CN101768610A - Method for efficiently cultivating lactobacillus and producing lactic acid - Google Patents
Method for efficiently cultivating lactobacillus and producing lactic acid Download PDFInfo
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- CN101768610A CN101768610A CN 201010120900 CN201010120900A CN101768610A CN 101768610 A CN101768610 A CN 101768610A CN 201010120900 CN201010120900 CN 201010120900 CN 201010120900 A CN201010120900 A CN 201010120900A CN 101768610 A CN101768610 A CN 101768610A
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 156
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 78
- 239000004310 lactic acid Substances 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title abstract description 14
- 241000186660 Lactobacillus Species 0.000 title abstract description 13
- 229940039696 lactobacillus Drugs 0.000 title abstract description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 37
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 19
- 229930006000 Sucrose Natural products 0.000 claims abstract description 19
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 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 abstract description 7
- 238000000855 fermentation Methods 0.000 claims abstract description 7
- 230000004151 fermentation Effects 0.000 claims abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract 4
- 235000017550 sodium carbonate Nutrition 0.000 claims abstract 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims abstract 2
- 241000894006 Bacteria Species 0.000 claims description 49
- 239000005720 sucrose Substances 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
- 230000012010 growth Effects 0.000 abstract description 20
- 239000003513 alkali Substances 0.000 abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 13
- 150000001720 carbohydrates Chemical class 0.000 abstract description 13
- 229910052799 carbon Inorganic materials 0.000 abstract description 13
- 239000000523 sample Substances 0.000 abstract description 2
- 239000011259 mixed solution Substances 0.000 abstract 2
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 229960004793 sucrose Drugs 0.000 abstract 1
- 239000013589 supplement Substances 0.000 abstract 1
- 229960000448 lactic acid Drugs 0.000 description 32
- 230000001360 synchronised effect Effects 0.000 description 9
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- 230000001276 controlling effect Effects 0.000 description 4
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- 230000000050 nutritive effect Effects 0.000 description 4
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- 238000011160 research Methods 0.000 description 3
- 238000012366 Fed-batch cultivation Methods 0.000 description 2
- 241000186610 Lactobacillus sp. Species 0.000 description 2
- 241000194036 Lactococcus Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000726221 Gemma Species 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 235000019846 buffering salt Nutrition 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
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- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
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- CEIZFXOZIQNICU-UHFFFAOYSA-N tenuazonic acid Chemical compound CCC(C)C1NC(=O)C(C(C)=O)=C1O CEIZFXOZIQNICU-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a method for efficiently cultivating lactobacillus and producing lactic acid, comprising the following steps: in the fermentation and cultivating process of lactobacillus, through adding the mixed solution of 1.0% to 50% of Na2CO3 or NaOH and 10%-50% of cane sugar or glucose, constantly controlling the pH value to be any value within the range of 5.0-8.0; and simultaneously, feeding saccharide required by the growth of lactobacillus to be served as a carbon source, thus enabling the pH to be controlled constantly and also providing a carbon-containing compound required by the growth of lactobacillus to be served as a carbon source. The invention has the advantages that in the method of the invention, the mixed solution of alkali controlling pH and carbon source saccharide feeding the growth of lactobacillus are taken as flowing liquid to control the pH value; the pH probe full-automatically and constantly controls the pH value, and simultaneous flows to supplement the carbon source required by the growth of lactobacillus, thus realizing efficient cultivating of lactobacillus and efficient production of lactic acid and having great application value in the industrial production of lactobacillus and lactic acid with high density.
Description
Technical field
The invention belongs to biological technical field, the method that is specifically related to a kind of high-efficient culture milk-acid bacteria and produces lactic acid has important use value and prospect aspect suitability for industrialized production high-density milk-acid bacteria and the lactic acid.
Background technology
Milk-acid bacteria (Lactobacillus) is that a class can utilize carbohydrate fermentation to produce the general name of lactic-acid-bacterium.Milk-acid bacteria is a lot of in the kind of occurring in nature, and distributed pole is wide, has found that at present the different lactobacillus inoculation of kind more than 200 divides at least on taxonomy 23 genus are arranged, and mainly comprises lactobacillus (Lactobacillus) and lactococcus (Lactococcus) etc.The lactic-acid bacteria cells form mainly contains elongated rod shape, rod-short and circular 3 kinds of forms, all belongs to gram-positive microorganism, does not form gemma.Milk-acid bacteria is a kind of facultative anaerobe, is adapted at oxygen level environment low or anaerobic and grows down, and 3.0 sour environments of the pH in the mammal stomach are had certain tolerance.Milk-acid bacteria except that carbon source, nitrogenous source, phosphorus source, sulphur source and inorganic salt, also needs somatomedins such as VITAMIN to nutritional requirement.
Milk-acid bacteria extensively is present in the enteron aisle of people, animal and fowl, studies show that in a large number milk-acid bacteria can regulate the body gastrointestinal tract normal microflora, keep microecological balance, improve the food digestion rate, reduce serum cholesterol, the controlling machine vivotoxin suppresses the generation of interior corrupt bacteria growing breeding of enteron aisle and spoilage product, make nutritive substance, stimulate tissue development, the control tumour takes place, anti-ageing and stress, be to be expected to replace antibiotic biotechnological formulation.In addition, milk-acid bacteria can also be improved Nutritive value of food, improves flavour of food products, improves the keepability and the added value of food, therefore is subjected to people's generally attention day by day.
Present Research shows both at home and abroad, and the method for milk-acid bacteria high-density culture mainly contains buffering salt method, chemical neutralisation, dialysis culture, cell cycle cultivation, membrane filtration cultivation, fed batch cultivation and microencapsulation cultivation etc.Lactobacillus-fermented is cultivated the main fed batch cultivation mode that adopts at present, just according to the characteristics of lactobacter growth and substratum, adds fresh culture in some way off and on or continuously at the different growth phases of milk-acid bacteria.Because lactobacillus-fermented belongs to the fermentation of growth coupling connection type, produce lactic acid in the fermenting process, a large amount of lactic acid accumulation makes the pH of fermented liquid reduce on the one hand, when dropping to 4.0, pH can produce serious restraining effect to the growth of milk-acid bacteria when following, product inhibition also can take place in the accumulation of lactic acid on the other hand, influences lactobacter growth.Therefore, in lactic acid bacteria fermentation process, must be by stream Ensure Liquid material, the methods such as pH value and eliminating meta-bolites of regulating to obtain highdensity milk-acid bacteria, its key is the control of feed supplement strategy and pH.Therefore determining and both can support the milk-acid bacteria nutritive substance fed-batch mode of growth fast, can keep the control strategy of the constant control of pH of fermented liquid again simultaneously, is the important topic that lactic acid was produced and produced in the industrialization of realization milk-acid bacteria.
At present, though have respectively both at home and abroad and adopt the feed supplement mode to cultivate to provide nutritive substance and constant control pH to provide the research report of suitable acid or alkali environment, the stream of constant control pH synchronous flow Ensure Liquid material is added application of policies do not see as yet that in the research of high-efficient culture milk-acid bacteria and lactic acid producing bibliographical information is arranged for lactobacter growth for milk-acid bacteria.
Summary of the invention
The object of the present invention is to provide a kind of can constant control fermented liquid pH value in the lactobacillus-fermented culturing process and replenish the method for carbohydrate synchronously as carbon source, with the purpose that reaches the high-efficient culture milk-acid bacteria and produce lactic acid.
The present invention tests the milk-acid bacteria USTB-08 (Lactobacillus sp.USTB-08) that used milk-acid bacteria is a strain oneself screening, liquid nutrient medium is joined in the fermentor tank, through 121 ℃ of sterilizations of 20 minutes, be cooled to 30-35 ℃ after the inoculating lactic acid bacterium culture; Culture condition is 200 rev/mins of fermentor tank mixing speed, and no aeration anaerobism is cultivated, temperature 25-45 ℃.Adopt the pH on-line Full controlling flow of popping one's head in to add 1.0%-50%Na
2CO
3Or the mixing solutions of NaOH and 10%-50% sucrose or glucose, both constant control pH any one value in the 5.0-8.0 scope, the needed carbon source of feed supplement lactobacter growth again, pH control and feed supplement controlled to be combined as a whole finish, reached that control is simple, the purpose of processing ease and high-efficient culture milk-acid bacteria and lactic acid producing, have important use and be worth.
The milk-acid bacteria substratum is composed as follows: peptone 10.0g, extractum carnis 10.0g, yeast extract paste 5.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, glucose 20.0g, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, tween 80 1.0mL, 1000ml deionized water.
The present invention will control pH and stream and add carbon source and be incorporated into one in the culturing process of milk-acid bacteria, successfully controlling culture pH constant simultaneously, synchronous flow with sucrose or glucose as carbon source, not only can realize the high-density culture of milk-acid bacteria, and can obtain a large amount of lactic acid, have important commercial application and be worth.
Principle of work of the present invention is: in the lactobacillus-fermented culturing process, milk-acid bacteria can utilize various saccharides to ferment and produce lactic acid, and the generation of lactic acid can make the quick reduction of culture pH, causes the growth of milk-acid bacteria to be suppressed.With 1.0%-50%Na
2CO
3Or the mixing solutions of NaOH and 10%-50% sucrose or glucose is as the stream liquid feeding of constant control culture pH, in constant control culture pH, also stream has added the needed carbohydrate of lactobacter growth, is that the simple and easy to do optimization stream that integrates the lactobacillus-fermented of pH control and stream Ensure Liquid material that kills two birds with one stone adds strategy.Because lactic acid is the primary metabolite of lactobacillus-fermented carbohydrate, lactobacter growth lactic acid fast more, that produce is many more, pH reduces fast more, therefore it is many more that the stream that causes being used for constant control pH adds the alkali lye amount, and it is also big more that synchronous flow adds the carbohydrate amount, therefore can high-density culture milk-acid bacteria and high lactic acid producing.
The invention has the advantages that: in the lactobacillus-fermented culturing process, alkali and the required carbon source carbohydrate of additional lactobacter growth of control pH are mixed as the stream liquid feeding of controlling pH, when the full-automatic stream of popping one's head in by pH adds constant control pH, synchronous flow adds and has replenished the needed carbon source of lactobacter growth, realized the high-efficient culture of milk-acid bacteria and the High-efficient Production of lactic acid, in suitability for industrialized production high-density milk-acid bacteria and lactic acid, had important use and be worth.
Description of drawings
Fig. 1 be based on stream add the constant control of alkali lye pH be 7.0 and synchronously the feed supplement sucrose cultivate and disposablely required carbohydrate is joined in the fermentor tank stream add alkali lye to control pH be under 7.0 two kinds of different conditions, the growth curve of milk-acid bacteria USTB-08, " △ " representative control pH7.0 synchronous flow is with sucrose; The disposable adding sucrose of " " representative control pH7.0;
Fig. 2 be based on stream add the constant control of alkali lye pH be 7.0 and synchronously the feed supplement sucrose cultivate and disposablely required carbohydrate is joined in the fermentor tank stream add alkali lye to control pH be under 7.02 kinds of different conditions, milk-acid bacteria USTB-08 produces the amount of lactic acid, and " △ " representative control pH7.0 synchronous flow is with sucrose; The disposable adding sucrose of " " representative control pH7.0.
Embodiment
Embodiment 1
1, the selection of lactic acid bacteria culturers and fermention medium
The present invention tests the milk-acid bacteria USTB-08 (Lactobacillus sp.USTB-08) that used bacterial classification is a strain we oneself screening.The milk-acid bacteria substratum is composed as follows: peptone 10.0g, extractum carnis 10.0g, yeast extract paste 5.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, glucose 20.0g, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, tween 80 1.0ml, 1000ml deionized water.
2, the milk-acid bacteria fed-batch fermentation is cultivated
Adopt 50 liters of full automatically controlled fermentors, add 30 liters of liquid nutrient mediums, after 121 ℃ of sterilizations of 20 minutes and being cooled to 35 ℃, the milk-acid bacteria cultivated in batches of inoculation 300ml is as bacterial classification, carries out anaerobism and cultivate under 35 ℃ of 200 rev/mins of mixing speed and temperature.Be inserted with pH probe and carry out on-line Full stream to add the constant control of stream liquid feeding pH be 7.0 in culture, control pH constant flows liquid feeding and consists of 200g/L Na
2CO
3With the mixing solutions of 500g/L sucrose, in constant control pH7.0, synchronous flow has added the carbon source of sucrose as lactobacter growth, cultivates the OD that represents the lactic acid bacteria biological amount after 4 day time
680nmReached (Fig. 1) more than 10, the lactic acid content that produces in the culture has reached 35g/L above (Fig. 2), has realized the purpose of high density fermentation cultivation milk-acid bacteria and high lactic acid producing, has important industrialization production application and is worth.
Fig. 1 be based on stream add the constant control of alkali lye pH be 7.0 and synchronously the feed supplement sucrose cultivate and disposablely required carbohydrate is joined in the fermentor tank stream add alkali lye to control pH be under 7.02 kinds of different conditions, the growth curve of milk-acid bacteria USTB-08.Wherein, X-coordinate is the time, unit: hour; Ordinate zou is the optical density value (OD of representative lactobacter growth
680nm).By demonstrating among Fig. 1: a stream in the disposable adding fermentor tank of sucrose is added under the constant control of the alkali lye pH condition, cultivate the OD in 4 days
680nOnly reached 6.7; And add Na by stream
2CO
3Constant and the synchronous flow of control fermented liquid pH is with sucrose, represents the OD of lactic acid bacteria biological amount in 4 days
680nmReached 10.2, improved 52.2% lactic acid bacteria biological amount.
Fig. 2 be based on stream add the constant control of alkali lye pH be 7.0 and synchronously the feed supplement sucrose cultivate and disposablely required carbohydrate joined in the fermentor tank stream to add alkali lye control pH be under 7.02 kinds of different conditions, the amount of milk-acid bacteria USTB-08 production lactic acid.As can be seen from Figure 2, a stream adds under the constant control of the alkali lye pH condition in the disposable adding fermentor tank of sucrose, cultivates that lactic acid has only reached 24.7g/L in 4 days; And add Na by stream
2CO
3Constant and the synchronous flow of control fermented liquid pH is with sucrose, and lactic acid concn presents ascendant trend always in 4 days, reaches 35.0g/L, and makes lactic acid content improve 41.7% in the 4th day.
Claims (1)
1. the high-efficient culture milk-acid bacteria and the method for producing lactic acid is characterized in that: in milk-acid bacteria cultivation and fermentation process, adopt the pH on-line Full control of popping one's head in, stream adds 1.0%-50% yellow soda ash Na simultaneously
2CO
3Or the mixing solutions of sodium hydroxide NaOH and 10%-50% sucrose or glucose, the pH of constant control fermented liquid is any one value in the 5.0-8.0 scope.
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| CN 201010120900 CN101768610A (en) | 2010-03-05 | 2010-03-05 | Method for efficiently cultivating lactobacillus and producing lactic acid |
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Cited By (5)
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| CN102191202A (en) * | 2011-04-08 | 2011-09-21 | 石家庄君乐宝乳业有限公司 | High-density culture method for lactic acid bacteria |
| CN106350473A (en) * | 2016-11-30 | 2017-01-25 | 北京市农林科学院 | High-density fermentation culture medium for feed lactobacillus brevis and fermentation method thereof |
| CN112088978A (en) * | 2020-09-16 | 2020-12-18 | 黄河三角洲京博化工研究院有限公司 | Lactic acid bacteria stabilizer and application method thereof |
| CN114958667A (en) * | 2022-05-27 | 2022-08-30 | 江苏大学 | Lactococcus capable of highly producing L-lactic acid and application thereof |
| CN119242520A (en) * | 2024-11-06 | 2025-01-03 | 天地成微生物技术(北京)有限公司 | A high-density culture method of lactic acid bacteria based on pH control system |
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| CN102191202B (en) * | 2011-04-08 | 2013-05-22 | 石家庄君乐宝乳业有限公司 | High-density culture method for lactic acid bacteria |
| CN106350473A (en) * | 2016-11-30 | 2017-01-25 | 北京市农林科学院 | High-density fermentation culture medium for feed lactobacillus brevis and fermentation method thereof |
| CN106350473B (en) * | 2016-11-30 | 2019-10-11 | 北京市农林科学院 | A kind of high-density fermentation medium and fermentation method thereof for feeding Lactobacillus brevis |
| CN112088978A (en) * | 2020-09-16 | 2020-12-18 | 黄河三角洲京博化工研究院有限公司 | Lactic acid bacteria stabilizer and application method thereof |
| CN114958667A (en) * | 2022-05-27 | 2022-08-30 | 江苏大学 | Lactococcus capable of highly producing L-lactic acid and application thereof |
| CN114958667B (en) * | 2022-05-27 | 2023-09-29 | 朱密 | Lactococcus for high yield of L-lactic acid and application thereof |
| CN119242520A (en) * | 2024-11-06 | 2025-01-03 | 天地成微生物技术(北京)有限公司 | A high-density culture method of lactic acid bacteria based on pH control system |
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