Preparation method of compound microbial lactic acid bacteria

Abstract

The invention relates to a preparation method of compound microbial lactic acid bacteria. The preparation method comprises the following specific operation steps: S1, activating bacterial strains; s2, preparing a seed solution; s3, fermentation culture; s4, collecting thalli; s5, preserving the thalli; adding a proper amount of a protective agent into the collected thalli to enable the final concentration of the thalli to reach 10-20%, fully and uniformly mixing the thalli, subpackaging the thalli into sterile cryopreservation tubes, and storing the sterile cryopreservation tubes in a refrigerator at the temperature of-80 DEG C for later use; by adopting the compound strains of enterococcus faecalis, lactobacillus paracasei, lactobacillus acidophilus and lactobacillus plantarum, the utilization range of a fermentation substrate is widened, the product diversity is improved, heterotype / homotype fermentation strains are matched, the lactic acid / acetic acid ratio is balanced, acid inhibition is avoided, the method is suitable for functional preparation development, compound flora competitively inhibits infectious microbes, and the sterilization cost is reduced; the fermentation success rate is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation and cultivation of composite microbial lactic acid bacteria, in particular to a preparation method of composite microbial lactic acid bacteria. Background Art

[0002] Composite microbial lactic acid bacteria (composed of a combination of multiple lactic acid bacteria, such as Enterococcus faecalis, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus plantarum, etc.) have wide application value in many fields due to their synergistic effects and multifunctional metabolic characteristics.

[0003] Existing composite microbial lactic acid bacteria use only a single strain, a single metabolic pathway, a single product, rely on strict sterilization, and are susceptible to contamination. At the same time, during cultivation, the initial pH is fixed and no adjustment is made later, and the dissolved oxygen is fixed throughout the process, relying only on experience or a single indicator. Summary of the Invention

[0004] The problem solved by the present invention is to provide a method for preparing composite microbial lactic acid bacteria, which solves the technical problems that existing composite microbial lactic acid bacteria only use a single strain, a single metabolic pathway, a single product, rely on strict sterilization, are susceptible to contamination, and at the same time, during cultivation, the initial pH is fixed, no adjustment is made later, the dissolved oxygen is fixed throughout the process, and only relies on experience or a single indicator.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The preparation method of composite microbial lactic acid bacteria, the specific operation steps of the preparation method are as follows:

[0007] S1: strain activation

[0008] Remove the lactic acid bacteria strain from the preserved freeze-dried tube or slant, inoculate it into a test tube containing 5 mL of seed culture medium, and culture it in a 37°C constant temperature incubator for 18-24 hours for activation. Transfer 1-2 loops of the activated strain to new seed culture medium and culture it under the same conditions. Repeat the activation 2-3 times to ensure the viability and purity of the strain.

[0009] S2: Seed solution preparation

[0010] Take the activated strain and inoculate it into a 500mL Erlenmeyer flask containing 100mL seed culture medium at an inoculum size of 2-5% (v / v). Incubate the culture in a shaking incubator at 37°C and 150-200 rpm for 18-24 hours to obtain the seed solution. During the culture process, regularly check the pH value and bacterial solution concentration to ensure the quality of the seed solution.

[0011] S3: Fermentation culture

[0012] The seed liquid is inoculated into a fermentation tank filled with fermentation medium at an inoculum size of 5-10% (v / v), and the fermentation conditions are controlled as follows: temperature 37° C., stirring speed 150-200 rpm, ventilation volume is adjusted as needed, and dissolved oxygen is maintained at 20-30%. During the fermentation process, samples are taken at regular intervals to test indicators such as pH value, bacterial liquid concentration, and metabolites. Fermentation parameters are adjusted based on the test results, and fermentation is terminated until the bacterial liquid concentration reaches a predetermined value, i.e., OD600 is greater than 1.5, or metabolite accumulation reaches a certain level, i.e., homofermentative lactic acid bacteria ≥8 g / L and heterofermentative lactic acid bacteria ≥5 g / L.

[0013] S4: Bacteria Collection

[0014] Centrifuge the fermentation broth at 4°C and 5000-8000 rpm for 10-15 minutes to collect the cells or directly collect them together with the metabolites. Wash the cells 2-3 times with sterile saline or buffer to remove impurities and culture medium components.

[0015] S5: Bacteria preservation

[0016] Add an appropriate amount of protective agent to the collected bacteria to make the final concentration reach 10-20%, mix thoroughly, and then divide into sterile cryopreservation tubes and store in a -80℃ refrigerator for later use.

[0017] Preferably, the lactic acid bacteria strains include Enterococcus faecalis, Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus plantarum.

[0018] Preferably, the seed culture medium is MRS medium, which includes 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 20 g of agar, 1000 mL of distilled water, and a pH of 6.2-6.4;

[0019] Fermentation medium: optimized MRS medium or other suitable medium.

[0020] Preferably, the protective agent includes glycerol and skim milk.

[0021] Preferably, the composite microbial lactic acid bacteria are subjected to morphological observation, bacterial liquid concentration detection and metabolite detection;

[0022] Morphological observation: Observe the bacterial morphology under a microscope to ensure that it is pure lactic acid bacteria;

[0023] Bacterial solution concentration detection: Use plate count method or spectrophotometer method to determine the bacterial solution concentration to ensure that a certain number of viable bacteria is reached;

[0024] Metabolite detection: such as detecting the content of metabolites such as lactic acid and acetic acid to evaluate the metabolic activity of the strain.

[0025] The beneficial effects of the present invention are as follows: the present application adopts a composite strain of Enterococcus faecalis, Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus plantarum, broadens the scope of fermentation substrate utilization, improves product diversity, combines heterotypic and homotypic fermentation strains, balances the lactic acid / acetic acid ratio, avoids acid inhibition, is suitable for the development of functional preparations, and the composite bacterial community competitively inhibits miscellaneous bacteria, reduces sterilization costs, and improves the fermentation success rate;

[0026] Real-time monitoring and alkali supplementation prolong the logarithmic growth phase, increase the viable bacterial count by 30%-50%, and adjust according to the stage, 20%-30% in the logarithmic phase and <10% in the stable phase, reduce oxidative stress, and increase the bacterial survival rate by 20%. Multiple indicators, including OD600, lactic acid, and viable bacterial count, avoid over-fermentation and maximize product yield;

[0027] The number of live bacteria increased by 50%-200%, the lactic acid production efficiency increased by more than 40%, the frozen survival rate was increased, and the shelf life of the bacterial agent was extended. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing experimental data of the activation stage of the strain of the present invention;

[0029] Figure 2 This is a graph showing experimental data from the seed solution preparation stage of the present invention;

[0030] Figure 3 This is a diagram of experimental data during the fermentation culture stage of the present invention;

[0031] Figure 4 This is a graph showing experimental data for the bacterial collection and preservation stages of the present invention;

[0032] Figure 5 This is the experimental data diagram of the final report stage of the quality inspection of the present invention;

[0033] Figure 6 This is a graph showing the experimental data of high-density culture and product efficiency improvement of the present invention. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] Specific examples are given below.

[0036] See also Figures 1 to 6 The preparation method of composite microbial lactic acid bacteria, the specific operation steps of the preparation method are as follows:

[0037] S1: strain activation

[0038] Remove the lactic acid bacteria strain from the preserved freeze-dried tube or slant, inoculate it into a test tube containing 5 mL of seed culture medium, and culture it in a 37°C constant temperature incubator for 18-24 hours for activation. Transfer 1-2 loops of the activated strain to new seed culture medium and culture it under the same conditions. Repeat the activation 2-3 times to ensure the viability and purity of the strain.

[0039] S2: Seed solution preparation

[0040] Take the activated strain and inoculate it into a 500mL Erlenmeyer flask containing 100mL seed culture medium at an inoculum size of 2-5% (v / v). Incubate the culture in a shaking incubator at 37°C and 150-200 rpm for 18-24 hours to obtain the seed solution. During the culture process, regularly check the pH value and bacterial solution concentration to ensure the quality of the seed solution.

[0041] S3: Fermentation culture

[0042] The seed liquid is inoculated into a fermentation tank filled with fermentation medium at an inoculum size of 5-10% (v / v), and the fermentation conditions are controlled as follows: temperature 37° C., stirring speed 150-200 rpm, ventilation volume is adjusted as needed, and dissolved oxygen is maintained at 20-30%. During the fermentation process, samples are taken at regular intervals to test indicators such as pH value, bacterial liquid concentration, and metabolites. Fermentation parameters are adjusted based on the test results, and fermentation is terminated until the bacterial liquid concentration reaches a predetermined value, i.e., OD600 is greater than 1.5, or metabolite accumulation reaches a certain level, i.e., homofermentative lactic acid bacteria ≥8 g / L and heterofermentative lactic acid bacteria ≥5 g / L.

[0043] S4: Bacteria Collection

[0044] Centrifuge the fermentation broth at 4°C and 5000-8000 rpm for 10-15 minutes to collect the cells or directly collect them together with the metabolites. Wash the cells 2-3 times with sterile saline or buffer to remove impurities and culture medium components.

[0045] S5: Bacteria preservation

[0046] The collected bacteria were added with an appropriate amount of protective agent, including glycerol and skim milk, to a final concentration of 10-20%. After thorough mixing, the cells were divided into sterile cryopreservation tubes and stored in a -80°C refrigerator for later use.

[0047] The lactic acid bacteria strains include Enterococcus faecalis, Lactobacillus paracasei, Lactobacillus acidophilus, and Lactobacillus plantarum. The seed culture medium is MRS medium, which includes 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 20 g of agar, and 1000 mL of distilled water, with a pH of 6.2-6.4.

[0048] Fermentation medium: optimized MRS medium or other suitable medium.

[0049] Conduct morphological observation, bacterial liquid concentration detection and metabolite detection on composite microbial lactic acid bacteria;

[0050] Morphological observation: Observe the bacterial morphology under a microscope to ensure that it is pure lactic acid bacteria;

[0051] Bacterial solution concentration detection: Use plate count method or spectrophotometer method to determine the bacterial solution concentration to ensure that a certain number of viable bacteria is reached;

[0052] Metabolite detection: such as detecting the content of metabolites such as lactic acid and acetic acid to evaluate the metabolic activity of the strain.

[0053] The present application uses a composite strain of Enterococcus faecalis, Lactobacillus paracasei, Lactobacillus acidophilus, and Lactobacillus plantarum to broaden the scope of fermentation substrate utilization, enhance product diversity, and combine heterotypic and homotypic fermentation strains to balance the lactic acid / acetic acid ratio, avoid acid inhibition, and be suitable for the development of functional preparations. The composite bacterial community competitively inhibits miscellaneous bacteria, reduces sterilization costs, and improves the fermentation success rate.

[0054] Real-time monitoring and alkali supplementation prolong the logarithmic growth phase, increase the viable bacterial count by 30%-50%, and adjust according to the stage, 20%-30% in the logarithmic phase and <10% in the stable phase, reduce oxidative stress, and increase the bacterial survival rate by 20%. Multiple indicators, including OD600, lactic acid, and viable bacterial count, avoid over-fermentation and maximize product yield;

[0055] The number of live bacteria increased by 50%-200%, the lactic acid production efficiency increased by more than 40%, the frozen survival rate was increased, and the shelf life of the bacterial agent was extended.

[0056] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a composite microbial lactic acid bacteria, characterized in that: The specific steps of the preparation method are as follows: S1: strain activation Remove the lactic acid bacteria strain from the preserved freeze-dried tube or slant, inoculate it into a test tube containing 5 mL of seed culture medium, and culture it in a 37°C constant temperature incubator for 18-24 hours for activation. Transfer 1-2 loops of the activated strain to new seed culture medium and culture it under the same conditions. Repeat the activation 2-3 times to ensure the viability and purity of the strain. S2: Seed solution preparation Take the activated strain and inoculate it into a 500mL Erlenmeyer flask containing 100mL seed culture medium at an inoculum size of 2-5% (v / v). Incubate the culture in a shaking incubator at 37°C and 150-200 rpm for 18-24 hours to obtain the seed solution. During the culture process, regularly check the pH value and bacterial solution concentration to ensure the quality of the seed solution. S3: Fermentation culture The seed liquid is inoculated into a fermentation tank filled with fermentation medium at an inoculum size of 5-10% (v / v), and the fermentation conditions are controlled as follows: temperature 37° C., stirring speed 150-200 rpm, ventilation volume is adjusted as needed, and dissolved oxygen is maintained at 20-30%. During the fermentation process, samples are taken at regular intervals to test indicators such as pH value, bacterial liquid concentration, and metabolites. Fermentation parameters are adjusted based on the test results, and fermentation is terminated until the bacterial liquid concentration reaches a predetermined value, i.e., OD600 is greater than 1.5, or metabolite accumulation reaches a certain level, i.e., homofermentative lactic acid bacteria ≥8 g / L and heterofermentative lactic acid bacteria ≥5 g / L. S4: Bacteria Collection Centrifuge the fermentation broth at 4°C and 5000-8000 rpm for 10-15 minutes to collect the cells or directly collect them together with the metabolites. Wash the cells 2-3 times with sterile saline or buffer to remove impurities and culture medium components. S5: Bacteria preservation Add an appropriate amount of protective agent to the collected bacteria to make the final concentration reach 10-20%, mix thoroughly, and then divide into sterile cryopreservation tubes and store in a -80℃ refrigerator for later use.

2. The method for preparing the composite microbial lactic acid bacteria according to claim 1, wherein The lactic acid bacteria strains include Enterococcus faecalis, Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus plantarum.

3. The method for preparing the composite microbial lactic acid bacteria according to claim 2, wherein: The seed culture medium is MRS culture medium, which includes 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 20g of agar, 1000mL of distilled water, and a pH of 6.2-6.4; Fermentation medium: optimized MRS medium or other suitable medium.

4. The method for preparing the composite microbial lactic acid bacteria according to claim 3, wherein: The protective agents include glycerin and skim milk.

5. The method for preparing the composite microbial lactic acid bacteria according to claim 4, characterized in that: Conduct morphological observation, bacterial liquid concentration detection and metabolite detection on composite microbial lactic acid bacteria; Morphological observation: Observe the bacterial morphology under a microscope to ensure that it is pure lactic acid bacteria; Bacterial solution concentration detection: Use plate count method or spectrophotometer method to determine the bacterial solution concentration to ensure that a certain number of viable bacteria is reached; Metabolite detection: such as detecting the content of metabolites such as lactic acid and acetic acid to evaluate the metabolic activity of the strain.

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