CN117946929A - High-density culture method of lactic acid bacteria - Google Patents
High-density culture method of lactic acid bacteria Download PDFInfo
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- CN117946929A CN117946929A CN202410151624.7A CN202410151624A CN117946929A CN 117946929 A CN117946929 A CN 117946929A CN 202410151624 A CN202410151624 A CN 202410151624A CN 117946929 A CN117946929 A CN 117946929A
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- 238000009825 accumulation Methods 0.000 claims description 5
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 4
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 4
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- 239000008101 lactose Substances 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
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- 229940089442 lacticare Drugs 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of lactic acid bacteria, and particularly relates to a high-density culture method of lactic acid bacteria, which comprises the following steps: step 1, determining the components and the formula of a culture medium; step 2, preparing and adjusting a culture medium; step 3, sterilizing and asepsis operation; step 4, preparing seed bacteria; step 5, inoculating operation; step 6, controlling culture conditions; step 7, culturing time and mode; step 8, separating and harvesting thalli; and 9, treating and preserving thalli. According to the invention, the components and the formula of the culture medium are determined according to the characteristics of the strain, the culture purpose and the characteristics of the required product, so that the culture effect and the yield are improved; preparing and adjusting a culture medium to ensure uniform dissolution and stable pH; sterilization and aseptic operation, ensuring a sterile environment; selecting pure seed bacterial liquid, and improving the purity of the bacterial strain; regulating temperature and oxygen supply to provide proper conditions; determining the optimal culture time, and separating and harvesting thalli; and (3) a proper thallus processing and preserving method is adopted, so that the quality and activity are maintained.
Description
Technical Field
The invention belongs to the technical field of lactic acid bacteria, and particularly relates to a high-density culture method of lactic acid bacteria.
Background
Lactic acid bacteria are a probiotic bacteria that are capable of fermenting carbohydrates into lactic acid and are thus named. Lactic acid bacteria are widely distributed and are commonly found in foods such as meat, milk and vegetables and products thereof. In addition, lactic acid bacteria are widely present in intestinal tracts of livestock and birds and few clinical samples, wherein lactic acid bacteria in the environments of oral cavity, intestinal tracts and the like of humans and other mammals are important members constituting normal microbial flora in specific areas, lactic acid bacteria are spore-free gram-staining positive bacteria of which fermentation sugar main products are lactic acid, the bacteria comprise a plurality of species, most of the lactic acid bacteria do not move, a few of the lactic acid bacteria do move at Zhou Mao, and bacterial cells are often arranged into chains. The streptococcus lactis family, the bacterial cells are spherical, usually paired or in chains. The lactobacillus family, the thallus rod-shaped, single or chain-formed, sometimes filiform, produces false branches, and common lactobacillus has extremely weak activity, can only survive in relatively limited environments, and can be subjected to death once being separated from the environments, and the lactobacillus can be classified into cocci and bacilli in the form of the lactobacillus family in the eubacterial schema. Wherein the spherical lactobacillus comprises Streptococcus, leuconostoc, pediococcus; the bacillus includes lactococcus, lactobacillus, bifidobacterium, etc.; the growth temperature can be classified into a high temperature type and a medium temperature type; from the fermentation type, it can be classified into homofermentation and heterofermentation; from the source, it can be largely classified into animal-derived lactic acid bacteria and plant-derived lactic acid bacteria.
The existing method for culturing probiotics adopts a universal culture medium to uniformly culture probiotics, is inconvenient to determine the components and the formula of the culture medium according to the characteristics of strains, the purpose of culture and the characteristics of required products, is unfavorable for improving the culture effect and the yield, has unstable purity of the strains and poor quality and activity of lactic acid bacteria, and therefore, a high-density culture method of the lactic acid bacteria is provided for solving the problems.
Disclosure of Invention
The invention aims to provide a high-density culture method of lactobacillus, which can determine the components and the formula of a culture medium according to the characteristics of strains, the culture purpose and the characteristics of required products, and improve the culture effect and the yield; preparing and adjusting a culture medium to ensure uniform dissolution and stable pH; sterilization and aseptic operation, ensuring a sterile environment; selecting pure seed bacterial liquid, and improving the purity of the bacterial strain; regulating temperature and oxygen supply to provide proper conditions; determining the optimal culture time, and separating and harvesting thalli; and (3) a proper thallus processing and preserving method is adopted, so that the quality and activity are maintained.
The technical scheme adopted by the invention is as follows:
a high-density culture method of lactic acid bacteria, the culture method comprising the steps of:
Step 1, determining the components and the formula of a culture medium;
Step 2, preparing and adjusting a culture medium;
Step 3, sterilizing and asepsis operation;
Step 4, preparing seed bacteria;
Step 5, inoculating operation;
Step 6, controlling culture conditions;
step 7, culturing time and mode;
Step 8, separating and harvesting thalli;
And 9, treating and preserving thalli.
In a preferred embodiment, the medium components and the formula are determined by considering the characteristics of the strain, the purpose of culture and the characteristics of the required product, wherein the medium components are carbon sources, nitrogen sources, inorganic salts and moisture, the carbon sources are one or more of glucose, lactose and maltose, the nitrogen sources are one or more of yeast extract, yeast extract and meat extract, the inorganic salts are one or more of phosphate, sulfate and chloride, and auxiliary components including vitamins and hormones are added to promote the growth of the thallus and the yield of the metabolic products.
In a preferred embodiment, the culture medium is formulated and adjusted such that the various components are added one by one to distilled water according to the formulation of the culture medium, to ensure uniform dissolution, the pH of the culture medium is adjusted and stabilized by adding a pH buffer, and an antioxidant or preservative is added to maintain the stability of the culture medium.
In a preferred embodiment, the sterilization and aseptic manipulation is to load the prepared culture medium into a culture container, wherein the culture container is one or more of a culture flask, a test tube and a culture dish, and perform high-pressure sterilization treatment, and sterilize the culture container for 20-30 minutes with high-pressure steam at 121 ℃, and during the manipulation, the aseptic manipulation needs to be ensured, and a sterile manipulation table, sterile gloves and other sterile vessels are used to avoid bacterial and fungal contamination.
In a preferred scheme, the seed bacteria are prepared by selecting pure seed bacteria liquid as a starting strain for culture, pre-culturing the seed bacteria liquid for one night in advance to enhance the activity of the seed bacteria liquid, and identifying and purifying strains after activating the seed bacteria liquid to ensure that the selected strain is the target lactobacillus.
In a preferred scheme, the inoculation operation is to add a proper amount of activated seed bacterial liquid into a culture medium, shake the activated seed bacterial liquid uniformly and spread the activated seed bacterial liquid in a culture container, ensure the uniform distribution of inoculated bacterial liquid, avoid local over-concentration or over-dilution of bacterial colonies, control the inoculation amount, cause slow bacterial colony growth by the too small inoculation amount, cause too dense bacterial colonies by the too large inoculation amount, influence the supply of oxygen and nutrients, and further influence the growth and yield of bacterial colonies.
In a preferred embodiment, the controlled culture conditions are a temperature of the culture and oxygen, the lactic acid bacteria are grown at a suitable temperature of 35-40 ℃, and the oxygen supply is adjusted by controlling the speed of the shaker, the aeration rate and the shape of the culture vessel to provide a suitable oxygen supply, and the anaerobic lactic acid bacteria select anaerobic culture conditions.
In a preferred scheme, the culture time and the culture mode are determined, the culture time is determined according to specific lactobacillus strains and requirements, the culture time of the lactobacillus is 24-48 hours, the culture time needs longer duration for some slow-growing or special-requirement lactobacillus, timely sampling and monitoring are carried out in the culture process, and the optimal harvest time is judged by observing the thallus growth curve, the change of pH value and the accumulation condition of metabolites.
In a preferred embodiment, the separation and harvest of the cells is performed by separating the cells from the culture medium by a centrifuge or a filter after the high-density culture is completed, washing the precipitated cells with a sterile substance to remove the residual culture medium, and concentrating the cells to a certain concentration by a proper concentration method, and removing the residual culture medium and other impurities by using a microporous filter or a centrifuge.
In a preferred embodiment, the cells are treated and stored as harvested cells for further treatment and utilization, the treatment being one or more of cryopreservation, dry storage, freeze drying, during storage, to ensure sterility of the cells and using suitable storage conditions and containers.
The invention has the technical effects that:
The components and the formula of the culture medium are determined according to the characteristics of the strain, the culture purpose and the characteristics of the required products, so that the requirements of different strains can be met, the culture effect and the product yield are improved, the components of the culture medium are ensured to be fully and uniformly dissolved, the pH value is stable by a method of preparing and adjusting the culture medium, an antioxidant or a preservative is added, the stability of the culture medium is maintained, and a proper environment is provided for the growth of thalli;
Sterilizing and aseptic operating to ensure the aseptic condition of the culture environment, using high-pressure steam sterilizing equipment, matching with aseptic vessels such as an aseptic operation table, aseptic gloves and the like, avoiding the pollution of bacteria and fungi, ensuring the purity of the culture process, in the preparation of seed bacteria, ensuring that the selected strain is target lactobacillus by selecting pure seed bacterial liquid as an initial strain, performing the steps of pre-culture, identification, purification and the like, and improving the culture effect and yield;
The method has the advantages that the proper growth environment is provided by adjusting the culture temperature and oxygen supply, anaerobic culture conditions are selected for the anaerobic lactic acid bacteria, the growth of the bacteria and the yield of metabolites are ensured, the optimal culture time is determined according to specific strains and requirements, the bacteria are ensured to be separated and harvested at the optimal harvesting time through timely sampling and monitoring, the effect of subsequent treatment and utilization is improved, the aseptic state of the bacteria is ensured through proper treatment methods such as freezing preservation, drying preservation and the like, and the proper preservation conditions and containers are used, so that the quality and activity of the bacteria are ensured to be maintained, and the subsequent utilization is facilitated.
Drawings
FIG. 1 is a schematic diagram of a method for high-density culture of lactic acid bacteria according to the present invention.
Description of the embodiments
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
Examples
Referring to FIG. 1, the invention provides a high-density culture method of lactobacillus, which comprises the following steps:
Step 1, determining the components and the formula of a culture medium;
Step 2, preparing and adjusting a culture medium;
Step 3, sterilizing and asepsis operation;
Step 4, preparing seed bacteria;
Step 5, inoculating operation;
Step 6, controlling culture conditions;
step 7, culturing time and mode;
Step 8, separating and harvesting thalli;
And 9, treating and preserving thalli.
Determining the components and the formula of a culture medium in consideration of the characteristics of strains, the purpose of culture and the characteristics of required products, wherein the components of the culture medium comprise a carbon source, a nitrogen source, inorganic salts and moisture, the carbon source comprises glucose and lactose, the nitrogen source comprises yeast extract and yeast extract, the inorganic salts comprise phosphate and sulfate, and auxiliary components comprising vitamins and hormone are added to promote the growth of thalli and the yield of metabolites;
The preparation and adjustment of the culture medium are that according to the formula of the culture medium, various components are added into distilled water one by one to ensure uniform dissolution, the pH value of the culture medium is regulated and stabilized by adding a pH buffer, and an antioxidant or a preservative is added to keep the stability of the culture medium;
The sterilization and aseptic operation is to fill the prepared culture medium into a culture container, wherein the culture container is a culture bottle and a test tube, and is subjected to high-pressure sterilization treatment, and the culture container is sterilized for 20-30 minutes by high-pressure steam at 121 ℃, and in the operation process, the aseptic operation is required to be ensured, and a sterile operation table, sterile gloves and other sterile vessels are used to avoid the pollution of bacteria and fungi;
The preparation of the seed bacteria is to select pure seed bacteria liquid as a cultured initial strain, pre-culture the seed bacteria liquid for one night in advance to enhance the activity of the seed bacteria liquid, and identify and purify strains after activating the seed bacteria liquid to ensure that the selected strains are target lactic acid bacteria;
The inoculation operation is to add a proper amount of activated seed bacterial liquid into a culture medium, shake the culture medium uniformly and spread the culture medium in a culture container to ensure the uniform distribution of the inoculated bacterial liquid, avoid the local over-concentration or over-dilution of bacterial colonies, control the inoculation amount, the too-low inoculation amount can cause slow growth of bacterial colonies, the too-high inoculation amount can cause too-dense bacterial colonies, influence the supply of oxygen and nutrients, and further influence the growth and the yield of bacterial colonies;
The culture conditions are controlled to adjust the culture temperature and oxygen, the lactobacillus grows at a proper temperature, the proper temperature is 35-40 ℃, the oxygen supply is adjusted by controlling the speed of a shaking table, the ventilation and the shape of a culture container so as to provide proper oxygen supply, and anaerobic lactobacillus selects anaerobic culture conditions;
The culture time and the culture mode are determined, the culture time is determined according to specific lactobacillus strains and requirements, the culture time of the lactobacillus is 24-48 hours, the culture time needs longer duration for some slow-growing or special-requirement lactobacillus, timely sampling and monitoring are carried out in the culture process, and the optimal harvesting time is judged by observing the change of a thallus growth curve and pH value and the accumulation condition of metabolites;
separating and harvesting thalli, namely separating thalli from a culture medium through a centrifuge or a filter membrane after high-density culture is finished, flushing the precipitated thalli with sterile substances to remove residual culture medium, obtaining the thalli to a certain concentration by adopting a proper concentration method, and removing the residual culture medium and other impurities by using a microporous filter membrane or the centrifuge;
The thallus is processed and preserved by freezing preservation and drying preservation, and the thallus is preserved in a sterile state and suitable preservation conditions and containers are used.
Examples
Referring to FIG. 1, the invention provides a high-density culture method of lactobacillus, which comprises the following steps:
Step 1, determining the components and the formula of a culture medium;
Step 2, preparing and adjusting a culture medium;
Step 3, sterilizing and asepsis operation;
Step 4, preparing seed bacteria;
Step 5, inoculating operation;
Step 6, controlling culture conditions;
step 7, culturing time and mode;
Step 8, separating and harvesting thalli;
And 9, treating and preserving thalli.
Determining the components and the formula of a culture medium in consideration of the characteristics of strains, the purpose of culture and the characteristics of required products, wherein the components of the culture medium comprise nitrogen sources, inorganic salts and moisture, the carbon sources comprise glucose, lactose and maltose, the nitrogen sources comprise yeast extracts and meat extracts, the inorganic salts comprise sulfate and chloride, and the auxiliary components comprise vitamins and hormone so as to promote the growth of thalli and the yield of metabolic products;
The preparation and adjustment of the culture medium are that according to the formula of the culture medium, various components are added into distilled water one by one to ensure uniform dissolution, the pH value of the culture medium is regulated and stabilized by adding a pH buffer, and an antioxidant or a preservative is added to keep the stability of the culture medium;
The sterilization and aseptic operation is to fill the prepared culture medium into a culture container, wherein the culture container is a test tube or a culture dish, and is subjected to high-pressure sterilization treatment, and the culture container is sterilized by high-pressure steam at 121 ℃ for 20-30 minutes, and in the operation process, the aseptic operation is required to be ensured, and a sterile operation table, sterile gloves and other sterile vessels are used to avoid the pollution of bacteria and fungi;
The preparation of the seed bacteria is to select pure seed bacteria liquid as a cultured initial strain, pre-culture the seed bacteria liquid for one night in advance to enhance the activity of the seed bacteria liquid, and identify and purify strains after activating the seed bacteria liquid to ensure that the selected strains are target lactic acid bacteria;
The inoculation operation is to add a proper amount of activated seed bacterial liquid into a culture medium, shake the culture medium uniformly and spread the culture medium in a culture container to ensure the uniform distribution of the inoculated bacterial liquid, avoid the local over-concentration or over-dilution of bacterial colonies, control the inoculation amount, the too-low inoculation amount can cause slow growth of bacterial colonies, the too-high inoculation amount can cause too-dense bacterial colonies, influence the supply of oxygen and nutrients, and further influence the growth and the yield of bacterial colonies;
The culture conditions are controlled to adjust the culture temperature and oxygen, the lactobacillus grows at a proper temperature, the proper temperature is 35-40 ℃, the oxygen supply is adjusted by controlling the speed of a shaking table, the ventilation and the shape of a culture container so as to provide proper oxygen supply, and anaerobic lactobacillus selects anaerobic culture conditions;
The culture time and the culture mode are determined, the culture time is determined according to specific lactobacillus strains and requirements, the culture time of the lactobacillus is 24-48 hours, the culture time needs longer duration for some slow-growing or special-requirement lactobacillus, timely sampling and monitoring are carried out in the culture process, and the optimal harvesting time is judged by observing the change of a thallus growth curve and pH value and the accumulation condition of metabolites;
separating and harvesting thalli, namely separating thalli from a culture medium through a centrifuge or a filter membrane after high-density culture is finished, flushing the precipitated thalli with sterile substances to remove residual culture medium, obtaining the thalli to a certain concentration by adopting a proper concentration method, and removing the residual culture medium and other impurities by using a microporous filter membrane or the centrifuge;
The thallus is processed and stored for further processing and utilization, and the processing method is drying storage and freeze drying, and during the storage, the thallus is ensured to be in a sterile state and proper storage conditions and containers are used.
Examples
Referring to FIG. 1, the invention provides a high-density culture method of lactobacillus, which comprises the following steps:
Step 1, determining the components and the formula of a culture medium;
Step 2, preparing and adjusting a culture medium;
Step 3, sterilizing and asepsis operation;
Step 4, preparing seed bacteria;
Step 5, inoculating operation;
Step 6, controlling culture conditions;
step 7, culturing time and mode;
Step 8, separating and harvesting thalli;
And 9, treating and preserving thalli.
Determining the components and the formula of a culture medium in consideration of the characteristics of strains, the purpose of culture and the characteristics of required products, wherein the components of the culture medium comprise a carbon source, a nitrogen source, inorganic salts and moisture, the carbon source comprises glucose and maltose, the nitrogen source comprises yeast extract and meat extract, the inorganic salts comprise phosphate and chloride, and the auxiliary components comprise vitamins and hormone so as to promote the growth of thalli and the yield of metabolites;
The preparation and adjustment of the culture medium are that according to the formula of the culture medium, various components are added into distilled water one by one to ensure uniform dissolution, the pH value of the culture medium is regulated and stabilized by adding a pH buffer, and an antioxidant or a preservative is added to keep the stability of the culture medium;
The sterilization and aseptic operation is to fill the prepared culture medium into a culture container, wherein the culture container is a culture bottle or a culture dish, and the culture container is subjected to high-pressure sterilization treatment, and is sterilized by high-pressure steam at 121 ℃ for 20-30 minutes, and in the operation process, the aseptic operation is required to be ensured, and a sterile operation table, sterile gloves and other sterile vessels are used to avoid the pollution of bacteria and fungi;
The preparation of the seed bacteria is to select pure seed bacteria liquid as a cultured initial strain, pre-culture the seed bacteria liquid for one night in advance to enhance the activity of the seed bacteria liquid, and identify and purify strains after activating the seed bacteria liquid to ensure that the selected strains are target lactic acid bacteria;
The inoculation operation is to add a proper amount of activated seed bacterial liquid into a culture medium, shake the culture medium uniformly and spread the culture medium in a culture container to ensure the uniform distribution of the inoculated bacterial liquid, avoid the local over-concentration or over-dilution of bacterial colonies, control the inoculation amount, the too-low inoculation amount can cause slow growth of bacterial colonies, the too-high inoculation amount can cause too-dense bacterial colonies, influence the supply of oxygen and nutrients, and further influence the growth and the yield of bacterial colonies;
The culture conditions are controlled to adjust the culture temperature and oxygen, the lactobacillus grows at a proper temperature, the proper temperature is 35-40 ℃, the oxygen supply is adjusted by controlling the speed of a shaking table, the ventilation and the shape of a culture container so as to provide proper oxygen supply, and anaerobic lactobacillus selects anaerobic culture conditions;
The culture time and the culture mode are determined, the culture time is determined according to specific lactobacillus strains and requirements, the culture time of the lactobacillus is 24-48 hours, the culture time needs longer duration for some slow-growing or special-requirement lactobacillus, timely sampling and monitoring are carried out in the culture process, and the optimal harvesting time is judged by observing the change of a thallus growth curve and pH value and the accumulation condition of metabolites;
separating and harvesting thalli, namely separating thalli from a culture medium through a centrifuge or a filter membrane after high-density culture is finished, flushing the precipitated thalli with sterile substances to remove residual culture medium, obtaining the thalli to a certain concentration by adopting a proper concentration method, and removing the residual culture medium and other impurities by using a microporous filter membrane or the centrifuge;
The thallus is processed and stored for further processing and utilization, and the processing method is freezing preservation and freeze drying, and during the preservation, the thallus is ensured to be in a sterile state and proper preservation conditions and containers are used.
According to the invention, the components and the formula of a culture medium are determined according to the characteristics of the strain, the culture purpose and the characteristics of required products, the requirements of different strains can be met, the culture effect and the product yield are improved, the culture effect and the product yield are ensured by preparing and adjusting the culture medium, the culture medium components are fully and uniformly dissolved, the pH value is stable, an antioxidant or preservative is added, the stability of the culture medium is maintained, the proper environment is provided for the growth of thalli, the sterilization and aseptic operation is ensured, the aseptic state of the culture environment is ensured, a high-pressure steam sterilization device is used, a sterile operation table, sterile gloves and other sterile vessels are matched, the pollution of bacteria and fungi is avoided, the purity of the culture process is ensured, in the preparation of seed bacteria, the pure seed bacteria liquid is selected as an initial strain, the steps of preculture and identification and purification are performed, the selected strain is ensured to be target lactobacillus, the culture effect and the yield are improved, the proper growth environment is provided by adjusting the culture temperature and oxygen supply, the anaerobic culture condition is selected for the anaerobic lactobacillus, the growth and the metabolite yield are ensured, the optimal culture time is determined according to the specific environment and requirements, the proper sample and strain is used for monitoring, the proper sample and time are ensured, the bacterial harvesting and the bacterial quality are separated at the optimal time, the subsequent harvest and the quality is ensured, the proper quality is ensured, the quality is ensured and the quality is convenient to be preserved and is ensured by the preservation and the quality is convenient to be preserved and preserved by the method.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention. Structures, devices and methods of operation not specifically described and illustrated herein, unless otherwise indicated and limited, are implemented according to conventional means in the art.
Claims (10)
1. A high-density culture method of lactic acid bacteria is characterized in that: the culture method comprises the following steps:
Step 1, determining the components and the formula of a culture medium;
Step 2, preparing and adjusting a culture medium;
Step 3, sterilizing and asepsis operation;
Step 4, preparing seed bacteria;
Step 5, inoculating operation;
Step 6, controlling culture conditions;
step 7, culturing time and mode;
Step 8, separating and harvesting thalli;
And 9, treating and preserving thalli.
2. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the method comprises the steps of determining culture medium components and formulas, wherein the characteristics of strains, the purpose of culture and the characteristics of required products are considered, the culture medium components comprise a carbon source, a nitrogen source, inorganic salts and moisture, the carbon source comprises one or more of glucose, lactose and maltose, the nitrogen source comprises one or more of yeast extract, yeast extract and meat extract, the inorganic salts comprise one or more of phosphate, sulfate and chloride, and the auxiliary components comprise vitamins and hormones so as to promote the growth of thalli and the yield of metabolic products.
3. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the preparation and adjustment of the culture medium are that various components are added into distilled water one by one according to the culture medium formula, so as to ensure uniform dissolution, the pH value of the culture medium is regulated and stabilized by adding a pH buffer, and an antioxidant or a preservative is added so as to keep the stability of the culture medium.
4. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the sterilization and aseptic operation is to fill the prepared culture medium into a culture container, wherein the culture container is one or more of a culture bottle, a test tube and a culture dish, and is subjected to high-pressure sterilization treatment, and the culture container is sterilized by high-pressure steam at 121 ℃ for 20-30 minutes, and during the operation, the aseptic operation is required to be ensured, and a sterile operation table, sterile gloves and other sterile vessels are used to avoid the pollution of bacteria and fungi.
5. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the preparation of the seed bacteria is to select pure seed bacteria liquid as a cultured initial strain, pre-culture the seed bacteria liquid for one night in advance to enhance the activity of the seed bacteria liquid, and identify and purify strains after activating the seed bacteria liquid to ensure that the selected strains are target lactic acid bacteria.
6. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the inoculation operation is to add a proper amount of activated seed bacterial liquid into a culture medium, shake the activated seed bacterial liquid uniformly and spread the activated seed bacterial liquid in a culture container, ensure the uniform distribution of inoculated bacterial liquid, avoid local over-concentration or over-dilution of bacterial colonies, control the inoculation amount, the too small inoculation amount can lead to slow growth of bacterial colonies, the too large inoculation amount can lead to too dense bacterial colonies, influence the supply of oxygen and nutrients, and further influence the growth and the yield of bacterial colonies.
7. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the culture conditions are that the culture temperature and oxygen are regulated, the lactobacillus grows at a proper temperature, the proper temperature is 35-40 ℃, and the oxygen supply is controlled by the methods of standing culture, filling inert gas and the like according to the characteristics of the lactobacillus preferring anaerobic environment.
8. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the culture time and the culture mode are determined, the culture time is determined according to different strains and requirements of specific lactic acid bacteria, the culture time of the lactic acid bacteria is 15-48 hours, the culture time needs longer duration for some lactic acid bacteria which grow slowly or are in special requirements, timely sampling and monitoring are carried out in the culture process, and the optimal harvesting time is judged by observing the growth curve of the thallus, the change of pH value and the accumulation condition of metabolites.
9. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: separating and harvesting the thalli, namely separating the thalli from the culture medium through a centrifugal machine or a filter membrane after the high-density culture is finished, flushing the precipitated thalli with sterile substances to remove the residual culture medium, adopting a proper concentration method to obtain the thalli with a certain concentration, and using the microporous filter membrane or the centrifugal machine to remove the residual culture medium and other impurities.
10. The method for high-density cultivation of lactic acid bacteria according to claim 1, wherein: the thallus is processed and stored for further processing and utilization, the processing method is one or more of freezing preservation, drying preservation and freeze drying, and during the preservation, the aseptic state of the thallus is ensured, and proper preservation conditions and containers are used.
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| CN105820989A (en) * | 2016-06-15 | 2016-08-03 | 苏州健世星生物科技有限公司 | High-density culture method of direct-feeding-type leavening agent probiotic lactic bacteria |
| CN115584356A (en) * | 2022-10-26 | 2023-01-10 | 广西工业职业技术学院 | Method for producing lactic acid by aerobic high-density fermentation of lactic acid bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820989A (en) * | 2016-06-15 | 2016-08-03 | 苏州健世星生物科技有限公司 | High-density culture method of direct-feeding-type leavening agent probiotic lactic bacteria |
| CN115584356A (en) * | 2022-10-26 | 2023-01-10 | 广西工业职业技术学院 | Method for producing lactic acid by aerobic high-density fermentation of lactic acid bacteria |
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