CN104371958A - Method for increasing biomass by virtue of mixed cultivation of enterococcus faecium and bacillus subtilis - Google Patents
Method for increasing biomass by virtue of mixed cultivation of enterococcus faecium and bacillus subtilis Download PDFInfo
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- CN104371958A CN104371958A CN201410653601.2A CN201410653601A CN104371958A CN 104371958 A CN104371958 A CN 104371958A CN 201410653601 A CN201410653601 A CN 201410653601A CN 104371958 A CN104371958 A CN 104371958A
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- enterococcus faecium
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000002028 Biomass Substances 0.000 title claims abstract description 24
- 244000063299 Bacillus subtilis Species 0.000 title abstract description 12
- 235000014469 Bacillus subtilis Nutrition 0.000 title abstract description 11
- 241000194031 Enterococcus faecium Species 0.000 title abstract description 7
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 238000011081 inoculation Methods 0.000 claims abstract description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 22
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 22
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 19
- 239000001888 Peptone Substances 0.000 claims description 16
- 108010080698 Peptones Proteins 0.000 claims description 16
- 235000019319 peptone Nutrition 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 16
- 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 11
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 11
- -1 citric acid hydrogen diamine Chemical class 0.000 claims description 11
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 239000001257 hydrogen Substances 0.000 claims description 11
- 229910052739 hydrogen Inorganic materials 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 11
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 11
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 11
- 239000001632 sodium acetate Substances 0.000 claims description 11
- 235000017281 sodium acetate Nutrition 0.000 claims description 11
- 239000007788 liquid Substances 0.000 abstract description 23
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 12
- 241000894006 Bacteria Species 0.000 abstract description 11
- 239000006041 probiotic Substances 0.000 abstract description 10
- 235000018291 probiotics Nutrition 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 241001465754 Metazoa Species 0.000 abstract description 6
- 235000014655 lactic acid Nutrition 0.000 abstract description 6
- 108010062877 Bacteriocins Proteins 0.000 abstract description 3
- 239000012533 medium component Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 2
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 239000004310 lactic acid Substances 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 2
- 239000002253 acid Substances 0.000 abstract 1
- 239000003674 animal food additive Substances 0.000 abstract 1
- 230000007935 neutral effect Effects 0.000 abstract 1
- 238000011160 research Methods 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 17
- 235000015097 nutrients Nutrition 0.000 description 14
- 239000012153 distilled water Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000009630 liquid culture Methods 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000007747 plating Methods 0.000 description 5
- 241000186660 Lactobacillus Species 0.000 description 4
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 210000003608 fece Anatomy 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 239000010871 livestock manure Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000186000 Bifidobacterium Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000002068 microbial inoculum Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 230000000529 probiotic effect Effects 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193422 Bacillus lentus Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 1
- 241000186012 Bifidobacterium breve Species 0.000 description 1
- 241000235646 Cyberlindnera jadinii Species 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 241000186840 Lactobacillus fermentum Species 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 241000191998 Pediococcus acidilactici Species 0.000 description 1
- 241000191996 Pediococcus pentosaceus Species 0.000 description 1
- 241000190950 Rhodopseudomonas palustris Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229940012969 lactobacillus fermentum Drugs 0.000 description 1
- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 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)
- Chemical & Material Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
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- Medicinal Chemistry (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)
Abstract
The invention discloses a method for increasing biomass by virtue of mixed cultivation of enterococcus faecium and bacillus subtilis. Enterococcus faecium and bacillus subtilis are important probiotics during breeding of animals and are widely used as microbial preparations in feed additives; enterococcus faecium is a lactic acid bacterium and can produce antibacterial substances such as acid and bacteriocin in a fermentation process, and bacillus subtilis is suitable for being cultured under a neutral condition; researches show that the biomass of harvested thallus is greatly influenced by different inoculation proportions, charge coefficients, dosages of medium components and the like in the mixed cultivation of enterococcus faecium and bacillus subtilis, and a method capable of remarkably increasing the total biomass of the mixed cultivation of enterococcus faecium and bacillus subtilis is finally explored by optimizing the medium components and culture conditions. The method is applicable to liquid fermentation production of microbial preparations and has the advantages that the total biomass is finally remarkably increased by virtue of the mixed cultivation of two strains with different growing and physiological features, furthermore, the proportion of biomasses of the two strains is approximate to 1 to 1, the utilization rate of equipment is increased, and the production cost is lowered.
Description
Technical field
The present invention relates to a kind of method of faecium and genus bacillus mixed culture, particularly relate to a kind of faecium and subtilis mixed culture raising biomass method, belong to biological technical field.
Background technology
Probiotics (Probitics) has another name called probiotic bacterium, live bacteria agent, probiotics etc., is the microorganism feed addictive of living.Per os or other mucosal route drop into, and are intended to the balance improving mucomembranous surface microorganism or enzyme, or stimulate specificity or nonspecific immunity mechanism, improve efficiency of feed utilization, strengthen animal disease resistant ability and improve the effects such as production performance.In fodder industry, people are using probiotics as one of the most effective substitute products of microbiotic, place hope on the animal products by probiotics production safety, strengthen the protection to environment simultaneously.In December, 2013 Ministry of Agriculture of China No. 2045 bulletins disclose " fodder additives kind catalogue (2013) ", wherein the microorganism of adding in its feed is allowed to have Bacillus licheniformis for cultivated animals, subtilis, bifidumbacterium bifidum, enterococcus faecalis, faecium, lactoenterococcus, Lactobacterium acidophilum, lactobacterium casei, moral formula Lactobacillus lactate subspecies, plant lactobacillus, pediococcus acidilactici, Pediococcus pentosaceus, Candida utilis, yeast saccharomyces cerevisiae, Rhodopseudomonas palustris, bifidobacteria infantis, bifidus longum bb, bifidobacterium breve, bifidobacterium adolescentis, thermophilus streptococcus, lactobacillus reuteri, animal bifidobacteria, aspergillus niger, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosas, lactobacillus fermentum, lactobacillus delbruockii subspecies bulgaricus.
Probiotics can be divided into single microbial inoculum and composite fungus agent again according to the composition of bacterial strain, and the research and development of single microbial inoculum is more, and current development trend is development composite fungus agent.Composite fungus agent can adapt to multiple condition and host, more can promote the growth of livestock and poultry and improve food conversion ratio than single bacteria preparation.Be applied to probiotic bacterium mainly milk-acid bacteria, genus bacillus and the yeast in livestock and poultry breeding industry probiotics at present.Milk-acid bacteria can be fermented and be produced lactic acid and reduce intestinal pH, and can secreting bacteria element thus suppress the growth of pathogenic bacterium.Genus bacillus can produce VITAMIN, various enzyme and multiple meta-bolites, digests and assimilates and the Nutrition and Metabolism of animal plays promoter action to the degraded of feed.Containing multiple nutritional components such as very rich in protein, vitamin B group, fat, sugar, enzymes in yeast thalline, raising animal immunizing power, production performance and minimizing stress etc. in all have effect.
Milk-acid bacteria is facultative anaerobe, genus bacillus is aerobic bacteria, both growth characteristics and nutritional requirement widely different, and milk-acid bacteria can produce the organic acid with antibacterial or sterilization effect in the process of cultivating, peroxidation oxygen, di-acetyl and bacteriocin, wherein bacteriocin has stronger restraining effect to gram-positive microorganism, affect the growth of subtilis, mixed culture Fungal biodiversity is lower, therefore usually single culture is adopted in traditional industrial fermentation, then decomposite production technique, cause plant factor low thus, cycle stretch-out, production cost increases, also there is no the report of faecium and subtilis mixed culture at present.
Summary of the invention
For the deficiency of existing production technique, a kind of faecium and subtilis mixed culture is the object of the present invention is to provide to improve the method for biomass.
The inventive method concrete steps are as follows:
(1) be inoculated in MRS liquid nutrient medium by the manure enterococcin strain that inclined-plane is preserved, 37 DEG C of quiescent culture 14h, breed three generations continuously; Be inoculated in LB liquid nutrient medium by the bacillus subtilis strain that inclined-plane is preserved and activate, 37 DEG C, 150rpm shaking table cultivation 14h, breed three generations continuously.
(2) by after the ratio mixing of the faecium that activated and subtilis 1:12-1:8 by volume, by volume the ratio of per-cent 3-6% by mixed strains inoculation in the fermentation medium, at 35-42 DEG C, coefficient (i.e. fermention medium and volume of a container ratio) is 1/8-1/10, under the condition of 150-180rpm, 16-20h acquisition cultivated by shaking table, wherein fermention medium consists of peptone 13.0-16.0g/L, extractum carnis 13.0-16.0g/L, yeast powder 6.0-8.0g/L, glucose 18.0-22.0g/L, dipotassium hydrogen phosphate 2.0g/L, citric acid hydrogen diamine 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.2g/L, manganous sulfate 0.04g/L, pH 6.2-6.8.
Above-mentioned faecium (
enterococcus faecium) be Enterococcus strain, subtilis (
bacillus subtilis) be Bacillus strain.
LB liquid culture based formulas used in the activation of above-mentioned faecium and subtilis is: peptone 10.0g/L, yeast powder 5.0 g/L, sodium-chlor (NaCl) 10.0 g/L.
Above-mentioned MRS liquid culture based formulas is: peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, tween ﹣ 80 1.0mL, distilled water 1000mL.
The inventive method, the liquid fermenting being applicable to probiotics is produced, the present invention utilizes the physilogical characteristics of two kinds of bacterial strains to carry out mixed culture, final total biomass has had and has significantly improved, at least improve 5 times than during single culture, and the ratio of the two biomass is close to 1:1, gemma rate is also more satisfactory, improve plant factor, reduce production cost.
Embodiment
Below by embodiment, the present invention is described in further detail, but protection scope of the present invention is not limited to described content.
Embodiment 1: faecium and subtilis mixed culture improve the comparison of different vaccination ratio in biomass method, and concrete operations are as follows:
(1) be inoculated in MRS liquid nutrient medium by the manure enterococcin strain that inclined-plane is preserved, 37 DEG C of quiescent culture 14h, breed three generations continuously; Be inoculated in LB liquid nutrient medium by the bacillus subtilis strain that inclined-plane is preserved, 37 DEG C, 150rpm shaking table cultivation 14h, breed three generations continuously.
(2) by the faecium that fully activates and subtilis according to volume ratio 1:12, in the ratio of inoculation volume percent 3%, mixed strains is inoculated in the fermentation medium, 37 DEG C, coefficient is the fermention medium of the bottled 50ml of fermentation of 1/10(and every 500ml), 180rpm shaking table cultivates 16h results, in control group, the two ratio is 1:5, and all the other process are all identical.
MRS liquid culture based formulas is peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, tween ﹣ 80 1.0mL, distilled water 1000mL, 115 DEG C of condition sterilizing 30min.
LB liquid culture based formulas is peptone 10.0g/L, yeast powder 5.0g/L, sodium-chlor (NaCl) 10.0g/L, 121 DEG C of condition sterilizing 20min.
Fermention medium consists of peptone 14.0g, extractum carnis 14.0g, yeast powder 7.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, distilled water 1000mL, pH 6.5.
Employing dilution plating procedure detects, and learns that in test group fermented liquid, faecium and subtilis viable count reach 3.91 × 10 respectively
10cFU/mL and 2.92 × 10
10cFU/mL, the biomass of the two is close to 1:1.Control group faecium and subtilis viable count difference 2.89 × 10
10cFU/mL and 1.41 × 10
10cFU/mL, control group biomass is lower than test group, and the ratio of two kinds of Fungal biodiversity is unbalance to some extent.
Embodiment 2: faecium and subtilis mixed culture improve the comparison of different liquid amount in biomass method, and concrete operations are as follows:
(1) be inoculated in MRS liquid nutrient medium by the manure enterococcin strain that inclined-plane is preserved, 37 DEG C of quiescent culture 14h, breed three generations continuously; Be inoculated in LB liquid nutrient medium by the bacillus subtilis strain that inclined-plane is preserved, 37 DEG C, 150rpm shaking table cultivation 14h, breed three generations continuously.
(2) by the faecium that fully activates and subtilis according to volume ratio 1:12, in the ratio of inoculation volume percent 3%, mixed strains is inoculated in the fermentation medium, 37 DEG C, coefficient is the fermention medium of the bottled 50ml of fermentation of 1/10(and every 500ml), 180rpm shaking table cultivates 16h results, the liquid amount of control group is 150/500mL, and all the other process are identical.
MRS liquid culture based formulas is peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, tween ﹣ 80 1.0mL, distilled water 1000mL, 115 DEG C of condition sterilizing 30min.
LB liquid culture based formulas is peptone 10.0g/L, yeast powder 5.0g/L, sodium-chlor (NaCl) 10.0g/L, 121 DEG C of condition sterilizing 20min.
Fermention medium consists of peptone 14.0g, extractum carnis 14.0g, yeast powder 7.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, distilled water 1000mL, pH 6.5.
Employing dilution plating procedure detects, and learns that in test group fermented liquid, faecium and subtilis viable count reach 3.91 × 10 respectively
10cFU/mL and 2.92 × 10
10cFU/mL, control group faecium and subtilis viable count difference 8.98 × 10
9cFU/mL and 5.23 × 10
9cFU/mL, the biomass of control group two kinds of bacterium is all starkly lower than test group, and the ratio of two kinds of Fungal biodiversity is unbalance to some extent.
Embodiment 3: faecium and subtilis mixed culture improve the comparison of medium component different amounts in biomass method, and concrete operations are as follows
(1) be inoculated in MRS liquid nutrient medium by the manure enterococcin strain that inclined-plane is preserved, 37 DEG C of quiescent culture 14h, breed three generations continuously; Be inoculated in LB liquid nutrient medium by the bacillus subtilis strain that inclined-plane is preserved, 37 DEG C, 150rpm shaking table cultivation 16h, breed three generations continuously.
(2) by the faecium that fully activates and subtilis according to volume ratio 1:12, in inoculation volume percent 3% ratio by mixed strains inoculation in the fermentation medium, 37 DEG C, coefficient is the fermention medium of the bottled 50ml of fermentation of 1/10(and every 500ml), 180rpm shaking table cultivates 16h results.
MRS liquid culture based formulas is peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, tween ﹣ 80 1.0mL, distilled water 1000mL, 115 DEG C of condition sterilizing 30min.
LB liquid culture based formulas is peptone 10.0g/L, yeast powder 5.0g/L, sodium-chlor (NaCl) 10.0g/L, 121 DEG C of condition sterilizing 20min.
Fermention medium consists of peptone 14.0g, extractum carnis 14.0g, yeast powder 7.0g, glucose (C
6h
12o
6h
2o) 20.0g, dipotassium hydrogen phosphate (K
2hPO
43H
20) 2.0g, citric acid hydrogen diamine [(NH
4)
2hC
6h
50
7] 2.0g, sodium acetate (CH
3cOONa3H
2o) 5.0g, magnesium sulfate (MgSO
47H
2o) 0.2g, manganous sulfate (MnSO
4h
20) 0.04g, distilled water 1000mL, pH 6.5.The fermention medium consumption of control group is peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, and all the other process are identical.
Employing dilution plating procedure detects, and learns that in test group fermented liquid, faecium and subtilis viable count reach 3.91 × 10 respectively
10cFU/mL and 2.92 × 10
10cFU/mL, the biomass of the two is close to 1:1.Control group faecium and subtilis viable count difference 2.47 × 10
10cFU/mL and 8.69 × 10
9cFU/mL, in control group, bacillus subtilis bacteria biomass is starkly lower than test group, and the proportional imbalance of two kinds of Fungal biodiversity is larger.
Respectively single culture test is carried out to faecium and subtilis simultaneously, substratum is MRS liquid nutrient medium and LB liquid nutrient medium, all the other treatment condition are identical with mixed culture, after carrying out standing and shaking table cultivation 16h respectively, faecium and subtilis viable count are respectively 1.35 × 10
10cFU/mL and 5.07 × 10
9cFU/mL, compares number of viable during mixed culture, and the viable count of single culture is obviously fewer, and the total biomass 6.83 × 10 during mixed culture
10faecium biomass 1.35 × 10 when CFU/mL is single culture
105 times of CFU/mL.
Embodiment 4: faecium and subtilis mixed culture improve the method for biomass, by after the ratio mixing of the faecium that activated and subtilis 1:10 by volume, by volume the ratio of per-cent 4% by mixed strains inoculation in the fermentation medium, at 35 DEG C, coefficient is the fermention medium of the bottled 62.5ml of fermentation of 1/8(and every 500ml) condition under, 150rpm shaking table is cultivated 20h and is obtained, wherein fermention medium consists of peptone 13.0g, extractum carnis 16.0g, yeast powder 6.0g, glucose 22.0g, dipotassium hydrogen phosphate 2.0g, citric acid hydrogen diamine 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganous sulfate 0.04g, distilled water 1000mL, pH 6.2.
Employing dilution plating procedure detects, and in mixed fermentation liquid, faecium and subtilis viable count reach 2.97 × 10 respectively
10cFU/mL and 1.89 × 10
10cFU/mL.
Respectively single culture test is carried out to faecium and subtilis simultaneously, substratum is MRS liquid nutrient medium and LB liquid nutrient medium, all the other process are identical with mixed culture, and after carrying out standing and shaking table cultivation 16h respectively, faecium and subtilis viable count are respectively 9.44 × 10
9cFU/mL and 4.18 × 10
9cFU/mL, number of viable during mixed culture is obviously more than single culture.
Embodiment 5: faecium and subtilis mixed culture improve the method for biomass, by after the ratio mixing of the faecium that activated and subtilis 1:8 by volume, by volume the ratio of per-cent 6% by mixed strains inoculation in the fermentation medium, at 42 DEG C, coefficient is the fermention medium of the bottled 55.6ml of fermentation of 1/9(and every 500ml) condition under 170rpm shaking table cultivate 17h obtain, wherein fermention medium consists of peptone 16.0g, extractum carnis 13.0g, yeast powder 8.0g, glucose 18.0g, dipotassium hydrogen phosphate 2.0g, citric acid hydrogen diamine 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganous sulfate 0.04g, distilled water 1000mL, pH 6.8.
Employing dilution plating procedure detects, and in mixed fermentation liquid, faecium and subtilis viable count reach 3.82 × 10 respectively
10cFU/mL and 2.66 × 10
10cFU/mL, the biomass of the two is close to 1:1.
Respectively single culture test is carried out to faecium and subtilis simultaneously, substratum is MRS liquid nutrient medium and LB liquid nutrient medium, all the other process are identical with mixed culture, and after carrying out standing and shaking table cultivation 16h respectively, faecium and subtilis viable count are respectively 1.07 × 10
10cFU/mL and 4.96 × 10
9cFU/mL, compares number of viable during mixed culture, and viable count when faecium and subtilis single culture has obvious gap.
Claims (1)
1. a faecium and subtilis mixed culture improve the method for biomass, it is characterized in that: after the ratio mixing of the faecium that activated and subtilis 1:12-1:8 by volume, by volume the ratio of per-cent 3-6% by mixed strains inoculation in the fermentation medium, at 35-42 DEG C, coefficient is 1/8-1/10, under the condition of 150-180rpm, 16-20h acquisition cultivated by shaking table, wherein fermention medium consists of peptone 13.0-16.0g/L, extractum carnis 13.0-16.0g/L, yeast powder 6.0-8.0g/L, glucose 18.0-22.0g/L, dipotassium hydrogen phosphate 2.0g/L, citric acid hydrogen diamine 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.2g/L, manganous sulfate 0.04g/L, pH 6.2-6.8.
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