CN104371958B - 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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- CN104371958B CN104371958B CN201410653601.2A CN201410653601A CN104371958B CN 104371958 B CN104371958 B CN 104371958B CN 201410653601 A CN201410653601 A CN 201410653601A CN 104371958 B CN104371958 B CN 104371958B
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- 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
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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 enterococcus faecalis and the method for bacillus cereus mixed culture, particularly relate to a kind of enterococcus faecalis and bacillus subtilis mixed culture improves biomass method, belong to biological technical field.
Background technology
Probiotics (Probitics) has another name called probiotic bacteria, live bacteria agent, microbial ecological agent etc., is the additive for microbe feedstuff lived.Per os or other mucosal route put into, it is intended to improve the balance of 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 feed industry, people are using microbial ecological agent as one of maximally effective substitute products of antibiotic, place hope on the animal products by microbial ecological agent production safety, strengthen the protection to environment simultaneously.No. 2045 bulletins of the Ministry of Agriculture of in December, 2013 China disclose " feed additive kind catalogue (2013) ", wherein the microorganism added in its feedstuff is allowed to have Bacillus licheniformis for cultivated animals, bacillus subtilis, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecalis, lactoenterococcus, bacillus acidophilus, lactobacillus casei, moral formula Lactobacillus lactate subspecies, Lactobacillus plantarum, pediococcus acidilactici, Pediococcus pentosaceus, Candida utilis, saccharomyces cerevisiae, Rhodopseudomonas palustris, bifidobacteria infantis, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, Lactobacillus reuteri, animal bifidobacteria, aspergillus niger, aspergillus oryzae, bacillus lentus, Bacillus pumilus, lactobacillus cellobiosas, Lactobacillus fermenti, lactobacillus delbruockii subspecies bulgaricus.
Microbial ecological agent can be divided into again single microbial inoculum and composite bacteria agent capable according to the composition of bacterial strain, and the research and development of single microbial inoculum is more, and current development trend is to develop composite bacteria agent capable.Composite bacteria agent capable adapts to multiple condition and host, more can promote the growth of poultry than single bacteria preparation and improve food conversion ratio.The probiotic bacteria being applied in livestock and poultry breeding industry microbial ecological agent at present is mainly lactic acid bacteria, bacillus cereus and yeast.Lactic acid bacteria can ferment generation lactic acid reduce intestinal pH, and can secreting bacteria element thus suppress the growth of pathogenic bacterium.Bacillus cereus can produce vitamin, various enzyme and multiple metabolite, and the degraded to feedstuff is digested and assimilated and the Nutrition and Metabolism of animal plays facilitation.Containing multiple nutritional components such as very rich in protein, vitamin B group, fat, sugar, enzymes in yeast thalline, improve animal immunizing power, production performance and minimizing stress etc. aspect all have effect.
Lactic acid bacteria is facultative anaerobe, bacillus cereus is aerobic bacteria, both growth characteristics and nutritional requirement are widely different, and lactic acid bacteria can produce the organic acid with antibacterial or bactericidal effect during cultivating, peroxidating oxygen, biacetyl and bacteriocin, wherein bacteriocin has stronger inhibitory action to gram positive bacteria, affect the growth of bacillus subtilis, mixed culture Fungal biodiversity is relatively low, the most traditional industrial fermentation generally uses single culture, the most decomposite production technology, thereby result in utilization rate of equipment and installations low, cycle stretch-out, production cost increases, there is presently no enterococcus faecalis and the report of bacillus subtilis mixed culture.
Summary of the invention
For the deficiency of existing production technology, it is an object of the invention to provide a kind of enterococcus faecalis and the method for bacillus subtilis mixed culture raising Biomass.
The inventive method specifically comprises the following steps that
(1) manure enterococcin strain that inclined-plane preserves is inoculated in MRS fluid medium, 37 DEG C of quiescent culture 14h, breeds three generations continuously；The bacillus subtilis strain that inclined-plane preserves is inoculated in LB fluid medium activation, 37 DEG C, 150rpm shaking table cultivation 14h, breeds three generations continuously.
(2) after the ratio of the enterococcus faecalis activated and bacillus subtilis 1:12-1:8 by volume being mixed, by volume mixed bacteria is inoculated in the fermentation medium by the ratio of percentage ratio 3-6%, 35-42 DEG C, coefficient (i.e. fermentation medium and volume of a container than) be 1/8-1/10
, shaking table is cultivated 16-20h and is obtained under conditions of 150-180rpm, wherein fermentation medium consists of peptone 13.0-16.0g/L, Carnis Bovis seu Bubali cream 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, manganese sulfate 0.04g/L
pH 6.2-6.8。
Above-mentioned enterococcus faecalis (Enterococcus faecium) it is Enterococcus strain, bacillus subtilis (Bacillus subtilis) it is Bacillus strain.
LB liquid culture based formulas used in the activation of above-mentioned enterococcus faecalis and bacillus subtilis is: peptone 10.0g/L, yeast powder 5.0
G/L, sodium chloride (NaCl) 10.0
g/L。
Above-mentioned MRS liquid culture based formulas is: peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast powder 5.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 0.04g, Tween 80 1.0mL, distilled water 1000mL.
The inventive method, the liquid fermentation being applicable to microbial ecological agent produces, the present invention utilizes the physiological characteristics of two kinds of bacterial strains to be mixed, final total biomass is obviously improved, at least improve 5 times during than single culture, and the ratio of the two Biomass is close to 1:1, spore rate is the most more satisfactory, improve utilization rate of equipment and installations, reduce production cost.
Detailed description of the invention
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: enterococcus faecalis and bacillus subtilis mixed culture improve the comparison of different vaccination ratio in biomass method, and concrete operations are as follows:
(1) manure enterococcin strain that inclined-plane preserves is inoculated in MRS fluid medium, 37 DEG C of quiescent culture 14h, breeds three generations continuously；The bacillus subtilis strain that inclined-plane preserves is inoculated in LB fluid medium, 37 DEG C, 150rpm shaking table cultivation 14h, breeds three generations continuously.
(2) by the enterococcus faecalis fully activated and bacillus subtilis according to volume ratio 1:12, in the ratio of inoculation percent by volume 3%, mixed bacteria is inoculated in the fermentation medium, 37 DEG C, coefficient be the fermentation medium of the bottled 50ml of fermentation of the most every 500ml of 1/10(), 180rpm shaking table cultivate 16h results, in matched group, the two ratio is 1:5, and remaining process is the most identical.
MRS liquid culture based formulas is peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast powder 5.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 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 chloride (NaCl) 10.0g/L, 121 DEG C of condition sterilizing 20min.
Fermentation medium consists of peptone 14.0g, Carnis Bovis seu Bubali cream 14.0g, yeast powder 7.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 0.04g, distilled water 1000mL, pH 6.5.
Employing dilution plating procedure detects, and learns that in test group fermentation liquid, enterococcus faecalis and bacillus subtilis viable count respectively reach 3.91 × 1010CFU/mL and 2.92 × 1010CFU/mL, the Biomass of the two is close to 1:1.Matched group enterococcus faecalis and bacillus subtilis viable count difference 2.89 × 1010CFU/mL and 1.41 × 1010CFU/mL, matched group Biomass is less than test group, and the ratio of two kinds of Fungal biodiversity is the most unbalance.
Embodiment 2: enterococcus faecalis and bacillus subtilis mixed culture improve the comparison of different liquid amounts in biomass method, and concrete operations are as follows:
(1) manure enterococcin strain that inclined-plane preserves is inoculated in MRS fluid medium, 37 DEG C of quiescent culture 14h, breeds three generations continuously；The bacillus subtilis strain that inclined-plane preserves is inoculated in LB fluid medium, 37 DEG C, 150rpm shaking table cultivation 14h, breeds three generations continuously.
(2) by the enterococcus faecalis fully activated and bacillus subtilis according to volume ratio 1:12, in the ratio of inoculation percent by volume 3%, mixed bacteria is inoculated in the fermentation medium, 37 DEG C, coefficient be the fermentation medium of the bottled 50ml of fermentation of the most every 500ml of 1/10(), 180rpm shaking table cultivate 16h results, the liquid amount of matched group is 150/500mL, and remaining process is identical.
MRS liquid culture based formulas is peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast powder 5.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 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 chloride (NaCl) 10.0g/L, 121 DEG C of condition sterilizing 20min.
Fermentation medium consists of peptone 14.0g, Carnis Bovis seu Bubali cream 14.0g, yeast powder 7.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 0.04g, distilled water 1000mL, pH 6.5.
Employing dilution plating procedure detects, and learns that in test group fermentation liquid, enterococcus faecalis and bacillus subtilis viable count respectively reach 3.91 × 1010CFU/mL and 2.92 × 1010CFU/mL, matched group enterococcus faecalis and bacillus subtilis viable count difference 8.98 × 109CFU/mL and 5.23 × 109CFU/mL, the Biomass of two kinds of bacterium of matched group is all significantly lower than test group, and the ratio of two kinds of Fungal biodiversity is the most unbalance.
Embodiment 3: enterococcus faecalis and bacillus subtilis mixed culture improve the comparison of medium component different amounts in biomass method, and concrete operations are as follows
(1) manure enterococcin strain that inclined-plane preserves is inoculated in MRS fluid medium, 37 DEG C of quiescent culture 14h, breeds three generations continuously；The bacillus subtilis strain that inclined-plane preserves is inoculated in LB fluid medium, 37 DEG C, 150rpm shaking table cultivation 16h, breeds three generations continuously.
(2) by the enterococcus faecalis fully activated and bacillus subtilis according to volume ratio 1:12, in the ratio of inoculation percent by volume 3%, mixed bacteria is inoculated in the fermentation medium, 37 DEG C, coefficient be the fermentation medium of the bottled 50ml of fermentation of the most every 500ml of 1/10(), 180rpm shaking table cultivates 16h and gathers in the crops.
MRS liquid culture based formulas is peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast powder 5.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 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 chloride (NaCl) 10.0g/L, 121 DEG C of condition sterilizing 20min.
Fermentation medium consists of peptone 14.0g, Carnis Bovis seu Bubali cream 14.0g, yeast powder 7.0g, glucose (C6H12O6·H2O) 20.0g, dipotassium hydrogen phosphate (K2HPO4·3H20) 2.0g, citric acid hydrogen diamine [(NH4)2HC6H507] 2.0g, sodium acetate (CH3COONa·3H2O) 5.0g, magnesium sulfate (MgSO4·7H2O) 0.2g, manganese sulfate (MnSO4·H20) 0.04g, distilled water 1000mL, pH 6.5.The fermentation medium consumption of matched group is peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast powder 5.0g, and remaining process is identical.
Employing dilution plating procedure detects, and learns that in test group fermentation liquid, enterococcus faecalis and bacillus subtilis viable count respectively reach 3.91 × 1010CFU/mL and 2.92 × 1010CFU/mL, the Biomass of the two is close to 1:1.Matched group enterococcus faecalis and bacillus subtilis viable count difference 2.47 × 1010CFU/mL and 8.69 × 109CFU/mL, in matched group, bacillus subtilis bacteria biomass is significantly lower than test group, and the proportional imbalance of two kinds of Fungal biodiversity is bigger.
Enterococcus faecalis and bacillus subtilis are carried out single culture test respectively simultaneously, culture medium is MRS fluid medium and LB fluid medium, remaining treatment conditions is identical with mixed culture, after carrying out standing and shaking table cultivation 16h respectively, enterococcus faecalis and bacillus subtilis viable count are respectively 1.35 × 1010CFU/mL and 5.07 × 109CFU/mL, compares number of viable during mixed culture, and the viable count of single culture is the most fewer, and the total biomass 6.83 × 10 during mixed culture10Enterococcus faecalis Biomass 1.35 × 10 when CFU/mL is single culture105 times of CFU/mL.
Embodiment 4: the method that enterococcus faecalis and bacillus subtilis mixed culture improve Biomass, it is by after the ratio mixing of the enterococcus faecalis activated and bacillus subtilis 1:10 by volume, by volume mixed bacteria is inoculated in the fermentation medium by the ratio of percentage ratio 4%, at 35 DEG C, coefficient is the fermentation medium of the bottled 62.5ml of fermentation of the most every 500ml of 1/8() under conditions of, 150rpm shaking table is cultivated 20h and is obtained, wherein fermentation medium consists of peptone 13.0g, Carnis Bovis seu Bubali cream 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, manganese sulfate 0.04g, distilled water 1000mL
, pH 6.2.
Employing dilution plating procedure detects, and in mixed fermentation liquid, enterococcus faecalis and bacillus subtilis viable count respectively reach 2.97 × 1010CFU/mL and 1.89 × 1010CFU/mL。
Enterococcus faecalis and bacillus subtilis are carried out single culture test respectively simultaneously, culture medium is MRS fluid medium and LB fluid medium, remaining processes and is mixed identical, and after carrying out standing and shaking table cultivation 16h respectively, enterococcus faecalis and bacillus subtilis viable count are respectively 9.44 × 109CFU/mL and 4.18 × 109CFU/mL, number of viable during mixed culture is significantly more than single culture.
Embodiment 5: the method that enterococcus faecalis and bacillus subtilis mixed culture improve Biomass, it is by after the ratio mixing of the enterococcus faecalis activated and bacillus subtilis 1:8 by volume, by volume mixed bacteria is inoculated in the fermentation medium by the ratio of percentage ratio 6%, at 42 DEG C, coefficient is the fermentation medium of the bottled 55.6ml of fermentation of the most every 500ml of 1/9() under conditions of 170rpm shaking table cultivate 17h obtain, wherein fermentation medium consists of peptone 16.0g, Carnis Bovis seu Bubali cream 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, manganese sulfate 0.04g, distilled water 1000mL
, pH 6.8.
Employing dilution plating procedure detects, and in mixed fermentation liquid, enterococcus faecalis and bacillus subtilis viable count respectively reach 3.82 × 1010CFU/mL and 2.66 × 1010CFU/mL, the Biomass of the two is close to 1:1.
Enterococcus faecalis and bacillus subtilis are carried out single culture test respectively simultaneously, culture medium is MRS fluid medium and LB fluid medium, remaining processes and is mixed identical, and after carrying out standing and shaking table cultivation 16h respectively, enterococcus faecalis and bacillus subtilis viable count are respectively 1.07 × 1010CFU/mL and 4.96 × 109CFU/mL, viable count when comparing number of viable during mixed culture, enterococcus faecalis and bacillus subtilis single culture has obvious gap.
Claims (1)
1. an enterococcus faecalis and bacillus subtilis mixed culture improve Biomass method, it is characterised in that: by activated enterococcus faecalis (Enterococcus faecium) and bacillus subtilis (Bacillus subtilis) by volume 1:12-1:8 ratio mixing after, by volume mixed bacteria is inoculated in the fermentation medium by the ratio of percentage ratio 3-6%, 35-42 DEG C, coefficient be 1/8-1/10,150-180rpm under conditions of shaking table cultivate 16-20h obtain, wherein fermentation medium consists of peptone 13.0-16.0g/L, Carnis Bovis seu Bubali cream 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, manganese sulfate 0.04g/L, pH 6.2-6.8.
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| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
| CN104996740B (en) * | 2015-08-25 | 2018-05-25 | 林州中农颖泰生物肽有限公司 | A kind of method that expansion drying prepares feed compound microecological powder |
| CN105296405A (en) * | 2015-12-02 | 2016-02-03 | 江苏绿科生物技术有限公司 | Compound microorganism liquid feed additive and preparation method thereof |
| CN105341425A (en) * | 2015-12-10 | 2016-02-24 | 北京科为博生物科技有限公司 | Pig feed containing bacteriostatic microbial ecological agent and application thereof |
| CN109943502B (en) * | 2019-03-04 | 2022-02-01 | 青岛海洋生物医药研究院 | Probiotics, leavening agent and application in Chinese herbal medicine preparation |
| CN111808764A (en) * | 2019-04-11 | 2020-10-23 | 河北维尔利动物药业集团有限公司 | Preparation method of bacillus subtilis composite liquid microbial inoculum for preventing and treating epidemic diseases of livestock and poultry |
| CN112175835B (en) * | 2019-07-05 | 2022-09-06 | 中粮生物科技股份有限公司 | Application of lactobacillus plantarum in storage of bacillus subtilis fermentation liquor |
| CN110628651A (en) * | 2019-10-21 | 2019-12-31 | 昆明理工大学 | Liquid submerged fermentation method for improving activity of paecilomyces lilacinus spores |
| CN113717899A (en) * | 2021-10-09 | 2021-11-30 | 广州金水动物保健品有限公司 | Preparation method of feeding enterococcus faecium raw powder |
| CN114908026A (en) * | 2022-06-30 | 2022-08-16 | 哈尔滨韶创生物科技有限公司 | Special liquid composite microecological preparation for rumination and preparation method thereof |
| CN116478882A (en) * | 2023-05-05 | 2023-07-25 | 石河子大学 | Preparation method of acid-producing mixed bacteria preparation capable of improving sheep daily gain and feed conversion rate |
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