CN105087420A - High-density fermentation medium and fermentation technology for forage-use enterococcus faecium - Google Patents

High-density fermentation medium and fermentation technology for forage-use enterococcus faecium Download PDF

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CN105087420A
CN105087420A CN201510145118.8A CN201510145118A CN105087420A CN 105087420 A CN105087420 A CN 105087420A CN 201510145118 A CN201510145118 A CN 201510145118A CN 105087420 A CN105087420 A CN 105087420A
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seed
fermentation
fermentor tank
liquid
medium
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乔琳
姚宏明
高长斌
刘蕊
金忠辉
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Beijing Vica Shengbang Biological Technology Co Ltd
BEIJING VICAREN BIOLOGICAL TECHNOLOGY Co Ltd
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Beijing Vica Shengbang Biological Technology Co Ltd
BEIJING VICAREN BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The invention relates to a high-density fermentation medium and a fermentation technology for forage-use enterococcus faecium. By adopting the high-density fermentation medium and an in-batch fed-batch fermentation technology provided by the invention, the highest viable count of enterococcus faecium WEI-10CGMCC No.7746 under a 50L fermentation tank scale condition can reach 2.5X10<10> CFU/mL, and the highest viable count of enterococcus faecium WEI-10CGMCC No.7746 under a 5-ton fermentation tank scale condition can reach 2.9X10<10> CFU/mL, which are respectively 34 times and 36 times of those of in-batch fermentation of the traditional MES medium under the same fermentation scale condition, while the cost of the high-density fermentation medium is obviously reduced compared with that of the MRS medium, and thus the low-cost and high-efficiency production of enterococcus faecium as the microecological preparation is realized.

Description

The high density fermentation culture medium of the feeding faecium of one strain and zymotechnique thereof
Technical field
The invention belongs to milk-acid bacteria culture technique field, relate to high density fermentation culture medium and the zymotechnique thereof of the feeding faecium of a strain.
Background technology
Faecium (EnterococcusFaecium) belongs to the normal microflora in people and animal intestinal, can effectively surely grow after entering enteron aisle.Faecium fermentation produces lactic acid, is conducive to reducing intestinal environment pH, suppresses the growth of harmful bacteria.Also can produce hydrogen peroxide in faecium metabolic process, the materials such as bacteriocin, have certain killing action to pathogenic bacterium, and it is residual that medicine does not occur.In view of the above characteristic of faecium, increasing producer selects it to carry out the fermentative production of probiotics.
Traditional lactobacillus-fermented multiselect MRS substratum, expensive, the viable count after fermentation is lower than 1 × 10 9cFU/mL, when the effective using dosage of probiotic bacterium is fixing, low fermentation level equals covert and adds cost.The substratum of low cost, the zymotechnique that can amplify efficiently has great guidance and practice significance for advancing the industrialization of probiotics fermention.
Summary of the invention
(1) goal of the invention:
First object of the present invention is to provide the low-cost high-density fermention medium of a kind of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746; Second object is to provide the high density liquid zymotechnique of a kind of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746.
(2) technical scheme:
Object of the present invention is obtained by following scheme and realizes:
1. the invention provides seed culture medium and the fermention medium of the high density fermentation of a kind of feeding faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746, its main component and content as follows:
Fish peptone 15-20g/L, sucrose 15-25g/L, sodium acetate trihydrate 3-7g/L, three hypophosphite monohydrate hydrogen dipotassium 1-4g/L, ammonium citrate 1-4g/L, bitter salt 0.05-0.4g/L, Manganous sulfate monohydrate 0.02-0.2g/L, tween 80 0.5-2g/L.
2. seed culture medium compound method provided by the invention is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
3. the compound method of fermention medium provided by the invention is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 50-70%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with tap water fermentor tank 2-4 rear emptying fermentor tank.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add the 50-70% of tap water to fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.11-0.16Mpa sterilizing 20-40 minute.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 37-42 DEG C, 50rpm, and under stuffiness condition, sky is trained to inoculation.
4. the cultural method of one-level shake-flask seed liquid provided by the invention:
(inclined-plane is MRS nutrient agar on faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, composition is with reference to GB478935-2010, lower same), be seeded in seed flask substratum, 37-42 DEG C of quiescent culture 10h is as one-level shake-flask seed liquid.
5. the high cell density fermentation under 50L fermentor tank scale condition provided by the invention is as follows:
(1) before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee without living contaminants.
(2) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after regulating the pH to 7.5-8.5 of fermention medium with 40% sodium hydroxide solution of sterilizing, according to the inoculum size of 0.5-4%, one-level shake-flask seed liquid is accessed in fermentor tank.
(3) ferment.Fermentation parameter is set as: temperature 37-42 DEG C; Rotating speed 100rpm; Stuffiness; Fermented liquid pH lower limit set is 6.4-6.8, and when pH is down to below set(ting)value, automatic makeup ammoniacal liquor makes pH maintain set(ting)value; Fermentation starts 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume; When the pH of fermented liquid no longer declines, second time mends the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume; The fermentation ends time is set as 12-18h.
6. the high cell density fermentation under 5 tons of fermentor tank scale conditions provided by the invention is as follows:
(1) cultivation of one-level shake-flask seed liquid is first carried out.
(2) in 100L fermentor tank, prepare secondary seed medium, before cultivating, microscopy is carried out to secondary seed medium and primary seed solution, guarantee without living contaminants.Before inoculation, 100L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting secondary seed medium pH to 7.5-8.5 with 40% sodium hydroxide solution of sterilizing, access in 100L fermentor tank according to the inoculum size of 0.5-2% by one-level shake-flask seed liquid, fermentation 10h terminates as secondary seed solution.
(3), before carrying out 5 tons of ferment tanks, microscopy is carried out to fermention medium and secondary seed solution, guarantees without living contaminants.Before inoculation, 5 tons of fermentor tank rotating speeds are adjusted to 100rpm, and after adjusting pH to 7.5-8.5 with 40% sodium hydroxide solution of sterilizing, secondary seed solution are all forwarded in 5 tons of fermentor tanks and ferment.Fermentation parameter is set as: temperature 37-42 DEG C; Rotating speed 100rpm; Stuffiness; Fermented liquid pH lower limit set is 6.4-6.8, and when pH is down to below set(ting)value, automatic makeup ammoniacal liquor makes pH maintain set(ting)value; Fermentation starts 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume; When the pH of fermented liquid no longer declines, second time mends the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume; The fermentation ends time is set as 12-18h.
(3) meaning:
Present invention optimizes seed culture medium and the fermention medium of the high density fermentation of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746, control fermented liquid pH by Feeding ammonia water in fermenting process and add the strategy of sucrose in batches, achieve the high density fermentation of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746, under 50L fermentor tank scale condition, viable count reaches 2.5 × 10 10cFU/mL, under 5 tons of fermentor tank scale conditions, viable count reaches 2.9 × 10 10cFU/mL, more than 34 times and 36 times of traditional MRS substratum batch fermentation viable count under identical fermentation-scale condition respectively, viable count is the highest in the faecium high density fermentation reported, culture medium cost reduces 23.17% than MRS substratum, achieves as the low cost of probiotics faecium, high efficiency production.
Accompanying drawing explanation
Fig. 1 is faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 comparison of Fermentative growth curve in high density fermentation culture medium and traditional MRS substratum respectively.
Fig. 2 is the Fermentative growth curve of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 under different alkaline pH neutralizing agent stream adds condition.
Fig. 3 is the Fermentative growth curve of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 under different controlled pH conditions.
Fig. 4 is the high density fermentation growth curve of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 under 5 tons of fermentor tank scale conditions.
Embodiment
Following specific embodiment can illustrate the present invention better, but range of application of the present invention is not limited in following examples.
Bacterial strain selected by the present invention is that our unit is separated and obtains from sodium selenite enteron aisle, through 16SrRNA and biochemical identification, is defined as faecium, called after WEI-10.And in Song China Microbial Culture Preservation Commission common micro-organisms center preservation June 19 in 2013, be numbered CGMCCNo.7746, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of Chinese Academy of Sciences postcode: 100101 phones: 86-10-64807596.
Embodiment one: the preparation of high-density seed culture medium and fermention medium and the comparison of fermentation viable count
1. the high density fermentation seed culture medium of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 and the main component of fermention medium and content as follows:
Fish peptone 15-20g/L, sucrose 15-25g/L, sodium acetate trihydrate 3-7g/L, three hypophosphite monohydrate hydrogen dipotassium 1-4g/L, ammonium citrate 1-4g/L, bitter salt 0.05-0.4g/L, Manganous sulfate monohydrate 0.02-0.2g/L, tween 80 0.5-2g/L.
The composition of preferred seed and fermention medium and consisting of: fish peptone 17g/L, sucrose 19g/L, sodium acetate trihydrate 5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, Manganous sulfate monohydrate 0.05g/L, tween 80 1g/L.
2. seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
3. fermentation tank culture medium compound method is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add tap water to 60% of fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.14Mpa sterilizing 40 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
The fermentation of 4.50L fermentor tank:
(1) the WEI-10 inclined-plane of picking fresh culture, is seeded in seed flask substratum, and 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.
(2) before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee pollution-free.
(3) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting pH to 8.0 with 40% sodium hydroxide solution of sterilizing, the inoculum size according to 2% is by one-level shake-flask seed liquid access fermentor tank.
(4) fermentation parameter setting: leavening temperature 40 DEG C, rotating speed 100rpm, stuffiness.Fermentation is stopped after fermentation 16h.
5. controlled trial:
(1) main component of traditional MRS substratum and content are:
Peptone 10g/L, beef powder 5g/L, yeast powder 4g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, seven hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, four anhydrous manganese 0.05g/L, tween 80 1mL/L.
(2) seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, adjust pH to 6.2 with Glacial acetic acid, be settled to volume required, 121 DEG C of sterilizings 15 minutes.
(3) fermentation tank culture medium compound method is as follows:
Load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.Take each component of substratum, add appropriate tap water and be stirred to abundant dissolving, add tap water to 60% of fermentor tank cumulative volume.40 DEG C, 100rpm is incubated 1h, after adjusting pH to 6.2 with Glacial acetic acid, and 0.12Mpa sterilizing 30 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.Substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
(4) fermentation of 50L fermentor tank:
Faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.Before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee pollution-free.Before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting pH to 6.2 with 40% sodium hydroxide solution of sterilizing, the inoculum size according to 2% is by one-level shake-flask seed liquid access fermentor tank.Leavening temperature 40 DEG C, rotating speed 100rpm, stuffiness, stops fermentation after fermentation 16h.
6. interpretation of result: the viable count of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 inclined-plane in the high-density seed optimized and fermention medium after 50L ferment tank reaches 5.4 × 10 9cFU/mL, and the viable count in traditional MRS substratum after 50L ferment tank is only 7.2 × 10 8cFU/mL, the former is 7.5 times of the latter, from culture medium cost, the former is than latter reducing 23.17%, therefore, no matter high-density seed culture medium provided by the invention and fermention medium are all better than traditional MRS substratum from viable bacteria output or production cost, tentatively achieve the high density fermentation of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746.On this basis, optimize the alkali of the benefit in batches supplying technics of this bacterium, the fermentation density of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 is improved further.
Embodiment two: the optimization of the suitableeest alkaline pH neutralizing agent
1. the composition of seed and fermention medium and preparation:
The main component of seed and fermention medium and content are: fish peptone 17g/L, sucrose 19g/L, sodium acetate trihydrate 5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, Manganous sulfate monohydrate 0.05g/L, tween 80 1g/L.
Seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
Fermentation tank culture medium compound method is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add tap water to 60% of fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.14Mpa sterilizing 40 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
2. the optimization of the suitableeest alkaline pH neutralizing agent:
(1) faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.
(2) before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee pollution-free.
(3) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting pH to 8.0 with 40% sodium hydroxide solution of sterilizing, the inoculum size according to 2% is by one-level shake-flask seed liquid access fermentor tank.
(4) fermentation parameter setting: leavening temperature 40 DEG C; Rotating speed 100rpm; Stuffiness; Fermented liquid pH lower limit set is 6.5.Select the NaOH of 40%, the KOH of 40% and ammoniacal liquor as alkaline pH neutralizing agent respectively, when pH is down to below set(ting)value, utilize the automatic makeup alkali function of fermentation system to carry out automatic control flow check to add, make pH maintain set(ting)value, not carry out batch fermentation that neutralizing agent stream adds in contrast.Fermentation is stopped after fermentation 16h.The parallel test of 3 batches is done under often kind of alkaline pH neutralizing agent condition.
3. interpretation of result: no matter adopt which kind of neutralizing agent, its fermentation viable count is all greater than not stream and adds the viable count of alkaline pH neutralizing agent.NaOH using the 40% and KOH of 40% as neutralizing agent, fermentation viable count no significant difference, but when flowing the added-time with 40%NaOH, maximum viable count is 7.7 × 10 9cFU/mL; Flow the added-time with 40%KOH, maximum viable count is 7.2 × 10 9cFU/mL, the former is a little more than the latter; And using ammoniacal liquor as neutralizing agent, fermentation viable count is apparently higher than the above two, and when fermentation 12h there is further rising in viable count, and maximum viable count reaches 1.1 × 10 10cFU/mL.Therefore, determine that ammoniacal liquor is as the suitableeest alkaline pH neutralizing agent of the present invention.
Embodiment three: the optimization of the suitableeest constant pH
1. the composition of seed and fermention medium and preparation:
The main component of seed and fermention medium and content are: fish peptone 17g/L, sucrose 19g/L, sodium acetate trihydrate 5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, Manganous sulfate monohydrate 0.05g/L, tween 80 1g/L.
Seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
Fermentation tank culture medium compound method is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add tap water to 60% of fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.14Mpa sterilizing 40 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
2. the optimization of the suitableeest constant pH
(1) faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.
(2) before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee pollution-free.
(3) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting pH to 8.0 with 40% sodium hydroxide solution of sterilizing, the inoculum size according to 2% is by one-level shake-flask seed liquid access fermentor tank.
(4) fermentation parameter setting: leavening temperature 40 DEG C; Rotating speed 100rpm; Stuffiness; Take ammoniacal liquor as neutralizing agent, the constant pH that selected stream adds process maintenance is respectively 6.0,6.5 and 7.0, when pH is down to below set(ting)value, utilizes the automatic makeup alkali function of fermentation system to carry out automatic control flow check and adds, make pH maintain set(ting)value, after fermentation 16h, stop fermentation.The parallel test of 3 batches is done under often kind of controlled pH conditions.
3. interpretation of result: constant pH be the condition bottom fermentation viable count of 6.5 apparently higher than pH6.0 and pH7.0, reach 1.1 × 10 10cFU/mL.Therefore, in order to realize the high density fermentation of bacterial strain WEI-10, the suitableeest constant pH maintained time stream adds pH neutralizing agent is 6.5.
Embodiment four: fed-batch fermentation is tested
Mend in the fermenting process of alkali, determine the total sugar content in fermented liquid, result shows in batches, and after fermentation starts 6h, the pH of fermented liquid no longer reduces, and sugar is almost totally consumed.Can infer thus, during fermentation 6h, the approach exhaustion of the carbon source in substratum, thalline cannot continued growth, and fermented liquid pH no longer reduces.Think thus, before culture medium carbon source exhausts, add carbon source, thalline further growth may be promoted, play the object improving viable count, so carried out fed-batch fermentation experiment, specific as follows:
1. the composition of seed and fermention medium and preparation:
The main component of seed and fermention medium and content are: fish peptone 17g/L, sucrose 19g/L, sodium acetate trihydrate 5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, Manganous sulfate monohydrate 0.05g/L, tween 80 1g/L.
Seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
Fermentation tank culture medium compound method is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add tap water to 60% of fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.14Mpa sterilizing 40 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
2. fed-batch fermentation
(1) faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.
(2) before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee pollution-free.
(3) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting pH to 8.0 with 40% sodium hydroxide solution of sterilizing, the inoculum size according to 2% is by one-level shake-flask seed liquid access fermentor tank.
(4) fermentation parameter setting: leavening temperature 40 DEG C; Rotating speed 100rpm; Stuffiness; Take ammoniacal liquor as neutralizing agent, the constant pH that selected stream adds process maintenance is 6.5, when pH is down to below set(ting)value, utilizes the automatic makeup alkali function of fermentation system to carry out automatic control flow check and adds, make pH maintain set(ting)value.Fermentation starts 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume, and now fermented liquid pH continues to decline, and ammoniacal liquor continues stream and adds; When the pH of fermented liquid no longer declines, second time mends the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume, and fermented liquid pH declines again, and ammoniacal liquor continues stream and adds, until pH no longer declines; Fermentation is stopped after fermentation 16h.
3, interpretation of result: by stream with sucrose concentrated solution is the further growth supplementary carbon source of faecium WEI-10CGMCCNo.7746, and the final maximum viable count obtained is 2.5 × 10 10cFU/mL, compared with the alkali of the benefit in batches technique not adding sucrose, viable count has had further raising.
Embodiment five: the high-density cultivation method under the 50L fermentor tank scale condition of faecium WEI-10CGMCCNo.7746
1. the composition of seed and fermention medium and preparation:
The main component of seed and fermention medium and content are: fish peptone 17g/L, sucrose 19g/L, sodium acetate trihydrate 5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, Manganous sulfate monohydrate 0.05g/L, tween 80 1g/L.
Seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
Fermentation tank culture medium compound method is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add tap water to 60% of fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.14Mpa sterilizing 40 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
High density fermentation under 2.50L fermentor tank scale condition
(1) faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.
(2) before fermentation, microscopy is carried out to fermention medium and one-level shake-flask seed liquid, guarantee pollution-free.
(3) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting pH to 8.0 with 40% sodium hydroxide solution of sterilizing, the inoculum size according to 2% is by one-level shake-flask seed liquid access fermentor tank.
(4) fermentation parameter setting: leavening temperature 40 DEG C; Rotating speed 100rpm; Stuffiness; Take ammoniacal liquor as neutralizing agent, the constant pH that selected stream adds process maintenance is 6.5, when pH is down to below set(ting)value, utilizes the automatic makeup alkali function of fermentation system to carry out automatic control flow check and adds, make pH maintain set(ting)value.Fermentation starts 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume, and now fermented liquid pH continues to decline, and ammoniacal liquor continues stream and adds; When the pH of fermented liquid no longer declines, second time mends the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume, and fermented liquid pH declines again, and ammoniacal liquor continues stream and adds, until pH no longer declines; Fermentation is stopped after fermentation 16h.
3. interpretation of result: under 50L fermentor tank scale condition, the viable count of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 high density fermentation reaches 2.5 × 10 10cFU/mL, be more than 34 times of traditional MRS substratum batch fermentation viable count, viable count is the highest in the faecium high density fermentation reported.
Embodiment six: the high-density cultivation method under 5 tons of fermentor tank scale conditions of faecium WEI-10CGMCCNo.7746
1. the composition of seed and fermention medium and preparation:
The main component of seed and fermention medium and content are: fish peptone 17g/L, sucrose 19g/L, sodium acetate trihydrate 5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, Manganous sulfate monohydrate 0.05g/L, tween 80 1g/L.
Seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
Fermentation tank culture medium compound method is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add tap water to 60% of fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, after adjusting pH to 8.0 with 40% sodium hydroxide solution, and 0.14Mpa sterilizing 40 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
2. seed liquor preparation
(1) faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as one-level shake-flask seed liquid.
(2) in 100L fermentor tank, prepare secondary seed medium, before cultivating, microscopy is carried out to secondary seed medium and one-level shake-flask seed liquid, guarantee without living contaminants.
(3) before inoculation, 100L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting secondary seed medium pH to 8.0 with 40% sodium hydroxide solution of sterilizing, primary seed solution is accessed in 100L fermentor tank by the inoculum size according to 1%.
(4) fermentation parameter setting: leavening temperature 40 DEG C; Rotating speed 100rpm; Stuffiness; Not Feeding ammonia water and sucrose, fermentation 10h terminates as secondary seed solution.
High density fermentation under 3.5 tons of fermentor tank scale conditions
(1) before fermentation, microscopy is carried out to fermention medium and secondary seed solution, guarantee pollution-free.
(2) before inoculation, 5 tons of fermentor tank rotating speeds are adjusted to 100rpm, and after adjusting pH to 8.0 with 40% sodium hydroxide solution of sterilizing, secondary seed solution are all forwarded in 5 tons of fermentor tanks and ferment.
(3) fermentation parameter is set as: temperature 40 DEG C; Rotating speed 100rpm; Stuffiness; Fermented liquid pH lower limit set is 6.5, and when pH is down to below set(ting)value, automatic makeup ammoniacal liquor makes pH maintain set(ting)value; Fermentation starts 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume; When the pH of fermented liquid no longer declines, second time mends the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume; The fermentation ends time is set as 16h.
4. controlled trial:
(1) main component of traditional MRS substratum and content are:
Peptone 10g/L, beef powder 5g/L, yeast powder 4g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, seven hypophosphite monohydrate hydrogen dipotassium 2g/L, ammonium citrate 2g/L, bitter salt 0.2g/L, four anhydrous manganese 0.05g/L, tween 80 1mL/L.
(2) seed flask substratum compound method is as follows:
Take each component of substratum, after fully dissolving, adjust pH to 6.2 with Glacial acetic acid, be settled to volume required, 121 DEG C of sterilizings 15 minutes.
(3) fermentation tank culture medium compound method is as follows:
Load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with emptying fermentor tank after tap water fermentor tank 2 times.Take each component of substratum, add appropriate tap water and be stirred to abundant dissolving, add tap water to 60% of fermentor tank cumulative volume.40 DEG C, 100rpm is incubated 1h, after adjusting pH to 6.2 with Glacial acetic acid, and 0.12Mpa sterilizing 30 minutes.When temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.Substratum after sterilizing is at 40 DEG C, and 50rpm, under stuffiness condition before empty training to inoculation.
The fermentation of (4) 5 tons of fermentor tanks:
Faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 40 DEG C of quiescent culture 10h are as primary seed solution.In 100L fermentor tank, prepare secondary seed medium, before cultivating, microscopy is carried out to secondary seed medium and primary seed solution, guarantee without living contaminants.Before fermentation, microscopy is carried out to fermention medium and secondary seed solution, guarantee pollution-free.Before inoculation, 5 tons of fermentor tank rotating speeds are adjusted to 100rpm, and after adjusting pH to 6.2 with 40% sodium hydroxide solution of sterilizing, secondary seed solution are all accessed in fermentor tank.Leavening temperature 40 DEG C, rotating speed 100rpm, stuffiness, stops fermentation after fermentation 16h.
5. interpretation of result: under 5 tons of fermentor tank scale conditions, the viable count of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 high density fermentation reaches 2.9 × 10 10cFU/mL, and the maximum viable count adopting batch fermentation manner to obtain in traditional MRS substratum is 8.0 × 10 8cFU/mL, therefore, the viable count of high density fermentation is more than 36 times of traditional MRS substratum batch fermentation viable count, meets the production requirement of faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746 as feeding lactobacillus completely.

Claims (5)

1. the high density fermentation seed culture medium of a feeding lactobacillus and fermention medium.Described feeding lactobacillus is faecium (Enterococcusfaecium) WEI-10CGMCCNo.7746, main component and the content of described high density fermentation seed culture medium and fermention medium are as follows: fish peptone 15-20g/L, sucrose 15-25g/L, sodium acetate trihydrate 3-7g/L, three hypophosphite monohydrate hydrogen dipotassium 1-4g/L, ammonium citrate 1-4g/L, bitter salt 0.05-0.4g/L, Manganous sulfate monohydrate 0.02-0.2g/L, tween 80 0.5-2g/L.
2. seed culture medium according to claim 1, is characterized in that the compound method of substratum is as follows:
Take each component of substratum, after fully dissolving, with 40% sodium hydroxide solution tune pH to 8.0, be settled to volume required, 121 DEG C of sterilizings 20 minutes.
3. fermention medium according to claim 1, is characterized in that the compound method of substratum is as follows:
(1) load 17g/L sodium hydroxide solution in fermentor tank, liquid amount is 50-70%, 0.11-0.16Mpa sterilizing 1h, tapping after cooling, with tap water fermentor tank 2-4 rear emptying fermentor tank.
(2) first take fish peptone, add appropriate tap water and be stirred to abundant dissolving.
(3) take successively and add other nutrient media components, add the 50-70% of tap water to fermentor tank cumulative volume.
(4) 40 DEG C, 100rpm is incubated 1h, and after adjusting pH to 8.0 with 40% sodium hydroxide solution, 0.11-0.16Mpa sterilizing 20-40 minute, when temperature-stable is at 40 DEG C, correcting dissolved oxygen is 100%.
(5) substratum after sterilizing is at 37-42 DEG C, 50rpm, and under stuffiness condition, sky is trained to inoculation.
4. the high density fermentation seed of feeding lactobacillus and the high density fermentation cultural method of fermention medium under 50L fermentor tank scale condition according to claim 1-3, is characterized in that key step is as follows:
(1) seed culture: faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 37-42 DEG C of quiescent culture 10h is as one-level shake-flask seed liquid.
(2), before carrying out 50L ferment tank, microscopy is carried out to fermention medium and primary seed solution, guarantees without living contaminants.
(3) before inoculation, 50L fermentor tank rotating speed is adjusted to 100rpm, and after readjusting the distribution ferment medium pH to 7.5-8.5 with 40% sodium hydroxide solution of sterilizing, according to the inoculum size of 0.5-4%, one-level shake-flask seed liquid is accessed in fermentor tank.
(4) ferment.Fermentation parameter is set as: temperature 37-42 DEG C; Rotating speed 100rpm; Stuffiness; Fermented liquid pH lower limit set is 6.4-6.8, and when pH is down to below set(ting)value, automatic makeup ammoniacal liquor makes pH maintain set(ting)value; Fermentation starts rear 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume; When the pH of fermented liquid no longer declines, second time adds the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume; The fermentation ends time is 12-18h.
5. the high density fermentation seed of feeding lactobacillus and the high density fermentation cultural method of fermention medium under 5 tons of fermentor tank scale conditions according to claim 1-3, is characterized in that key step is as follows:
(1) one-level shake-flask seed liquid: faecium (Enterococcusfaecium) the WEI-10CGMCCNo.7746 inclined-plane of picking fresh culture, be seeded in seed flask substratum, 37-42 DEG C of quiescent culture 10h is as one-level shake-flask seed liquid.
(2) secondary seed nutrient solution: prepare secondary seed medium according to claim 1 and claim 3 in 100L fermentor tank, carries out microscopy to secondary seed medium and one-level shake-flask seed liquid before cultivating, guarantees without living contaminants.Before inoculation, 100L fermentor tank rotating speed is adjusted to 100rpm, and after adjusting secondary seed medium pH to 7.5-8.5 with 40% sodium hydroxide solution of sterilizing, access in 100L fermentor tank according to the inoculum size of 0.5-2% by one-level shake-flask seed liquid, fermentation 10h terminates as secondary seed solution.
(3), before carrying out 5 tons of ferment tanks, microscopy is carried out to fermention medium and secondary seed solution, guarantees without living contaminants.Before inoculation, 5 tons of fermentor tank rotating speeds are adjusted to 100rpm, and after adjusting pH to 7.5-8.5 with 40% sodium hydroxide solution of sterilizing, secondary seed solution are all forwarded in 5 tons of fermentor tanks and ferment; Fermentation parameter is set as: temperature 37-42 DEG C; Rotating speed 100rpm; Stuffiness; Fermented liquid pH lower limit set is 6.4-6.8, and when pH is down to below set(ting)value, automatic makeup ammoniacal liquor makes pH maintain set(ting)value; Fermentation starts 4.5-5.5h, and add the sucrose of 500g/L for the first time, additional amount is 2% of fermentating liquid volume; When the pH of fermented liquid no longer declines, second time adds the sucrose of 500g/L, and additional amount is 2% of fermentating liquid volume; The fermentation ends time is 12-18h.
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CN105754889A (en) * 2015-12-11 2016-07-13 清华大学 Enterococcus faecium and application of enterococcus faecium to producing hydrogen by means of anaerobic fermentation
CN106978369A (en) * 2017-04-12 2017-07-25 福建奥姆龙生物工程有限公司 A kind of high-purity, resistance to storage at normal temperature VREF solid state fermentation culture process
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