CN102417892B - Feeding lactobacillus johnsonii high-density fermentation medium and application thereof - Google Patents
Feeding lactobacillus johnsonii high-density fermentation medium and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of lactobacillus culture, and relates to a feeding lactobacillus johnsonii high-density fermentation medium and application thereof. By using the high-density fermentation medium and the lactobacillus johnsonii fermented by a fermentation method for the fermentation medium, the bacterial density is remarkably improved relative to common fermentation, the number of viable bacteria is not less than 1,010cfu/ml and is improved by over 80 times compared with the viable bacteria cultured by a common MRS culture medium, and high-density fermentation of the lactobacillus johnsonii is realized; and according to the fermentation medium and the application thereof, the separation cost of biomass is reduced, the production period is shortened, the production cost is reduced, and the production efficiency is improved.
Description
Technical field
The invention belongs to milk-acid bacteria culture technique field, relate to a kind of feeding lactobacillus johnsonii high-density fermentation medium and application thereof.
Background technology
Milk-acid bacteria is the normal bacteria often having in animal digestive tract, applies the history of such bacterium development probiotic agent the earliest, and this bacterium has been widely used in leavened food, animal-feed and protective foods.
Lactobacillus johnsonii (Lactobacillus johnsonii) is the one in milk-acid bacteria, the comprehensive current bibliographical information about Lactobacillus johnsonii, its prebiotic function to animal is mainly reflected in following two aspects, on the one hand, Lactobacillus johnsonii has regulating effect to natural animal system of defense and immunity function; On the other hand, Lactobacillus johnsonii shows the specificity prophylactic effect to animal dermatitis.The discoveries such as Tanaka, mouse atopic dermatitis (atopic dermatitis, the AD) sickness rate that gavages Lactobacillus johnsonii reduces, and can effectively suppress mouse skin hyperplasia simultaneously.
In the fermented liquid that traditional standing for fermentation method obtains, the fermentation density of thalline is lower, Lactobacillus johnsonii viable count is in 5-10 hundred million left and right, in order to obtain more viable count, just need to increase the volume of bio-reactor or increase fermentation times etc., thereby increase the separation costs of biomass, extend manufacture cycle, thereby improve production cost, reduction production efficiency.
Lactobacillus johnsonii is still difficult to realize high density fermentation at present, the subject matter that is difficult to realize is fermention medium and fermentation process thereof, fermentation later stage nutritive substance is not enough and meta-bolites (being mainly lactic acid) accumulation suppresses thalli growth, causes high density fermentation to be difficult to realize.
Summary of the invention
(1) technical problem that will solve
The object of this invention is to provide a kind of feeding lactobacillus johnsonii high-density fermentation medium.
Another object of the present invention is to provide the application of this high density fermentation culture medium in Lactobacillus johnsonii fermentation.
(2) technical scheme
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of feeding Lactobacillus johnsonii (Lactobacillus johnsonii) high density fermentation culture medium, main ingredient and the content thereof of this substratum are as follows: glucose 20～30g/L, Tryptones 10～15g/L, extractum carnis or beef powder 6～15g/L, yeast powder 5～10g/L, K
2hPO
42～3g/L, CH
3cOONa 5～8g/L, Trisodium Citrate 2～4g/L, Mg
2+water-soluble cpds 0.2～0.4g/L, Fe
2+water-soluble cpds 0.1～0.2g/L and Mn
2+water-soluble cpds 45～55mg/L.
Wherein, described Mg
2+water-soluble cpds is MgSO
4, MgCl
2in any; Described Fe
2+water-soluble cpds is FeSO
4, FeCl
2, (NH
4)
2fe (SO
4)
2in any; Described Mn
2+water-soluble cpds is MnSO
4.
The compound method of fermention medium of the present invention is as follows:
(1) take respectively each component by content, with distilled water dissolving, wherein glucose dissolves separately;
(2) 115 ℃ of independent sterilizings of glucose 20 minutes, other component packs in fermentor tank 121 ℃ of sterilizings 30 minutes into;
(3) before inoculation, by aseptic technique, glucose is added in fermentor tank, stirring and evenly mixing, to obtain final product.
Feeding lactobacillus johnsonii high-density fermentation medium of the present invention can be applied in the fermentation of Lactobacillus johnsonii, is the effect of maximum performance substratum of the present invention, and while using substratum fermentation Lactobacillus johnsonii of the present invention, its fermenting process is as follows:
(1) Lactobacillus johnsonii of freezing preservation is activated three times at MRS agar plate, picking list colony inoculation is in deep layer MRS liquid tube, cultivate after 10 hours for 37 ℃, transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 8～10 hours as seed liquor;
(2) preparation Lactobacillus johnsonii high density fermentation culture medium, substratum is down to when temperature required, connects dissolved oxygen electrode, and demarcating dissolved oxygen is 100%;
(3) with 5%～10% inoculum size, the seed liquor of step (1) gained is inoculated in the fermention medium of step (2);
(4) fermentation, in fermenting process, maintaining fermented liquid pH value is 6～6.5, in the time that pH exceedes set(ting)value, adds feed liquid; When pH is during lower than set(ting)value, any in auto-feeding alkaline neutraliser ammoniacal liquor, KOH, NaOH; At 37～39 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 10～14 hours, fermenting process stuffiness or pass into N
2, maintain dissolved oxygen and be less than 1%;
(5) in the time adding that material liquid pH no longer reduces or the light absorption value of fermented liquid under 600nm wavelength no longer increases, stop fermentation, obtain Lactobacillus johnsonii viable count and reach 10
10fermented liquid more than cfu/ml.
Wherein, the main ingredient of described feed liquid and content thereof are as follows: sucrose 500g/L, peptone 150g/L, beef powder or extractum carnis 150g/L, yeast extract paste or yeast soak powder 75g/L.
High density fermentation culture medium of the present invention and fermentation process thereof also can be applicable to other milk-acid bacteria, but for to reach best invention effect, are preferably applicable to Lactobacillus johnsonii (Lactobacillusjohnsonii).
(3) beneficial effect
The present invention adopts pH feedback control fed-batch fermentation strategy, reduce the concentration of Lactic Acid from Fermentation Broth simultaneously, thereby remove the feedback inhibition of lactic acid, use the Lactobacillus johnsonii of high density fermentation culture medium of the present invention and fermentation process fermentation thereof, cell density significantly improves compared with common fermentation, and viable count is not less than 10
10cfu/ml, this is the unapproachable fermentation standard of existing technology, more common MRS culture medium culturing viable count 5.1 × 10
8cfu/ml, improves more than 80 times, has realized the high density fermentation of Lactobacillus johnsonii; The separation costs that fermention medium of the present invention and application thereof have also reduced biomass, shortens the production cycle, thereby reduces production costs, enhances productivity.
Embodiment
, it should be understood that described embodiment is only for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
The following example of the present invention instrument model, reagent and source thereof used is as follows:
Fermentor tank: the BioFlo/Celligen115 of NewBrunswick company type;
Recirculated water machine: Beijing DX-208 of Chang Liu scientific instrument company type;
Air compressor: Hospitality Franchise, Beijing mechanical & electrical technology Application and Development HF-6100C of company limited type;
Spectrophotometer: the 721N of Shanghai Precision Scientific Apparatus Co., Ltd type;
Microscope: OLYMPUS CX31 type;
The safe and sound SW-CJ-2FD type of super clean bench: Su Jing;
Biochemical cultivation case: the SPX-250B-Z of Medical Equipment Plant of Shanghai Bo Xun Industrial Co., Ltd. type
Yeast extract paste, beef powder, Tryptones are purchased from Beijing extensive and profound in meaning star biotechnology responsibility company limited, and other reagent are all purchased from Beijing chemical reagents corporation.
Lactobacillus johnsonii: derive from Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.4926.
The preparation of embodiment 1 fermention medium and fermentation are relatively
1, Lactobacillus johnsonii high density fermentation culture medium component and content thereof are:
Glucose: 20g/L, Tryptones: 10g/L, extractum carnis: 10g/L, yeast powder: 5g/L, K
2hPO
4: 2g/L, CH
3cOONa:5g/L, Trisodium Citrate: 2g/L, MgSO
4: 0.2g/L, FeSO
4: 0.1g/L, MnSO
4: 55mg/L.
2, substratum compound method:
(1) take respectively each component by above-mentioned content, with distilled water dissolving, wherein glucose dissolves separately;
(2) 115 ℃ of independent sterilizings of glucose 20 minutes, other components pack in fermentor tank 121 ℃ of sterilizings 30 minutes into;
(3) before inoculation, by aseptic technique, glucose is added in fermentor tank, stirring and evenly mixing, to obtain final product.
3, the comparison of high density fermentation and prior art fermentation
3.1 high density fermentation key steps are as follows
(1) Lactobacillus johnsonii of freezing preservation is activated three times at MRS agar plate, picking list colony inoculation, in deep layer MRS liquid tube, is cultivated after 10 hours for 37 ℃, transfers in MRS liquid nutrient medium with 1% inoculum size, cultivates after 10 hours as seed liquor;
(2) preparation fermention medium, substratum is down to when temperature required, connects dissolved oxygen electrode, and demarcating dissolved oxygen is 100%;
(3) with 5% inoculum size, the seed liquor of step (1) gained is inoculated in the fermention medium of step (2);
(4) fermentation, in fermenting process, maintaining fermented liquid pH value is 6.0, in the time that pH exceedes 6.0, adds the about 100ml of feed liquid; When pH is lower than 6.0 time, auto-feeding alkaline neutraliser ammoniacal liquor; At 37 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 10 hours, fermenting process stuffiness, maintains dissolved oxygen and is less than 1%;
(5) in the time adding material liquid pH and no longer reduce, stop fermentation, obtain Lactobacillus johnsonii viable count and reach 4.3 × 10
10the fermented liquid of cfu/ml.
3.2 control experiment
(1) Lactobacillus johnsonii fermention medium (MRS substratum) its component and content are:
Glucose: 20g/L, peptone: 10g/L, extractum carnis: 5g/L, yeast powder: 4g/L, K
2hPO
4: 2g/L, CH
3cOONa:5g/L, Trisodium Citrate: 2g/L, MgSO
4: 0.2g/L, MnSO
4: 50mg/L, tween 80: 1ml.
(2) fermentation
By the Lactobacillus johnsonii of freezing preservation, MRS agar plate activation three times, picking list colony inoculation, in deep layer MRS liquid tube, is cultivated after 10 hours for 37 ℃.Transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 10 hours as seed liquor, with 10% inoculum size inoculation fermentation substratum.37 ℃ leave standstill cultivation 10 hours, stop fermentation, and obtaining Lactobacillus johnsonii viable count is 5.1 × 10
8cfu/ml.
3.3 interpretation of result
Apply substratum of the present invention and fermentation process thereof, can obtain Lactobacillus johnsonii viable count and reach 4.3 × 10
10the fermented liquid of cfu/ml, and apply existing fermention medium (MRS substratum) and fermentation process thereof, the Lactobacillus johnsonii viable count obtaining is only 5.1 × 10
8cfu/ml.Experimental results show that nearly 80 times that Lactobacillus johnsonii viable count that substratum of the present invention and fermentation process thereof obtain is prior art, realized the high density fermentation of Lactobacillus johnsonii.
The preparation of embodiment 2 fermention mediums and fermentation are relatively
1, its component of Lactobacillus johnsonii high density fermentation culture medium and content are:
Glucose: 25g/L, Tryptones: 10g/L, beef powder: 15g/L, yeast powder: 10g/L, K
2hPO
4: 2g/L, CH
3cOONa:7g/L, Trisodium Citrate: 3g/L, MgCl
2: 0.2g/L, FeCl
2: 0.1g/L, MnSO
4: 50mg/L.
2, substratum compound method:
(1) take respectively each component by content, with distilled water dissolving, wherein glucose dissolves separately;
(2) 115 ℃ of independent sterilizings of glucose 20 minutes, other components pack in fermentor tank 121 ℃ of sterilizings 30 minutes into;
(3) before inoculation, by aseptic technique, glucose is added in fermentor tank, stirring and evenly mixing, to obtain final product.3, the comparison of high density fermentation and prior art fermentation
3.1 high density fermentation key steps are as follows
(1) Lactobacillus johnsonii of freezing preservation is activated three times at MRS agar plate, picking list colony inoculation, in deep layer MRS liquid tube, is cultivated after 10 hours for 37 ℃, transfers in MRS liquid nutrient medium with 1% inoculum size, cultivates after 8 hours as seed liquor;
(2) preparation fermention medium, substratum is down to when temperature required, connects dissolved oxygen electrode, and demarcating dissolved oxygen is 100%;
(3) with 5% inoculum size, the seed liquor of step (1) gained is inoculated in the fermention medium of step (2);
(4) fermentation, in fermenting process, maintaining fermented liquid pH value is 6.3, in the time that pH exceedes set(ting)value, adds the about 100ml of feed liquid; When pH is during lower than set(ting)value, auto-feeding alkaline neutraliser ammoniacal liquor; At 38 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 12 hours, fermenting process stuffiness, maintains dissolved oxygen and is less than 1%;
(5) when adding feed liquid fermented liquid OD
600while no longer increasing, stop fermentation, obtain Lactobacillus johnsonii viable count and reach 5.9 × 10
10the fermented liquid of cfu/ml.
3.2 control experiments:
(1) Lactobacillus johnsonii fermention medium (MRS substratum) its component and content are:
Glucose: 20g/L, peptone: 10g/L, beef powder: 5g/L, yeast powder: 4g/L, K
2hPO
4: 2g/L, CH
3cOONa:5g/L, Trisodium Citrate: 2g/L, MgSO
4: 0.2g/L, MnSO
4: 50mg/L, tween 80: 1ml.
(2) fermentation:
By the Lactobacillus johnsonii of freezing preservation, MRS agar plate activation three times, picking list colony inoculation, in deep layer MRS liquid tube, is cultivated after 10 hours for 37 ℃.Transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 10 hours as seed liquor, with 10% inoculum size inoculation fermentation substratum.38 ℃ leave standstill cultivation 12 hours, stop fermentation, and obtaining described Lactobacillus johnsonii viable count is 6.0 × 10
8cfu/ml.
3.3 interpretation of result
Apply substratum of the present invention and fermentation process thereof, can obtain Lactobacillus johnsonii viable count and reach 5.9 × 10
10the fermented liquid of cfu/ml, and apply existing fermention medium (MRS substratum) and fermentation process thereof, the Lactobacillus johnsonii viable count obtaining is only 6.0 × 10
8cfu/ml.Experimental results show that 98 times that Lactobacillus johnsonii viable count that substratum of the present invention and fermentation process thereof obtain is prior art, realized the high density fermentation of Lactobacillus johnsonii.
The preparation of embodiment 3 fermention mediums and fermentation are relatively
1, its component of Lactobacillus johnsonii high density fermentation culture medium and content are:
Glucose: 30g/L, Tryptones: 15g/L, extractum carnis: 6g/L, yeast powder: 6g/L, K
2hPO
4: 3g/L, CH
3cOONa:8g/L, Trisodium Citrate: 4g/L, (NH
4)
2fe (SO
4)
2: 0.2g/L, MgSO
4: 0.4g/L, MnSO
4: 45mg/L.
2, substratum compound method:
(1) take respectively each component by content, with distilled water dissolving, wherein glucose dissolves separately;
(2) 115 ℃ of independent sterilizings of glucose 20 minutes, other components pack in fermentor tank 121 ℃ of sterilizings 30 minutes into;
(3) before inoculation, by aseptic technique, glucose is added in fermentor tank, stirring and evenly mixing, to obtain final product.
3, the comparison of high density fermentation and prior art fermentation
3.1 high density fermentation key steps are as follows
(1) Lactobacillus johnsonii of freezing preservation is activated three times at MRS agar plate, picking list colony inoculation, in deep layer MRS liquid tube, is cultivated after 10 hours for 37 ℃, transfers in MRS liquid nutrient medium with 1% inoculum size, cultivates after 10 hours as seed liquor;
(2) preparation fermention medium, substratum is down to when temperature required, connects dissolved oxygen electrode, and demarcating dissolved oxygen is 100%;
(3) with 10% inoculum size, the seed liquor of step (1) gained is inoculated in the fermention medium of step (2);
(4) fermentation, in fermenting process, maintaining fermented liquid pH value is 6.5, in the time that pH exceedes set(ting)value, adds the about 100ml of feed liquid; When pH is during lower than set(ting)value, auto-feeding alkaline neutraliser ammoniacal liquor; At 39 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 14 hours, fermenting process passes into nitrogen, maintains dissolved oxygen and is less than 1%;
(5) in the time adding material liquid pH and no longer reduce, stop fermentation, obtain Lactobacillus johnsonii viable count and reach 4.9 × 10
10the fermented liquid of cfu/ml.
3.2 control experiments:
(1) its component of Lactobacillus johnsonii fermention medium (MRS substratum) is:
Glucose: 20g/L, peptone: 10g/L, extractum carnis: 5g/L, yeast powder: 4g/L, K
2hPO
4: 2g/L, CH
3cOONa:5g/L, Trisodium Citrate: 2g/L, MgSO
4: 0.2g/L, MnSO
4: 50mg/L, tween 80: 1ml.
(2) fermentation
By the Lactobacillus johnsonii of freezing preservation, MRS agar plate activation three times, picking list colony inoculation, in deep layer MRS liquid tube, is cultivated after 10 hours for 37 ℃.Transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 10 hours as seed liquor, with 10% inoculum size inoculation fermentation substratum.39 ℃ leave standstill cultivation 14 hours, stop fermentation, and obtaining Lactobacillus johnsonii viable count is 4.8 × 10
8cfu/ml.
3.3 interpretation of result
Apply substratum of the present invention and fermentation process thereof, can obtain Lactobacillus johnsonii viable count and reach 4.9 × 10
10the fermented liquid of cfu/ml, and apply existing fermention medium (MRS substratum) and fermentation process thereof, the Lactobacillus johnsonii viable count obtaining is only 4.8 × 10
8cfu/ml.Experimental results show that nearly 102 times that Lactobacillus johnsonii viable count that substratum of the present invention and fermentation process thereof obtain is prior art, realized the high density fermentation of Lactobacillus johnsonii.In production, the realization of high density fermentation can reduce the separation costs of biomass, shortens the production cycle, reduces production costs, enhances productivity etc., and this is extremely important in energy-efficient contemporary society.
Claims (2)
1. feeding Lactobacillus johnsonii (Lactobacillus johnsonii) CGMCC No.4926 fermentation process in high density, is characterized in that, fermenting process is as follows:
(1) Lactobacillus johnsonii of freezing preservation is activated three times at MRS agar plate, picking list colony inoculation is in deep layer MRS liquid tube, cultivate after 10 hours for 37 ℃, transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 8～10 hours as seed liquor;
(2) preparation high density fermentation culture medium, substratum is down to when temperature required, connects dissolved oxygen electrode, and demarcating dissolved oxygen is 100%;
(3) with 5%～10% inoculum size, the seed liquor of step (1) gained is inoculated in the fermention medium of step (2);
(4) fermentation, in fermenting process, maintaining fermented liquid pH value is 6～6.5, in the time that pH exceedes set(ting)value, adds feed liquid; When pH is during lower than set(ting)value, any in auto-feeding alkaline neutraliser ammoniacal liquor, KOH, NaOH; At 37～39 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 10～14 hours, fermenting process stuffiness or pass into N
2, maintain dissolved oxygen and be less than 1%;
(5) in the time adding that material liquid pH no longer reduces or the light absorption value of fermented liquid under 600nm wavelength no longer increases, stop fermentation, obtain Lactobacillus johnsonii viable count and reach 10
10fermented liquid more than cfu/ml,
Wherein, the main ingredient of described high density fermentation culture medium and content thereof are as follows: glucose 20～30g/L, Tryptones 10～15g/L, extractum carnis or beef powder 6～15g/L, yeast powder 5～10g/L, K
2hPO
42～3g/L, CH
3cOONa 5～8g/L, Trisodium Citrate 2～4g/L, Mg
2+water-soluble cpds 0.2～0.4g/L, Fe
2+water-soluble cpds 0.1～0.2g/L and Mn
2+water-soluble cpds 45～55mg/L,
Wherein, the main ingredient of described feed liquid and content thereof are as follows: sucrose 500g/L, peptone 150g/L, beef powder or extractum carnis 150g/L, yeast extract paste or yeast soak powder 75g/L.
2. fermentation process in high density as claimed in claim 1, is characterized in that, described Mg
2+water-soluble cpds is MgSO
4, MgCl
2in any; Described Fe
2+water-soluble cpds is FeSO
4, FeCl
2, (NH
4)
2fe (SO
4)
2in any; Described Mn
2+water-soluble cpds is MnSO
4.
3. fermentation process in high density as claimed in claim 1 or 2, is characterized in that, the compound method of fermention medium is as follows:
(1) take respectively each component by content, with distilled water dissolving, wherein glucose dissolves separately;
(2) 115 ℃ of independent sterilizings of glucose 20 minutes, other component packs in fermentor tank 121 ℃ of sterilizings 30 minutes into;
(3) before inoculation, by aseptic technique, glucose is added in fermentor tank, stirring and evenly mixing, to obtain final product.
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| CN102783579A (en) * | 2012-08-22 | 2012-11-21 | 北京大北农科技集团股份有限公司 | Application of Lactobacillus johnsonii in improving production performance of laying chickens |
| CN104263671A (en) * | 2014-07-16 | 2015-01-07 | 江南大学 | Method for increasing viable count of Lactobacillus brevis for pickles by two-stage dissolved oxygen control strategy |
| CN106148213A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of forage plant lactobacillus high density fermentation culture medium and method |
| CN106260517A (en) * | 2015-05-22 | 2017-01-04 | 北京大北农科技集团股份有限公司 | A kind of method of Lactobacillus johnsonii fermented feed and feedstuff |
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| CN101418270A (en) * | 2008-11-06 | 2009-04-29 | 内蒙古农业大学 | The Lactobacillus casei Zhang high-density cultivation method, use them to prepare the method for freeze-dried vaccine powder and resulting freeze-dried vaccine powder and uses thereof |
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| CN101356950A (en) * | 2008-09-24 | 2009-02-04 | 东北农业大学 | Preparation method of composite bacteria fermentation bed |
| CN101418270A (en) * | 2008-11-06 | 2009-04-29 | 内蒙古农业大学 | The Lactobacillus casei Zhang high-density cultivation method, use them to prepare the method for freeze-dried vaccine powder and resulting freeze-dried vaccine powder and uses thereof |
| CN102226165A (en) * | 2011-04-22 | 2011-10-26 | 华中农业大学 | Engineering bacterium for producing Phospholipase A2 (PLA2) and applications thereof |
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