CN113278554A - Method for improving acid resistance of lactic acid bacteria by using mixed bacteria biological membrane - Google Patents
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Abstract
The invention discloses a method for improving acid resistance of lactic acid bacteria by using a mixed bacteria biomembrane, which sequentially comprises the steps of preparing a lactic acid bacteria culture medium and a yeast bacteria culture medium; respectively culturing lactic acid bacteria and yeast by using a lactic acid bacteria culture medium and a yeast culture medium to obtain lactic acid bacteria and yeast seed liquid; inoculating lactobacillus and yeast seed liquid into a lactobacillus culture medium in proportion, mixing, and culturing a mixed bacteria biofilm; and carrying out lactic acid bacteria acid stress treatment on the mixed bacteria biomembrane. The mixed bacteria biomembrane co-cultured by the lactic acid bacteria and the saccharomycetes is used for protecting the lactic acid bacteria from acid stress, and the survival rate of the lactic acid bacteria in the mixed bacteria biomembrane is obviously higher than that of the lactic acid bacteria in a single bacteria biomembrane after the lactic acid bacteria are subjected to the acid stress. The invention can be used for improving the stress resistance of the lactic acid bacteria and ensuring the biomass and metabolic capability of the lactic acid bacteria under the acidic condition. Solves the problem that the survival rate of the lactobacillus cultured by the single-bacterium biomembrane is low after the acid stress treatment, which affects the production efficiency of the traditional fermented food in the prior art.
Description
Technical Field
The invention relates to a method for improving acid resistance of lactic acid bacteria, in particular to a method for improving acid resistance of lactic acid bacteria by using a mixed bacteria biomembrane.
Background
The halophilic tetragenococcus is widely used as a halophilic lactic acid bacterium in the production of high-salt fermented foods such as soy sauce, jam and the like. The halophilic tetragenococcus is also one of important bacterial strains in fermentation production, is a main microorganism for generating volatile substances, and has the functions of promoting the generation of flavor components of products, improving the quality and the taste of the products and the like. The saccharomyces rouxii belongs to zygosaccharomyces in the saccharomyces family, is an important production strain for traditional fermentation, and is widely present in salted foods such as soy sauce, bean paste and the like. The zygosaccharomyces rouxii plays an important role in the fermentation period of the soy sauce, flavor substances such as ethanol, higher alcohols and the like are mainly produced, glutaminase in the zygosaccharomyces rouxii can convert a substrate to generate glutamic acid so as to enhance the delicate flavor of the soy sauce, and plays an active role in flavor fermentation of the seasoning.
In the production process of traditional fermented food, hydrolysis of macromolecular substances in raw materials under the action of microorganisms and reproductive metabolism of acid-producing bacteria such as lactic acid bacteria and the like can enable acidic substances such as lactic acid, acetic acid, amino acid and the like to be accumulated in a fermentation system, and the pH value in the system is reduced. Generally, bacteria require a pH between 4 and 8 for growth and survival. Some acids can passively diffuse into cells in an undissociated form, producing free protons. The increase in acidity results in inhibition of cell growth and metabolism, which in turn affects the integrity of purine bases, resulting in changes in essential enzymes within the cell, thereby affecting the efficiency of food production.
In the prior art, a method for improving the acid resistance of lactic acid bacteria by preparing a mixed bacteria biomembrane by using the lactic acid bacteria and yeast cannot be realized. In recent years, beneficial microorganisms have attracted more and more attention, and the capability of forming lactic acid bacteria biofilms has been studied more and more. The formation of the biofilm can not only improve the adhesion and utilization of the propagation metabolism of the lactic acid bacteria on the surface of the raw material, but also protect the cells from various adverse environmental factors including the stress of acid. In addition, research shows that the co-culture of lactic acid bacteria and yeast can effectively improve the biomass and metabolic capacity of two microorganisms. Therefore, it is required to prepare lactic acid bacteria with high acid resistance by using mixed bacteria biomembrane under coculture of lactic acid bacteria and yeast to solve the above technical problems.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for improving the acid resistance of lactic acid bacteria by using a mixed bacterial biomembrane, and aims to solve the problems that the survival rate of lactic acid bacteria cultured by a single bacterial biomembrane in the prior art is low after acid stress treatment, and the production efficiency of traditional fermented food is influenced.
In order to achieve the purpose, the invention adopts the technical scheme that: the method for improving the acid resistance of the lactic acid bacteria by using the mixed bacteria biomembrane comprises the following steps:
s1: preparing a lactic acid bacteria culture medium and a yeast bacteria culture medium, wherein the lactic acid bacteria culture medium comprises peptone, beef extract, yeast powder, glucose, sorbitol monooleate, dipotassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate and sodium chloride;
the yeast culture medium comprises yeast powder, peptone, glucose and sodium chloride;
s2: respectively culturing lactic acid bacteria and yeast by using a lactic acid bacteria culture medium and a yeast culture medium to obtain lactic acid bacteria and yeast seed liquid;
s3: inoculating lactobacillus and yeast seed liquid into a lactobacillus culture medium in proportion, mixing, and culturing a mixed bacteria biofilm;
s4: and carrying out lactic acid bacteria acid stress treatment on the mixed bacteria biomembrane.
The technical scheme has the advantages of simple method and convenient operation, and can be used for improving the stress resistance of the lactic acid bacteria. The lactobacillus and the saccharomycetes are co-cultured to effectively improve the biomass and metabolic capacity of two microorganisms, the mixed bacteria biomembrane under the co-culture of the saccharomycetes and the lactobacilli is adopted to protect the lactobacilli from acid stress, the biomass and metabolic capacity of the lactobacilli under the acidic condition are guaranteed, the food fermentation efficiency is further improved, and the method has important significance for traditional fermented food production.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the pH value of the lactic acid bacteria culture medium is 6.0-6.4, and the concentration of each component is as follows: 8-12 g/L of peptone, 6-10 g/L of beef extract, 2-6 g/L of yeast powder, 18-22 g/L of glucose, 0.08-0.12 v/v% of sorbitol monooleate, 1-3 g/L of dipotassium phosphate, 3-7 g/L of sodium acetate trihydrate, 1-3 g/L of triammonium citrate, 0.1-0.3 g/L of magnesium sulfate heptahydrate, 0.1-0.3 g/L of manganese sulfate tetrahydrate and 55-65 g/L of sodium chloride; the pH value of the yeast culture medium is 5.8-6.2, and the concentration of each component is as follows: 8-12 g/L yeast powder, 18-22 g/L peptone, 18-22 g/L glucose and 55-65 g/L sodium chloride.
Further, the pH of the lactic acid bacteria culture medium is preferably 6.2, and the concentration of each component is preferably: 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 0.1 v/v% of sorbitol monooleate, 2g/L of dipotassium phosphate, 5g/L of sodium acetate trihydrate, 2g/L of ammonium citrate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.2g/L of manganese sulfate tetrahydrate and 60g/L of sodium chloride; the pH of the yeast culture medium is preferably 6.0, and the concentration of each component is preferably: 10g/L of yeast powder, 20g/L of peptone, 20g/L of glucose and 60g/L of sodium chloride.
Further, the cultivation of the seed liquid in S2 includes the following steps:
respectively taking lactic acid bacteria and yeast preserved at the temperature of minus 80 ℃, respectively inoculating the lactic acid bacteria and the yeast into 4-6 mL of the lactic acid bacteria culture medium and the yeast culture medium according to the inoculation amount of 18-22%, and performing static culture for 24 hours at the temperature of 25-35 ℃ to obtain the lactobacillus and yeast.
Further, the preferable steps of culturing the seed liquid in S2 are as follows:
respectively taking the lactic acid bacteria and the yeast preserved at the temperature of minus 80 ℃, respectively inoculating the lactic acid bacteria and the yeast into 5mL of lactic acid bacteria culture medium and yeast culture medium according to the inoculation amount of 20%, and standing and culturing for 24h at the temperature of 30 ℃ to obtain the lactobacillus and yeast.
Further, the lactobacillus is Tetragenococcus halophilus CGMCC3792, and the yeast is Saccharomyces rouxii CGMCC 3791. The two strains are widely applied to the domestic seasonings, have important significance on flavor regulation and fermentation of the seasonings, and have practical life application value when being used as a mixed bacterium film for cultivation. The halophilic tetragenococcus is preserved in CGMCC-China general microbiological culture Collection center, and the preservation addresses are as follows: no.3 of Xilu No. 1 of Beijing, Chaoyang, the morning area, the preservation number is CGMCC NO.3792, the preservation date is 29 days 04 months 2010, and the SZ-B is named as Tetragenococcus haiophilus SZ-B-2 in a classified manner; the Saccharomyces rouxii is preserved in CGMCC-China general microbiological culture Collection center, and the preservation addresses are as follows: the No.3 Xilu No. 1 Beijing, Chaoyang, the morning area, has a preservation number of CGMCC NO.3791, a preservation date of 2010, 04-29 days, and is classified and named as Zygosaccharomyces rouxii SZ-1.
Further, the culture of the mixed bacteria biofilm in the S3 comprises the following steps:
uniformly mixing the lactobacillus seed liquid, the saccharomycete seed liquid and the lactobacillus culture medium according to the volume ratio of 1: 0.4-0.6: 90-110, and putting 3mL of the uniformly mixed liquid into a 5mL centrifuge tube for standing culture for 48 hours to obtain the lactobacillus culture medium.
Further, the culture step of the mixed bacterial biofilm in S3 is preferably:
uniformly mixing the lactobacillus seed liquid, the saccharomycete seed liquid and the lactobacillus culture medium according to the volume ratio of 1:0.5:100, and putting 3mL of the uniformly mixed liquid into a 5mL centrifuge tube for standing culture for 48 hours to obtain the lactobacillus culture medium.
Further, the lactic acid stress treatment in S4 comprises the following steps:
removing the upper layer fermentation liquid of the mixed bacteria biomembrane, adding into the lactic acid bacteria culture medium, standing at 30 deg.C and stressing for 90 min; the pH value of the lactobacillus culture medium is 4.1-4.3.
The invention has the beneficial effects that: according to the invention, the mixed bacteria biomembrane is used for improving the acid resistance of the lactic acid bacteria, preferably the lactic acid bacteria is halophilic tetragenococcus, preferably the zygosaccharomyces rouxii is used as the yeast for preparation, a lactic acid bacteria culture medium and a yeast culture medium are prepared in a proper proportion and inoculation mode, a lactic acid bacteria and yeast seed liquid, a lactic acid bacteria and yeast mixed bacteria biomembrane and a lactic acid bacteria colony subjected to acid stress treatment are prepared, the method is simple and convenient to operate, the stress resistance of the lactic acid bacteria is effectively improved, the survival rate of the lactic acid bacteria in the mixed bacteria biomembrane after acid stress is obviously higher than that of the lactic acid bacteria in a single bacteria biomembrane, and the industrial production capacity and the product quality of the food fermentation industry are further improved.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1:
a method for improving acid resistance of lactic acid bacteria by using a mixed bacteria biomembrane comprises the following steps:
s1: preparing a lactic acid bacteria culture medium and a yeast bacteria culture medium. Preparing a lactic acid bacteria culture medium: the pH value of the lactobacillus culture medium is 6.2, and the concentration of each component is as follows: 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 0.1 v/v% of sorbitol monooleate, 2g/L of dipotassium phosphate, 5g/L of sodium acetate trihydrate, 2g/L of ammonium citrate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.2g/L of manganese sulfate tetrahydrate and 60g/L of sodium chloride; preparing a yeast culture medium: the pH value of the yeast culture medium is 6.0, and the concentration of each component is as follows: 10g/L of yeast powder, 20g/L of peptone, 20g/L of glucose and 60g/L of sodium chloride.
S2: respectively culturing lactic acid bacteria and yeast by using a lactic acid bacteria culture medium and a yeast culture medium to obtain lactic acid bacteria and yeast seed liquid. Respectively inoculating lactobacillus and yeast preserved at-80 deg.C in 5mL of lactobacillus culture medium and yeast culture medium according to 20% inoculation amount, and standing at 30 deg.C for 24 hr. Wherein the lactobacillus is Tetragenococcus halophilus CGMCC 3792; the yeast is Zygosaccharomyces rouxii CGMCC 3791.
S3: inoculating lactobacillus and yeast seed liquid into lactobacillus culture medium in proportion, mixing, and culturing mixed bacteria biomembrane. Inoculating 1mL of the lactic acid bacteria seed solution prepared in S2 and 0.5mL of the yeast seed solution into 100mL of the lactic acid bacteria culture medium prepared in S1, and mixing uniformly. And 3mL of the uniformly mixed solution is respectively put into 2 groups of 5mL centrifuge tubes for standing culture for 48 hours to obtain the lactic acid bacteria mixed bacteria biomembrane.
S4: and carrying out lactic acid bacteria acid stress treatment on the mixed bacteria biomembrane. Removing the upper fermentation broth of the single-bacterium biofilm prepared by the S3, adding 3mL of lactic acid bacterium culture medium prepared by the S1 into the mixed-bacterium biofilm of the group 1, and adjusting the pH value of the lactic acid bacterium culture medium to 4.15 by adopting hydrochloric acid; the 1 group mixed bacterial biomembrane is added with 3mL of the lactic acid bacteria culture medium (namely the natural pH lactic acid bacteria culture medium) prepared by S1, and is kept stand and stressed for 90min at the temperature of 30 ℃.
Example 2:
a method for improving acid resistance of lactic acid bacteria by using a mixed bacteria biomembrane comprises the following steps:
s1: preparing a lactic acid bacteria culture medium and a yeast bacteria culture medium. Preparing a lactic acid bacteria culture medium: the pH value of the lactobacillus culture medium is 6.0, and the concentration of each component is as follows: 8g/L of peptone, 6g/L of beef extract, 2g/L of yeast powder, 18g/L of glucose, 0.08 v/v% of sorbitol monooleate, 1g/L of dipotassium phosphate, 3g/L of sodium acetate trihydrate, 1g/L of ammonium citrate trihydrate, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of manganese sulfate tetrahydrate and 55g/L of sodium chloride; preparing a yeast culture medium: the pH value of the yeast culture medium is 5.8, and the concentration of each component is as follows: 8g/L of yeast powder, 18g/L of peptone, 18g/L of glucose and 55g/L of sodium chloride.
S2: respectively culturing lactic acid bacteria and yeast by using a lactic acid bacteria culture medium and a yeast culture medium to obtain lactic acid bacteria and yeast seed liquid. Respectively inoculating 18% of lactobacillus and yeast preserved at-80 deg.C into 4mL of lactobacillus culture medium and yeast culture medium, and standing at 25 deg.C for 24 hr. Wherein the lactobacillus is Tetragenococcus halophilus CGMCC 3792; the yeast is Zygosaccharomyces rouxii CGMCC 3791.
S3: inoculating lactobacillus and yeast seed liquid into lactobacillus culture medium in proportion, mixing, and culturing mixed bacteria biomembrane. Inoculating 1mL of the lactic acid bacteria seed solution prepared in S2 and 0.4mL of the yeast seed solution into 90mL of the lactic acid bacteria culture medium prepared in S1, and mixing uniformly. And 3mL of the uniformly mixed solution is respectively put into 2 groups of 5mL centrifuge tubes for standing culture for 48 hours to obtain the lactic acid bacteria mixed bacteria biomembrane.
S4: and carrying out lactic acid bacteria acid stress treatment on the mixed bacteria biomembrane. Removing the upper fermentation broth of the single-bacterium biofilm prepared by the S3, adding 3mL of lactic acid bacterium culture medium prepared by the S1 into the mixed-bacterium biofilm of the group 1, and adjusting the pH value of the lactic acid bacterium culture medium to 4.1 by hydrochloric acid; the 1 group mixed bacterial biomembrane is added with 3mL of the lactic acid bacteria culture medium (namely the natural pH lactic acid bacteria culture medium) prepared by S1, and is kept stand and stressed for 90min at the temperature of 30 ℃.
Example 3:
a method for improving acid resistance of lactic acid bacteria by using a mixed bacteria biomembrane comprises the following steps:
s1: preparing a lactic acid bacteria culture medium and a yeast bacteria culture medium. Preparing a lactic acid bacteria culture medium: the pH value of the lactobacillus culture medium is 6.4, and the concentration of each component is as follows: 12g/L of peptone, 10g/L of beef extract, 6g/L of yeast powder, 22g/L of glucose, 0.12 v/v% of sorbitol monooleate, 3g/L of dipotassium phosphate, 7g/L of sodium acetate trihydrate, 3g/L of ammonium citrate trihydrate, 0.3g/L of magnesium sulfate heptahydrate, 0.3g/L of manganese sulfate tetrahydrate and 65g/L of sodium chloride; preparing a yeast culture medium: the pH value of the yeast culture medium is 6.2, and the concentration of each component is as follows: yeast powder 12g/L, peptone 22g/L, glucose 22g/L, and sodium chloride 65 g/L.
S2: respectively culturing lactic acid bacteria and yeast by using a lactic acid bacteria culture medium and a yeast culture medium to obtain lactic acid bacteria and yeast seed liquid. Respectively inoculating lactobacillus and yeast preserved at-80 deg.C in 6mL lactobacillus culture medium and yeast culture medium according to 22% inoculation amount, and standing at 35 deg.C for 24 hr. Wherein the lactobacillus is Tetragenococcus halophilus CGMCC 3792; the yeast is Zygosaccharomyces rouxii CGMCC 3791.
S3: inoculating lactobacillus and yeast seed liquid into lactobacillus culture medium in proportion, mixing, and culturing mixed bacteria biomembrane. Inoculating 1mL of the lactic acid bacteria seed solution prepared in S2 and 0.6mL of the yeast seed solution into 110mL of the lactic acid bacteria culture medium prepared in S1, and mixing uniformly. And 3mL of the uniformly mixed solution is respectively put into 2 groups of 5mL centrifuge tubes for standing culture for 48 hours to obtain the lactic acid bacteria mixed bacteria biomembrane.
S4: and carrying out lactic acid bacteria acid stress treatment on the mixed bacteria biomembrane. Removing the upper fermentation broth of the single-bacterium biofilm prepared by the S3, adding 3mL of lactic acid bacterium culture medium prepared by the S1 into the mixed-bacterium biofilm of the group 1, and adjusting the pH value of the lactic acid bacterium culture medium to 4.3 by hydrochloric acid; the 1 group mixed bacterial biomembrane is added with 3mL of the lactic acid bacteria culture medium (namely the natural pH lactic acid bacteria culture medium) prepared by S1, and is kept stand and stressed for 90min at the temperature of 30 ℃.
Example 4 (comparative):
a method for improving acid resistance of lactic acid bacteria by using a single-bacterium biomembrane comprises the following steps:
s1: preparing a lactic acid bacteria culture medium: the pH value of the lactobacillus culture medium is 6.2, and the concentration of each component is as follows: 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 0.1 v/v% of sorbitol monooleate, 2g/L of dipotassium phosphate, 5g/L of sodium acetate trihydrate, 2g/L of ammonium citrate tribasic, 0.2g/L of magnesium sulfate heptahydrate, 0.2g/L of manganese sulfate tetrahydrate and 60g/L of sodium chloride.
S2: culturing lactobacillus with lactobacillus culture medium to obtain lactobacillus seed solution. Inoculating lactobacillus preserved at-80 deg.C in 5mL lactobacillus culture medium at an inoculum size of 20%, and standing at 30 deg.C for 24 hr. Wherein the lactobacillus is Tetragenococcus halophilius CGMCC 3792.
S3: and culturing the single-bacterium biofilm. Inoculating the lactobacillus seed solution prepared in S2 into 100mL of lactobacillus culture medium prepared in S1, and mixing. 3mL of the uniformly mixed solution is respectively put into 2 groups of 5mL centrifuge tubes for standing culture for 48h, and the lactobacillus single-bacterium biomembrane is obtained.
S4: and carrying out lactic acid bacteria acid stress treatment on the single-bacterium biomembrane. Removing the upper layer fermentation liquid of the single-bacterium biomembrane prepared in the step S3, adding 3ml of lactic acid bacteria culture medium prepared in the step S1 into the single-bacterium biomembrane of the group 1, and adjusting the pH value of the lactic acid bacteria culture medium to 4.15 by adopting hydrochloric acid; 3mL of the lactic acid bacteria culture medium (i.e., the natural pH lactic acid bacteria culture medium) prepared by S1 was added to the single-bacterium biofilm of group 1, and the mixture was allowed to stand at 30 ℃ for 90min under stress.
The lactobacillus treated in the above examples was centrifuged and washed 3 times at 8000rpm and 4 ℃, the cells were collected and resuspended in 1mL of sterile water for dilution, 10. mu.L of the diluted cell suspension was aspirated and inoculated onto a solid selective medium containing 0.005% nystatin for lactic acid bacteria, and the number of colonies of lactic acid bacteria was counted after incubation for 48h at 30 ℃. And then calculating the survival rate of the lactobacillus, wherein the survival rate calculation process takes the number of the lactobacillus colonies treated by the natural pH lactobacillus culture medium as the number of the contrasted colonies. The survival rate calculation formula is as follows:
survival rate (%). the number of colonies (one/mL) after acid treatment)/the number of colonies (one/mL) after natural pH lactobacillus medium treatment X100%
The test results are shown in table 1 below:
TABLE 1 acid stress treatment test results of lactic acid bacteria Table
| Acid stress treatment pH value | Standing culture time (h) | Survival rate (%) | |
| Example 1 | 4.15 | 48 | 17.63 |
| Example 2 | 4.10 | 48 | 13.19 |
As can be seen from the above table, the survival rate of the lactic acid bacteria cultured by the single-bacterium biomembrane in the prior art is low after the treatment of acid stress; the survival rate of the lactic acid bacteria cultured by the mixed bacteria biomembrane is obviously higher than that of the lactic acid bacteria cultured by the common single bacteria biomembrane after the acid stress treatment, and the survival rate of the lactic acid bacteria cultured by the mixed bacteria biomembrane can be more than two times of that of the lactic acid bacteria cultured by the single bacteria biomembrane after the acid stress treatment. According to the invention, the mixed bacteria biomembrane co-cultured with the yeast is used for protecting the lactic acid bacteria from acid stress, the biomass and metabolic capacity of the lactic acid bacteria under the acidic condition are ensured, the food fermentation efficiency is further improved, and the method has an important significance for traditional fermented food production.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and alterations that can be made by a person skilled in the art without inventive step within the scope of what is described in the claims are still protected by the present patent.
Claims (9)
1. A method for improving acid resistance of lactic acid bacteria by using a mixed bacteria biomembrane is characterized by comprising the following steps:
s1: preparing a lactic acid bacteria culture medium and a yeast bacteria culture medium, wherein the lactic acid bacteria culture medium comprises peptone, beef extract, yeast powder, glucose, sorbitol monooleate, dipotassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate and sodium chloride;
the yeast culture medium comprises yeast powder, peptone, glucose and sodium chloride;
s2: respectively culturing lactic acid bacteria and yeast by using a lactic acid bacteria culture medium and a yeast culture medium to obtain lactic acid bacteria and yeast seed liquid;
s3: inoculating lactobacillus and yeast seed liquid into a lactobacillus culture medium in proportion, mixing, and culturing a mixed bacteria biofilm;
s4: and carrying out lactic acid bacteria acid stress treatment on the mixed bacteria biomembrane.
2. The method for improving acid resistance of lactic acid bacteria by using mixed bacteria biofilm as claimed in claim 1, wherein: the pH value of the lactic acid bacteria culture medium is 6.0-6.4, and the concentration of each component is as follows: 8-12 g/L of peptone, 6-10 g/L of beef extract, 2-6 g/L of yeast powder, 18-22 g/L of glucose, 0.08-0.12 v/v% of sorbitol monooleate, 1-3 g/L of dipotassium phosphate, 3-7 g/L of sodium acetate trihydrate, 1-3 g/L of triammonium citrate, 0.1-0.3 g/L of magnesium sulfate heptahydrate, 0.1-0.3 g/L of manganese sulfate tetrahydrate and 55-65 g/L of sodium chloride; the pH value of the yeast culture medium is 5.8-6.2, and the concentration of each component is as follows: 8-12 g/L yeast powder, 18-22 g/L peptone, 18-22 g/L glucose and 55-65 g/L sodium chloride.
3. The method for improving the acid resistance of lactic acid bacteria by using the mixed bacteria biofilm as claimed in claim 2, wherein the acid resistance of lactic acid bacteria is as follows: the pH value of the lactic acid bacteria culture medium is preferably 6.2, and the concentration of each component is preferably as follows: 10g/L of peptone, 8g/L of beef extract, 4g/L of yeast powder, 20g/L of glucose, 0.1 v/v% of sorbitol monooleate, 2g/L of dipotassium phosphate, 5g/L of sodium acetate trihydrate, 2g/L of ammonium citrate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.2g/L of manganese sulfate tetrahydrate and 60g/L of sodium chloride; the pH value of the yeast culture medium is preferably 6.0, and the concentration of each component is preferably as follows: 10g/L of yeast powder, 20g/L of peptone, 20g/L of glucose and 60g/L of sodium chloride.
4. The method for improving the acid resistance of lactic acid bacteria by using the mixed bacteria biofilm as claimed in claim 1, wherein the culturing of the seed solution in S2 comprises the following steps:
respectively taking lactic acid bacteria and yeast preserved at the temperature of minus 80 ℃, respectively inoculating the lactic acid bacteria and the yeast into 4-6 mL of the lactic acid bacteria culture medium and the yeast culture medium according to the inoculation amount of 18-22%, and performing static culture for 24 hours at the temperature of 25-35 ℃ to obtain the lactobacillus and yeast.
5. The method for improving the acid resistance of lactic acid bacteria by using the mixed bacteria biofilm as claimed in claim 1 or 4, wherein the preferable steps of culturing the seed solution in S2 are as follows:
respectively taking lactic acid bacteria and yeast preserved at the temperature of minus 80 ℃, respectively inoculating the lactic acid bacteria and the yeast into 5mL of the lactic acid bacteria culture medium and the yeast culture medium according to the inoculation amount of 20%, and standing and culturing for 24 hours at the temperature of 30 ℃ to obtain the lactobacillus and yeast.
6. The method for improving acid resistance of lactic acid bacteria by using mixed bacteria biofilm as claimed in claim 1 or 4, wherein: the lactobacillus is Tetragenococcus halophilus CGMCC3792, and the yeast is Zygosaccharomyces rouxii CGMCC 3791.
7. The method for improving the acid resistance of lactic acid bacteria by using the mixed bacteria biofilm as claimed in claim 1, wherein the culturing of the mixed bacteria biofilm in S3 comprises the following steps:
uniformly mixing the lactobacillus seed liquid, the saccharomycete seed liquid and the lactobacillus culture medium according to the volume ratio of 1: 0.4-0.6: 90-110, and putting 3mL of the uniformly mixed liquid into a 5mL centrifuge tube for standing culture for 48 hours to obtain the lactobacillus culture medium.
8. The method for improving acid resistance of lactic acid bacteria by using mixed bacteria biofilm as claimed in claim 1 or 7, wherein the culturing step of the mixed bacteria biofilm in S3 is preferably:
uniformly mixing the lactobacillus seed liquid, the saccharomycete seed liquid and the lactobacillus culture medium according to the volume ratio of 1:0.5:100, and putting 3mL of the uniformly mixed liquid into a 5mL centrifuge tube for standing culture for 48 hours to obtain the lactobacillus culture medium.
9. The method for improving the acid resistance of lactic acid bacteria by using the mixed bacteria biofilm as claimed in claim 1, wherein the acid stress treatment of lactic acid bacteria in S4 comprises the following steps:
removing the upper fermentation broth of the mixed bacteria biomembrane, adding into the lactic acid bacteria culture medium, and standing at 30 deg.C for 90 min; the pH value of the lactic acid bacteria culture medium is 4.1-4.3.
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