in temperature, the 750 ~ 850m of 36 ~ 38 DEG C
3air flow, the agitation condition bottom fermentation of/h cultivate 11 ~ 13h (being preferably 12h);
Then, the Bacillus licheniformis liquid of 140 ~ 160L is dropped in temperature, the 750 ~ 850m of 36 ~ 38 DEG C
3fermentation culture 15 ~ 17h (being preferably 16h) is continued under the air flow of/h, agitation condition;
Then, stop stirring and ventilation, at the temperature of 36 ~ 38 DEG C, continue fermentation culture 2h;
Finally, the clostridium butylicum bacterium liquid dropping into 140 ~ 160L leaves standstill anaerobically fermenting in the temperature of 36 ~ 38 DEG C and cultivates 23 ~ 25h (being preferably 24h);
4), step 3) gained filtering fermentation liquor after centrifugal concentrating to 28 ~ 32% of original volume; Then spray-dried, obtain composite probiotics preparations.
Improvement as the continuous fermentation method of complex microorganism of the present invention:
The preparation method of substratum III is: glucose 20g, Semen Maydis powder 10g, soyflour 40g, yeast powder 8g, 1g silicone oil (H201-100), Calcium hydrogen carbonate 0.5g, calcium chloride 0.015g, ammonium sulfate 1g, magnesium sulfate 0.15g, manganous sulfate 0.15g, add water 1000ml, uses NaHCO
3regulate pH to 7.0, sterilize 30-35min under 121-128 DEG C of condition.
Further improvement as the continuous fermentation method of complex microorganism of the present invention:
The preparation method of subtilis bacterium liquid is:
Be 50 × 10 by the concentration of 3.0ml
8the subtilis of cfu/ml (
preservationnumber: CGMCC No.9383) bacterium liquid is inoculated in the substratum I of 1.9 ~ 2.1L, cultivates 17 ~ 19h (being preferably 18h), then drop into the substratum III of 280 ~ 320L in 36 ~ 38 DEG C, 9 ~ 11m in 36 ~ 38 DEG C of shaking tables
3under the air flow of/h, agitation condition, enlarged culturing cultivates 17 ~ 19h (being preferably 18h); Obtain subtilis bacterium liquid;
The preparation method of Bacillus licheniformis liquid is:
Be 50 × 10 by the concentration of 3.0ml
8the Bacillus licheniformis of cfu/ml (
preservationnumber: CGMCC No.9385) bacterium liquid is inoculated in the substratum I of 1.9 ~ 2.1L, cultivates 17 ~ 19h (being preferably 18h), then drop into the substratum III of 280 ~ 320L in 36 ~ 38 DEG C, 9 ~ 11m in 36 ~ 38 DEG C of shaking tables
3enlarged culturing 17 ~ 19h (being preferably 18h) under the air flow of/h, agitation condition; Obtain Bacillus licheniformis liquid;
The preparation method of clostridium butylicum bacterium liquid is:
Be 2.0 × 10 by the concentration of 3.0ml
8the clostridium butylicum of cfu/ml (
preservationnumber: CGMCC No.9386) bacterium liquid is inoculated in the substratum II of 1.9 ~ 2.1L, in 36 ~ 38 DEG C of standing Anaerobic culturel 17 ~ 19h (being preferably 18h), then the substratum III dropping into 280 ~ 320L, in 36 ~ 38 DEG C of standing anaerobism enlarged culturing 17 ~ 19h (being preferably 18h), obtains clostridium butylicum bacterium liquid;
Described substratum I is beef extract-peptone Shake flask medium, and described substratum II is RCM substratum.Remarks illustrate: these 2 kinds of substratum are conventional medium.
Further improvement as the continuous fermentation method of complex microorganism of the present invention:
Described step 4) in be filtered into through 100 orders filter; Spray-dired inlet temperature 210 DEG C ± 5 DEG C, air outlet temperature 95 DEG C ± 5 DEG C.
The present invention also provides simultaneously and utilizes above-mentioned either method to prepare and the composite probiotics preparations (that is, composite fungus agent) that obtains.
In the product (composite probiotics preparations) that the present invention obtains, subtilis, bacillus licheniformis and butyric acid acid bacterial content are about 1.5 × 10 respectively
9cfu/g, 1.8 × 10
9cfu/g, 1.2 × 10
7cfu/g.Total count is about 3.3 × 10
9cfu/g.
Remarks illustrate, 3 kinds of bacterial strains involved in the present invention
preservationinformation is specific as follows:
Bacillus subtilis strain HJBA058,
preservationname is called: subtilis (Bacillus subtilis),
preservationunit: Chinese microorganism strain
preservationmanagement committee's common micro-organisms center,
preservationaddress: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica,
preservationnumber: CGMCC No.9383,
preservationdate: on 06 25th, 2014.
Lichem bacillus strain HJBL008,
preservationname is called: Bacillus licheniformis (Bacillus licheniformis),
preservationunit: Chinese microorganism strain
preservationmanagement committee's common micro-organisms center,
preservationaddress: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica,
preservationnumber: CGMCC No.9385,
preservationdate: on 06 25th, 2014.
Clostridium butylicum bacterial strain HJCB998,
preservationname is called: clostridium butylicum (Clostridium butyricum),
preservationunit: Chinese microorganism strain
preservationmanagement committee's common micro-organisms center,
preservationaddress: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica,
preservationnumber: CGMCC No.9386,
preservationdate: on 06 25th, 2014.
The principal feature of present invention process is:
(1) three inoculations, three fermenting processs, are had, first time inoculation subtilis bacterium liquid carries out the fermentation of bacillus subtilis spore, second time inoculation Bacillus licheniformis liquid carries out the fermentation of Bacillus licheniformis gemma, and third time inoculation clostridium butylicum bacterium liquid carries out the fermentation (anaerobically fermenting) of clostridium butylicum gemma;
(2), subtilis is aerobic bacteria, Bacillus licheniformis is facultative anaerobe, in the later stage of subtilis and Bacillus licheniformis combined ferment, stop ventilation, subtilis and Bacillus licheniformis can exhaust rapidly oxygen remaining in fermented liquid, for clostridium butylicum provides anaerobic condition, thus save and drive oxygen cost.
(3), first time is only needed to drop into basal fermentation liquid in fermenting process, point different time sections enters the inoculation of three kinds of bacterium liquid, the fermentative production of three kinds of bacterium can be completed in a fermentor tank, iterative cycles thus, avoid the preparatory process procedure that the clear tank, material loading, sterilizing, cooling etc. of intermittent zymotechnique are complicated, ensure that in completely in airtight environment and carry out, thus effectively reduce opportunities for contamination, reduce production cost.
Embodiment
Embodiment 1: the fermentation technique of compound probiotic
Substratum I: the preparation method of beef extract-peptone Shake flask medium is (routine techniques): add 1000mL water in 5g extractum carnis, 10g peptone, 5g NaCl; Regulate pH 7.0, to set high in warm Autoclave sterilizing 30min under 121 DEG C of conditions.
The preparation method of substratum II: RCM substratum is (routine techniques): in Tryptones 10g, yeast powder 3g, beef extract powder 10g, glucose 5g, Zulkovsky starch 1g, sodium-chlor 5g, anhydrous sodium acetate 1.5g, cysteine salt 0.5g, add 1000mL water, regulate pH7.5, adding food-level white oil (about 1-2cm) sets high in 115 DEG C, 20min sterilizing in warm Autoclave, for subsequent use.
Substratum III: seeding tank (fermentor tank) medium preparation method is: glucose 20g, Semen Maydis powder 10g, soyflour 40g, yeast powder 8g, 1g silicone oil (H201-100), Calcium hydrogen carbonate 0.5g, calcium chloride 0.015g, ammonium sulfate 1g, magnesium sulfate 0.15g, manganous sulfate 0.15g, add water 1000ml, uses NaHCO
3regulate pH to 7.0, sterilize 30-35min under 121-128 DEG C of condition.
Remarks illustrate: in following steps, shaking table refers to the rotating speed of 25-35 rev/min; Stir the rotating speed referring to 70-80 rev/min.
Step one, by subtilis (
preservationnumber: CGMCC No.9383) glycerine pipe 2 (often props up and is about 50 × 10 containing concentration
8the bacterium liquid 1.5ml of cfu/ml) be inoculated in (in-built 2L substratum I) in 1 5L triangular flask, cultivate 18h in 37 DEG C of shaking tables, be then fed into enlarged culturing 18h in 500L (loading amount is 300L substratum III) seeding tank and (culture temperature 37 DEG C, open stirring, air flow 10m
3/ h), so that also this obtains subtilis bacterium liquid.
By Bacillus licheniformis (
preservationnumber: CGMCC No.9385) (often prop up containing concentration is 50 × 10 to glycerine pipe 2
8the bacterium liquid 1.5ml of cfu/ml) be inoculated in (in-built 2L substratum I) in 1 5L triangular flask, cultivate 18h in 37 DEG C of shaking tables, be then fed into enlarged culturing 18h in 500L (loading amount is 300L substratum III) seeding tank and (culture temperature 37 DEG C, open stirring, air flow 10m
3/ h), obtain Bacillus licheniformis liquid with this.
By clostridium butylicum bacterial strain (
preservationnumber: CGMCC No.9386) (often prop up containing concentration is 2.0 × 10 to glycerine cryopreservation tube 2
8the bacterium liquid 1.5ml of cfu/ml) be inoculated in (in-built 2L substratum II) in 1 5L triangular flask, 37 DEG C of standing Anaerobic culturel 18h, then be fed into enlarged culturing 18h in 500L (loading amount is 300L substratum III) seeding tank (temperature 37 DEG C is not opened stirring, left standstill Anaerobic culturel) and obtain clostridium butylicum bacterium liquid with this.
15 tons of substratum III are dropped into 20T fermentor tank by step 2, based on fermented liquid.
Remarks illustrate: this step of 30-35min of sterilizing under 121-128 DEG C of condition can complete in above-mentioned fermentor tank.
Step 3, when basal fermentation liquid temp is 37 DEG C, controls tank pressure 0.01Mpa, adds subtilis liquid obtained in 150L step one, in culture temperature 37 DEG C, open stirring, air flow 800m
3the fermentation of bacillus subtilis spore is carried out under the culture condition of/h, after fermentation 12h, drop into Bacillus licheniformis liquid obtained in 150L step one, culture condition is constant, continue fermentation 16h, then close stirring, stop ventilation, insulation continues fermentation 2h, finally drop into clostridium butylicum bacterium liquid obtained in 150L step one to ferment, under the culture condition of culture temperature 37 DEG C, pass stirring, stuffiness, standing Anaerobic culturel, continue fermentation 24h.
Step 4, after fermented liquid 100 order obtained in step 3 is filtered, enter disk centrifugal separator centrifugal, be concentrated into 30% of original volume, then spray-dried (inlet temperature 210 DEG C ± 5 DEG C, air outlet temperature 95 DEG C ± 5 DEG C) obtains finished product (powder)---composite probiotics preparations.
Remarks illustrate: microorganism plate and test tube detect, and in the composite fungus agent of gained, the content of subtilis, Bacillus licheniformis, clostridium butylicum is respectively 1.5 × 10
9cfu/g, 1.8 × 10
9cfu/g, 1.2 × 10
7cfu/g.
Experiment 1: the application of complex microorganism preparations on animal
Choose Du × length × large piglet 216 of 21 age in days wean, be divided into 3 treatment group, each group 6 repetitions, each repetition 12, blank group is fed basal diet, microbiotic group adds 20mg/kg colistine sulfate in basal diet, complex ferment probiotic group adds 300mg/kg (the compound probiotic agent of embodiment 1 gained in basal diet, the mixture of the subtilis namely produced by complex ferment technology of the present invention, Bacillus licheniformis and clostridium butylicum), 21 days trial periods.Experimental session is observed each group of swinery and is searched for food and drinking-water situation, record feed consumption rate, measures the wean body weight of latter 21 days, calculates average daily gain, average daily ingestion amount and feed-weight ratio; When collecting off-test, rectum gets excrement, measures the quantity of wherein intestinal bacteria, lactobacillus and bifidus bacillus.Experimental result
as table 1with
table 2.
table 1composite probiotics preparations is on the impact of Growth Performance of Weaning Piglets
| Group | Blank group | Microbiotic group | Composite probiotics preparations group |
| Average starting weight (kg) | 7.05 | 7.08 | 6.96 |
| Average end heavy (kg) | 11.01 | 11.95 | 12.11 |
| Average daily gain (g) | 189 b | 232 a | 245 a |
| Average daily ingestion amount (g) | 302 | 354 | 372 |
| Feed-weight ratio | 1.61 a | 1.53 b | 1.53 b |
Note:
tablemiddle data mark different letter representation significant difference (P<0.05), lower same.
table 1result show: add the average daily gain that complex ferment probiotics preparation of the present invention can significantly improve weanling pig, reduce feed-weight ratio, complex ferment probiotics preparation and microbiotic group growth performance and feed-weight ratio are without significant difference.
table 2composite probiotics preparations is on the impact of weanling pig fecal microorganism flora
| Group | Blank group | Microbiotic group | Composite probiotics preparations group |
| Intestinal bacteria (lgcfu/g) | 8.26 a | 7.23 b | 7.47 b |
| Lactobacillus (lgcfu/g) | 8.76 b | 8.83 b | 9.77 a |
| Bifidus bacillus (lgcfu/g) | 9.47 b | 8.56 c | 10.72 a |
table 2result show: complex ferment probiotics preparation of the present invention significantly can reduce weanling pig fecal coli quantity, improves lactobacillus and bifidobacteria, is conducive to intestinal microflora balance.
Comparative example 1, three kind of probiotic bacterium are fermented respectively, are then mixed in proportion:
The preparation method of substratum I, substratum II, substratum III is with embodiment 1.
(1), fermentation of bacillus subtilis step:
Step one, by subtilis (
preservationnumber: CGMCC No.9383) glycerine pipe 2 (often props up and is about 50 × 10 containing concentration
8the bacterium liquid 1.5ml of cfu/ml) be inoculated in (in-built 2L substratum I) in 1 5L triangular flask, cultivate 18h in 37 DEG C of shaking tables, be then fed into enlarged culturing 18h in 500L (loading amount is 300L substratum III) seeding tank and (culture temperature 37 DEG C, open stirring, air flow 10m
3/ h), so that also this obtains subtilis bacterium liquid.
Step 2, drops into 15T substratum III in 20T fermentor tank, based on fermented liquid.
Remarks illustrate: this step of 30-35min of sterilizing under 121-128 DEG C of condition can complete in above-mentioned fermentor tank.
Step 3, when basal fermentation liquid temp is 37 DEG C, controls tank pressure 0.01Mpa, adds subtilis liquid obtained in 150L step one, in culture temperature 37 DEG C, open stirring, air flow 800m
3carry out the fermentation of bacillus subtilis spore under the culture condition of/h, fermentation time is 28h.
Step 4, after fermented liquid 100 order obtained in 3rd step is filtered, enter disk centrifugal separator centrifugal, be concentrated into 30% of original volume, then spray-dried (inlet temperature 210 DEG C ± 5 DEG C, air outlet temperature 95 DEG C ± 5 DEG C) obtains finished product (powder)---bacillus subtilis formulation.
Remarks illustrate: microorganism plate and test tube detect, and the bacillus subtilis bacterial content of gained is respectively 1.2 × 10
9cfu/g.Lower than the bacillus subtilis bacterial content of complex ferment explained hereafter.
(2), the lichen bacillus ferments step:
Step one, by Bacillus licheniformis (
preservationnumber: CGMCC No.9385) (often prop up containing concentration is 50 × 10 to glycerine pipe 2
8the bacterium liquid 1.5ml of cfu/ml) be inoculated in (in-built 2L substratum I) in 1 5L triangular flask, cultivate 18h in 37 DEG C of shaking tables, be then fed into enlarged culturing 18h in 500L (loading amount is 300L substratum III) seeding tank and (culture temperature 37 DEG C, open stirring, air flow 10m
3/ h), obtain Bacillus licheniformis liquid with this.
Step 2, drops into 15T substratum III in 20T fermentor tank, based on fermented liquid.
Remarks illustrate: this step of 30-35min of sterilizing under 121-128 DEG C of condition can complete in above-mentioned fermentor tank.
Step 3, when basal fermentation liquid temp is 37 DEG C, controls tank pressure 0.01Mpa, adds Bacillus licheniformis liquid obtained in 150L step one, in culture temperature 37 DEG C, open stirring, air flow 800m
3carry out the fermentation of Bacillus licheniformis under the culture condition of/h, fermentation time is 24h.
Step 4, after fermented liquid 100 order obtained in step 3 is filtered, enter disk centrifugal separator centrifugal, be concentrated into 30% of original volume, then spray-dried (inlet temperature 210 DEG C ± 5 DEG C, air outlet temperature 95 DEG C ± 5 DEG C) obtains finished product (powder)---Bacillus licheniformis preparation.
Remarks illustrate: microorganism plate and test tube detect, and the Bacillus licheniformis content of gained is respectively 1.5 × 10
9cfu/g.Lower than the Bacillus licheniformis content of complex ferment explained hereafter.
(3), clostridium butylicum strain fermentation step:
Step one, by clostridium butylicum bacterial strain (
preservationnumber: CGMCC No.9386) (often prop up containing concentration is 2.0 × 10 to glycerine cryopreservation tube 2
8the bacterium liquid 1.5ml of cfu/ml) be inoculated in (in-built 2L substratum II) in 1 5L triangular flask, 37 DEG C of standing Anaerobic culturel 18h, then be fed into enlarged culturing 18h in 500L (loading amount is 300L substratum III) seeding tank (temperature 37 DEG C is not opened stirring, left standstill Anaerobic culturel) and obtain clostridium butylicum bacterium liquid with this.
Step 2, drops into 15T substratum III in 20T fermentor tank, based on fermented liquid.
Remarks illustrate: this step of 30-35min of sterilizing under 121-128 DEG C of condition can complete in above-mentioned fermentor tank.
Step 3, when basal fermentation liquid temp is 37 DEG C, controls tank pressure 0.01Mpa, adds clostridium butylicum bacterium liquid obtained in 150L step one, and in culture temperature 37 DEG C, do not stir, carry out under airproof culture condition the fermentation of clostridium butylicum, fermentation time is 24h.
Step 4, after fermented liquid 100 order obtained in step 3 is filtered, enter disk centrifugal separator centrifugal, be concentrated into 30% of original volume, then spray-dried (inlet temperature 210 DEG C ± 5 DEG C, air outlet temperature 95 DEG C ± 5 DEG C) obtains finished product (powder)---clostridium butylicum preparation.
Remarks illustrate: microorganism plate and test tube detect, and the clostridium butylicum content of gained is respectively 1.0 × 10
7cfu/g, lower than the clostridium butylicum content of complex ferment explained hereafter.
Contrast experiment:
Above-mentioned bacillus subtilis formulation, Bacillus licheniformis preparation, clostridium butylicum preparation are mixed according to the mass ratio of 1:1:1, obtains microbial inoculum mixture; Test with the compound probiotic agent (addition is constant, still for 300mg/kg) in this microbial inoculum mixture replacing experiment 1, acquired results as
following table 3with
table 4shown in.
table 3, impact on Growth Performance of Weaning Piglets
| Group | Microbial inoculum mixture |
| Average starting weight (kg) | 7.08 |
| Average end heavy (kg) | 11.44 |
| Average daily gain (g) | 207 b |
| Average daily ingestion amount (g) | 330 |
| Feed-weight ratio | 1.59 b |
Note:
tablemiddle data mark different letter representation significant difference (P<0.05), lower same.
table 4, impact on weanling pig fecal microorganism flora
| Group | Microbial inoculum mixture |
| Intestinal bacteria (lgcfu/g) | 7.86 b |
| Lactobacillus (lgcfu/g) | 9.07 a |
| Bifidus bacillus (lgcfu/g) | 10.03 a |
In the preparation method of comparative example 2, substratum III, cancel the use of " Calcium hydrogen carbonate 0.5g, calcium chloride 0.015g, ammonium sulfate 1g, magnesium sulfate 0.15g, manganous sulfate 0.15g ", all the other are with embodiment 1.All the other contents are equal to embodiment 1.
Detect in the composite fungus agent of gained, the content of subtilis, Bacillus licheniformis, clostridium butylicum is respectively 2 × 10
8cfu/g, 1 × 10
8cfu/g, 2 × 10
6cfu/g.Probiotic bacterium content is far below the content of embodiment 1.
The release sequence of Bacillus licheniformis liquid and subtilis liquid in comparative example 3, change embodiment 1, namely, make into first to throw in Bacillus licheniformis liquid fermentation 16h, and then throw in subtilis liquid fermentation 12h, then close stirring, stop ventilation, continue fermentation 2h, finally drop into clostridium butylicum bacterium liquid and carry out fermentation 24h.All the other contents are equal to embodiment 1.
Detect in the composite fungus agent of gained, the content of subtilis, Bacillus licheniformis, clostridium butylicum is respectively 4.2 × 10
8cfu/g, 2 × 10
8cfu/g, 7.2 × 10
6cfu/g.Subtilis, Bacillus licheniformis and clostridium butylicum content are all far below the content of embodiment 1.Because Bacillus licheniformis helps its state bottom fermentation viable count driving oxygen to be affected not having subtilis, and such release sequence causes the tunning of Bacillus licheniformis to suppress the fermentation of subtilis and clostridium butylicum.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (4)
1. the continuous fermentation method of complex microorganism, is characterized in that comprising the following steps:
1), subtilis bacterium liquid, Bacillus licheniformis liquid and clostridium butylicum bacterium liquid is prepared respectively;
2), fermented liquid based on 15 tons of substratum III will be added in fermentor tank;
3), the temperature that controls basal fermentation liquid is 36 ~ 38 DEG C, controls tank pressure 0.01Mpa, drops into the subtilis liquid of 140 ~ 160L in temperature, the 750 ~ 850m of 36 ~ 38 DEG C
3air flow, the agitation condition bottom fermentation of/h cultivate 11 ~ 13h;
Then, the Bacillus licheniformis liquid of 140 ~ 160L is dropped in temperature, the 750 ~ 850m of 36 ~ 38 DEG C
3fermentation culture 15 ~ 17h is continued under the air flow of/h, agitation condition;
Then, stop stirring and ventilation, at the temperature of 36 ~ 38 DEG C, continue fermentation culture 2h;
Finally, the clostridium butylicum bacterium liquid dropping into 140 ~ 160L leaves standstill anaerobically fermenting in the temperature of 36 ~ 38 DEG C and cultivates 23 ~ 25h;
4), step 3) gained filtering fermentation liquor after centrifugal concentrating to 28 ~ 32% of original volume; Then spray-dried, obtain composite probiotics preparations.
2. the continuous fermentation method of complex microorganism according to claim 1, is characterized in that:
The preparation method of substratum III is: glucose 20g, Semen Maydis powder 10g, soyflour 40g, yeast powder 8g, 1g silicone oil, and Calcium hydrogen carbonate 0.5g, calcium chloride 0.015g, ammonium sulfate 1g, magnesium sulfate 0.15g, manganous sulfate 0.15g, add water 1000ml, uses NaHCO
3regulate pH to 7.0, sterilize 30-35min under 121-128 DEG C of condition.
3. the continuous fermentation method of complex microorganism according to claim 2, is characterized in that:
The preparation method of subtilis bacterium liquid is:
Be 50 × 10 by the concentration of 3.0ml
8cfu/ml, preserving number are that the bacterium liquid of the subtilis of CGMCC No.9383 is inoculated in the substratum I of 1.9 ~ 2.1L, cultivate 17 ~ 19h, then drop into the substratum III of 280 ~ 320L in 36 ~ 38 DEG C, 9 ~ 11m in 36 ~ 38 DEG C of shaking tables
3under the air flow of/h, agitation condition, enlarged culturing cultivates 17 ~ 19h; Obtain subtilis bacterium liquid;
The preparation method of Bacillus licheniformis liquid is:
Be 50 × 10 by the concentration of 3.0ml
8cfu/ml, preserving number are that the bacterium liquid of the Bacillus licheniformis of CGMCC No.9385 is inoculated in the substratum I of 1.9 ~ 2.1L, cultivate 17 ~ 19h, then drop into the substratum III of 280 ~ 320L in 36 ~ 38 DEG C, 9 ~ 11m in 36 ~ 38 DEG C of shaking tables
3enlarged culturing 17 ~ 19h under the air flow of/h, agitation condition; Obtain Bacillus licheniformis liquid;
The preparation method of clostridium butylicum bacterium liquid is:
Be 2.0 × 10 by the concentration of 3.0ml
8cfu/ml, preserving number are that the bacterium liquid of the clostridium butylicum of CGMCC No.9386 is inoculated in the substratum II of 1.9 ~ 2.1L, in 36 ~ 38 DEG C of standing Anaerobic culturel 17 ~ 19h, then the substratum III dropping into 280 ~ 320L, in 36 ~ 38 DEG C of standing anaerobism enlarged culturing 17 ~ 19h, obtains clostridium butylicum bacterium liquid;
Described substratum I is beef extract-peptone Shake flask medium, and described substratum II is RCM substratum.
4. the continuous fermentation method of complex microorganism according to claim 3, is characterized in that:
Described step 4) in be filtered into through 100 orders filter; Spray-dired inlet temperature 210 DEG C ± 5 DEG C, air outlet temperature 95 DEG C ± 5 DEG C.
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| CN107557322A (en) * | 2017-10-24 | 2018-01-09 | 山西大禹生物工程股份有限公司 | A kind of bacillus licheniformis and the preparation method of Miyarisan composite bacteria agent |
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