Method for regulating metabolism of high-density fermentation of lactic acid bacteria

Abstract

The application belongs to the technical field of microbial fermentation, and particularly relates to a high-density fermentation metabolic regulation method of lactic acid bacteria. The application provides a high-density culture method of lactic acid bacteria. The formula of the high-density fermentation culture medium is as follows: 10-20 g / L of proteose peptone, 10-15 g / L of beef extract, 5-10 g / L of yeast powder, 8-15 g / L of D-sorbitol, 5-10 g / L of L-proline, 3-7 g / L of sodium chloride, 0.3-0.6 g / L of magnesium sulfate 7H2O, 0.1-0.2 g / L of manganese sulfate 1H2O, 0.1-0.2 g / L of ferrous sulfate 7H2O, and pH is 6.0-7.0. The fermentation culture medium significantly improves the viable cell count of Lactobacillus fermentum CQPC04, Lactobacillus rhamnosus 4F225, Lactobacillus plantarum H6 and Lactobacillus gasseri GS18, realizes high-density culture of lactic acid bacteria, and lays a foundation for industrialized production of lactic acid bacteria.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to a high-density fermentation metabolic regulation method of lactic acid bacteria. BACKGROUND

[0002] Lactic acid bacteria (LAB) is a collective term for bacteria that produce large amounts of lactic acid by utilizing fermentable carbohydrates. This type of bacteria is widely distributed in nature and has rich species diversity. Except for a few, most of them are essential and have important physiological functions in the human body, and are widely present in the intestinal tract of the human body.

[0003] The organic acids, special enzyme systems, acid bacteriocins and other substances produced by lactic acid bacteria through fermentation have special physiological functions. A large amount of research data shows that lactic acid bacteria can promote animal growth, regulate normal flora in the gastrointestinal tract, maintain microecological balance, thereby improving gastrointestinal function, increasing food digestibility and biological value, reducing serum cholesterol, controlling endotoxins, inhibiting the growth of putrefactive bacteria in the intestinal tract, and improving the body's immunity. Lactic acid bacteria are fermentation bacteria with one or several inherent functional characteristics. Their various advantages in the senses, technology, nutrition or health make them have great potential in the food fermentation industry. They can be used for food preservation, improving food safety, improving food texture and flavor, accelerating cheese ripening, producing functional factors, removing toxic and side factors, and thus making food have health care efficacy. Among them, lactic acid bacteriocin is a kind of antibacterial polypeptide or protein synthesized and secreted by lactic acid bacteria during metabolism, which has a lytic effect. Because lactic acid bacteria are considered as food-grade microorganisms recognized worldwide, the antibacterial substances produced by them can be directly applied in the food industry as natural food preservatives.

[0004] Chinese patent CN 112126599 B discloses a high-density culture method of Lactobacillus helveticus, preparation of high-vigor bacterial powder and application thereof, which comprises the following steps: (1) inoculating Lactobacillus helveticus strain into the activated seed culture medium to obtain seed liquid; (2) inoculating the seed liquid into the high-density fermentation culture medium of Lactobacillus helveticus, and performing fermentation culture under the conditions of 35-43℃ and anaerobic stirring, to obtain live bacterial fermentation liquid after the fermentation is completed. The high-density whey proliferation culture medium provided by the application comprises 10-50g of whey powder, 1-10g of whey protein concentrate, 20-60g of soy peptone, 5-50g of concentrated tomato juice, 1-15g of yeast powder, 0.28-0.3g of manganese sulfate, 0.58-0.6g of magnesium sulfate, 1-5g of dipotassium hydrogen phosphate and 1-10g of sodium citrate; and Lactobacillus helveticus WHH2580 is subjected to high-density culture, so that a large number of bacterial bodies can be obtained.

[0005] The application discloses a high-density fermentation process for fermenting lactic acid bacteria and belongs to the technical field of lactic acid bacteria culture.

[0006] In view of the extremely high practical application value of lactic acid bacteria, other methods for increasing the number of live lactic acid bacteria in fermentation are needed to meet the application requirements of lactic acid bacteria. SUMMARY

[0007] To solve the above problems, the application provides a high-density culture method for lactic acid bacteria, and the formula of the high-density fermentation medium is as follows: 10-20 g / L of proteose peptone, 10-15 g / L of beef extract, 5-10 g / L of yeast powder, 8-15 g / L of D-sorbitol, 5-10 g / L of L-proline, 3-7 g / L of sodium chloride, 0.3-0.6 g / L of magnesium sulfate·7H2O, 0.1-0.2 g / L of manganese sulfate·1H2O, 0.1-0.2 g / L of ferrous sulfate·7H2O, and pH is 6.0-7.0.

[0008] In one aspect, the application provides a high-density culture method for lactic acid bacteria, which comprises the following steps:

[0009] (1) inoculating lactic acid bacteria into an activation culture medium to obtain a seed liquid through culture;

[0010] (2) inoculating the seed liquid obtained in step (1) into a high-density fermentation medium and carrying out stirring fermentation under anaerobic conditions.

[0011] The formula of the high-density fermentation medium is as follows: 10-20 g / L of proteose peptone, 10-15 g / L of beef extract, 5-10 g / L of yeast powder, 8-15 g / L of D-sorbitol, 5-10 g / L of L-proline, 3-7 g / L of sodium chloride, 0.3-0.6 g / L of magnesium sulfate·7H2O, 0.1-0.2 g / L of manganese sulfate·1H2O, 0.1-0.2 g / L of ferrous sulfate·7H2O, and pH is 6.0-7.0.

[0012] Specifically, the inoculation amount of the seed liquid in step (2) can be 2-10% by volume.

[0013] Specifically, the anaerobic condition in step (2) is to introduce N2 and CO2 mixed gas into the fermenter; and the stirring speed can be 50-80 rpm.

[0014] Further specifically, the ratio of N2 and CO2 is 1:4.

[0015] Specifically, the fermentation temperature in step (2) can be 35-40℃, the pH in fermentation can be 5.5-6.0, and the fermentation time can be 8-12h.

[0016] Further specifically, the pH in fermentation is controlled by adding a regulator.

[0017] Preferably, the regulator includes but is not limited to NaOH and / or Na2CO3.

[0018] Specifically, the lactic acid bacteria include but are not limited to Lactobacillus fermentum CQPC04 and / or Lactobacillus rhamnosus 4F225; the accession number of Lactobacillus fermentum CQPC04 is CGMCC NO.14493, and the accession number of Lactobacillus rhamnosus 4F225 is CGMCC NO.26437.

[0019] Further specifically, the formula of the high-density fermentation medium of Lactobacillus fermentum CQPC04 is: peptone 10g / L, beef extract 15g / L, yeast powder 5g / L, D-sorbitol 8g / L, L-proline 10g / L, sodium chloride 3g / L, magnesium sulfate·7H2O 0.6g / L, manganese sulfate·1H2O 0.1g / L, ferrous sulfate·7H2O 0.1g / L, and pH is 7.0.

[0020] Preferably, the fermentation temperature is 37℃, and the fermentation time is 10h.

[0021] Further specifically, the formula of the high-density fermentation medium of Lactobacillus rhamnosus 4F225 is: peptone 20g / L, beef extract 10g / L, yeast powder 10g / L, D-sorbitol 15g / L, L-proline 5g / L, sodium chloride 7g / L, magnesium sulfate·7H2O 0.3g / L, manganese sulfate·1H2O 0.2g / L, ferrous sulfate·7H2O 0.2g / L, and pH is 6.5.

[0022] Preferably, the fermentation temperature is 37℃, and the fermentation time is 10h.

[0023] Further specifically, the high-density fermentation medium of the Lactobacillus plantarum H6 has the following formulation: 15 g / L of proteose peptone, 12 g / L of beef extract, 8 g / L of yeast powder, 10 g / L of D-sorbitol, 7 g / L of L-proline, 5 g / L of sodium chloride, 0.5 g / L of magnesium sulfate 7H2O, 0.15 g / L of manganese sulfate 1H2O, 0.15 g / L of ferrous sulfate 7H2O, and pH 6.0.

[0024] Preferably, the fermentation temperature is 37℃, and the fermentation time is 10 h.

[0025] Further specifically, the high-density fermentation medium of the Lactobacillus gasseri GS18 has the following formulation: 10 g / L of proteose peptone, 15 g / L of beef extract, 5 g / L of yeast powder, 8 g / L of D-sorbitol, 10 g / L of L-proline, 3 g / L of sodium chloride, 0.6 g / L of magnesium sulfate 7H2O, 0.1 g / L of manganese sulfate 1H2O, 0.1 g / L of ferrous sulfate 7H2O, and pH 7.0.

[0026] Preferably, the fermentation temperature is 37℃, and the fermentation time is 10 h.

[0027] In another aspect, the present application provides the use of the lactic acid bacteria cultured by the aforementioned method in the production of lactic acid.

[0028] In another aspect, the present application provides the use of the lactic acid bacteria cultured by the aforementioned method in the production of lactic acid bacteria.

[0029] In another aspect, the present application provides the use of the lactic acid bacteria cultured by the aforementioned method in the production of lactic acid bacteria.

[0030] Specifically, the dairy products include, but are not limited to, yogurt, cheese, fermented buttermilk, lactic acid bacteria beverage, kefir, sour cream, and / or kumis.

[0031] In another aspect, the present application provides the use of the lactic acid bacteria cultured by the aforementioned method in the production of silage.

[0032] Specifically, the raw materials of the silage are selected from corn, blackgrass, smooth bromegrass, alfalfa, clover, Chinese milk vetch, sweet potato, pumpkin, and / or amaranth.

[0033] In another aspect, the present application provides the use of the lactic acid bacteria cultured by the aforementioned method in the improvement of the production performance of poultry and livestock.

[0034] Specifically, the production performance of poultry and livestock includes, but is not limited to, the survival rate of chicks and / or the survival rate of weaned piglets.

[0035] In yet another aspect, the present application provides the use of the lactic acid bacteria cultured by the aforementioned method in aquaculture.

[0036] Specifically, the use is mainly embodied in water quality improvement, disease treatment, and immunization of aquatic animals.

[0037] Technical effects achieved by the present application:

[0038] (1) Short fermentation time, the fermentation time is 8-12h.

[0039] (2) High viable count, the viable count of Lactobacillus fermentum CQPC04, Lactobacillus rhamnosus 4F225, Lactobacillus plantarum H6 and Lactobacillus gasseri GS18 is improved, realizing high-density fermentation of lactic acid bacteria.

[0040] (3) The production cost is reduced, and technical support is provided for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The figure is the viable count result of Lactobacillus fermentum CQPC04 Example 1 and Comparative Examples 1-3.

[0042] Figure 2 The figure is the viable count result of Lactobacillus rhamnosus 4F225 Example 1 and Comparative Examples 1-3.

[0043] Figure 3 The figure is the viable count result of Lactobacillus plantarum H6 Example 1 and Comparative Examples 1-3.

[0044] Figure 4 The figure is the viable count result of Lactobacillus gasseri GS18 Example 1 and Comparative Examples 1-3. DETAILED DESCRIPTION

[0045] The present application will be further described in detail below in combination with specific examples, and the following examples are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0046] The terms "comprises" or "comprising" as used herein are meant to be open-ended, i.e., to specify the presence of any stated features, elements, integers, steps, or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the terms "consisting of" and "consisting essentially of." In one embodiment, the term "comprising" as used throughout the specification and in claims, can be replaced with the term "consisting of."

[0047] The terms "optional", "any", "any of", or "any one of" as used herein means that the event or circumstance subsequently described can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence can or can not be present.

[0048] The term "and / or", as used herein, should be understood to mean either of the items it connects or any combination of the items it connects.

[0049] Example 1

[0050] 1.1 Strains

[0051] Lactobacillus fermentum CQPC04, with the preservation number of CGMCC NO. 14493; Lactobacillus rhamnosus 4F225, with the preservation number of CGMCC NO. 26437; Lactobacillus plantarum H6, with the preservation number of CGMCC No. 18205; Lactobacillus gasseri GS18, with the preservation number of CGMCC No. 23187.

[0052] 1.2 Methods

[0053] 1.2.1 Activation of strains

[0054] The strain cryovials preserved at -80°C were taken out, thawed at room temperature, mixed, inoculated into the activation medium at a volume fraction of 5%, and cultured at 37°C for 24 h. The seed liquid was obtained by subculturing for 2-3 generations.

[0055] The formula of the activation medium was: 15 g / L of proteose peptone, 10 g / L of beef extract, 5 g / L of yeast powder, and 5 g / L of sodium chloride. The pH value was adjusted to 6.0-6.4 with 0.1 mol / L sodium hydroxide or 0.1 mol / L hydrochloric acid.

[0056] 1.2.2 High-density fermentation

[0057] The seed solution of 1.2.1 was inoculated into a 10 L fermenter at an inoculum amount of 2-10% (v / v), the fermentation temperature was 35-40°C (preferably 37°C), N2and CO2mixed gas (N2:CO2=1:4) was supplied to the fermenter, the stirring speed was 50-80 rpm, the pH in the fermentation was 5.5-6.0 (adjusted by NaOH solution), the fermentation was carried out for 8-12 h (preferably 10 h), and the viable cell count in the fermentation broth after the fermentation was calculated.

[0058] The high-density fermentation medium was prepared as follows: peptone 20 g / L, beef extract 10 g / L, yeast powder 10 g / L, D-sorbitol 15 g / L, L-proline 5 g / L, sodium chloride 7 g / L, magnesium sulfate-7H2O 0.3 g / L, manganese sulfate-1H2O 0.2 g / L, ferrous sulfate-7H2O 0.2 g / L, and pH 6.5.

[0059] Example 2

[0060] The high-density fermentation medium was prepared as follows: peptone 10 g / L, beef extract 15 g / L, yeast powder 5 g / L, D-sorbitol 8 g / L, L-proline 10 g / L, sodium chloride 3 g / L, magnesium sulfate-7H2O 0.6 g / L, manganese sulfate-1H2O 0.1 g / L, ferrous sulfate-7H2O 0.1 g / L, and pH 7.0.

[0061] Example 3

[0062] The high-density fermentation medium was prepared as follows: peptone 15 g / L, beef extract 12 g / L, yeast powder 8 g / L, D-sorbitol 10 g / L, L-proline 7 g / L, sodium chloride 5 g / L, magnesium sulfate-7H2O 0.5 g / L, manganese sulfate-1H2O 0.15 g / L, ferrous sulfate-7H2O 0.15 g / L, and pH 6.0.

[0063] The viable cell count in the fermentation broth of Example 1 is shown in Table 1.

[0064] Table 1

[0065]

[0066] Comparative Example

[0067] A comparative example was prepared according to the procedure of Reference Example 1, and the preparation and results are shown in Table 2.

[0068] Table 2

[0069]

[0070]

[0071] As can be seen from the results of Table 1 and Table 2, the culture medium of Examples 1-3 can significantly increase the viable cell count of Lactobacillus fermentum CQPC04, Lactobacillus rhamnosus 4F225, Lactobacillus plantarum H6 and Lactobacillus gasseri GS18; especially, it can increase the viable cell count of Lactobacillus rhamnosus 4F225 in anaerobic fermentation. The culture of lactic acid bacteria by using the culture medium of the present embodiment not only has a short fermentation time, but also has a high viable cell count, realizes high-density fermentation of lactic acid bacteria, and reduces the production cost comprehensively.

Claims

1. A method for high-density culturing of lactic acid bacteria, characterized by, The method comprises the following steps: (1) inoculating lactic acid bacteria into an activated culture medium to obtain a seed liquid; (2) inoculating the seed liquid of step (1) into a high-density fermentation culture medium and stirring and fermenting under anaerobic conditions; The high-density fermentation culture medium comprises 10-20 g / L of proteose peptone, 10-15 g / L of beef extract, 5-10 g / L of yeast powder, 8-15 g / L of D-sorbitol, 5-10 g / L of L-proline, 3-7 g / L of sodium chloride, 0.3-0.6 g / L of magnesium sulfate 7H2O, 0.1-0.2 g / L of manganese sulfate 1H2O, 0.1-0.2 g / L of ferrous sulfate 7H2O, and has a pH of 6.0-7.

0. The lactic acid bacteria are Lactobacillus fermentum CQPC04, Lactobacillus rhamnosus 4F225, Lactobacillus plantarum H6 and / or Lactobacillus gasseri GS18; the preservation number of Lactobacillus fermentum CQPC04 is CGMCC NO. 14493, the preservation number of Lactobacillus rhamnosus 4F225 is CGMCC NO. 26437, the preservation number of Lactobacillus plantarum H6 is CGMCC No. 18205, and the preservation number of Lactobacillus gasseri GS18 is CGMCC No. 23187.

2. The method of claim 1, wherein, The inoculation amount of the seed liquid of step (2) is 2-10% by volume.

3. The method of claim 1, wherein, The anaerobic conditions of step (2) are that N2 and CO2 mixed gas is introduced into the fermentation tank; and the stirring speed is 50-80 rpm.

4. The method of claim 1, wherein, The fermentation temperature of step (2) is 35-40°C, the pH during fermentation is 5.5-6.0, and the fermentation time is 8-12 h.

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