A method for improving the tolerance of lactobacillus kefiranofaciens to extreme acid stress and application thereof
By adding glycerophosphate choline and lysophosphatidylcholine to Lactobacillus aceticus and combining it with stress acclimatization technology, the survival ability and metabolic stability of the strain in extremely acidic environments were improved, solving the problem of decreased strain activity during fermentation and achieving improved stability of the fermentation process and product quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MOUTAI INST
- Filing Date
- 2026-04-21
- Publication Date
- 2026-05-29
AI Technical Summary
Lactobacillus acetic acid in Jinshan suffers from decreased metabolic activity and reduced survival capacity due to the extremely acidic environment during the fermentation of baijiu and vinegar, which affects the stability of the fermentation process and the consistency of product quality.
By adding precursors such as glycerophosphate choline and lysophosphatidylcholine, and combining this with a stress acclimatization method that gradually reduces pH, the acid tolerance mechanism of the strain is enhanced, simulating the industrial fermentation environment and enabling the strain to adapt to extreme acidic conditions.
It significantly improves the abundance and survival rate of the strain in extremely acidic environments, enhances the stability of the fermentation process and the consistency of product flavor, and is suitable for traditional solid-state fermentation processes without the need for equipment modification.
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Figure CN122104474A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and specifically relates to a method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress and its application. Background Technology
[0002] Jinshan Lactobacillus acetic acid ( Acetilactobacillus jinshanensis Lactobacillus aceticus is a core dominant strain in the anaerobic stage of solid-state fermentation of baijiu (Chinese liquor) and vinegar. It exerts a crucial influence on the stability of the fermentation process and the flavor and quality of the product through multiple pathways, including driving environmental acidification, producing antibacterial active substances, and regulating community structure. However, during the fermentation of baijiu and vinegar, the system pH gradually drops to an extreme acidic level of 2.5–3.5, accompanied by the accumulation of high concentrations of organic acids such as lactic acid and acetic acid, as well as ethanol. This creates a multi-stress environment, leading to a decrease in the metabolic activity and survival ability of Lactobacillus aceticus, thus affecting its dominant position and the full performance of its fermentation function.
[0003] Existing research indicates that *Lactobacillus aceticus* copes with acid stress by remodeling membrane lipid composition and reconstructing amino acid metabolism. However, these natural tolerance mechanisms are insufficient to fully adapt to the extreme stress environments of industrial fermentation, leading to significant fluctuations in strain abundance and batch-to-batch product quality differences. Therefore, developing an efficient method to enhance the tolerance of *Lactobacillus aceticus* and strengthen its survival ability and metabolic stability in extremely acidic environments is of great significance for promoting the standardization and large-scale production of solid-state fermented foods. Summary of the Invention
[0004] The present invention aims to provide a method for improving the tolerance of Lactobacillus aceticus to extreme acid stress, so as to enhance the survival ability and metabolic stability of Lactobacillus aceticus in extreme acidic environments.
[0005] One method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress in this protocol includes the following steps: Step 1, Strain activation: Add glycerophosphate choline to the modified MRS medium, then inoculate with Lactobacillus acetic acid for activation culture to obtain activated bacterial solution; Step 2, stress adaptation culture: The activated bacterial solution obtained in Step 1 is inoculated into the stress adaptation culture medium, and the pH is gradually reduced to carry out stress acclimatization. Lysophosphatidylcholine is added during the acclimatization process, and culture is carried out to obtain the acclimatized bacterial solution. Step 3: Fermentation system enhancement: Inoculate the domesticated bacterial culture obtained in Step 2 into the target fermentation system, and supplement the fermentation system with glycerophosphate choline or its hydrochloride to carry out fermentation.
[0006] Furthermore, in step one, the modified MRS medium consists of: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate, and 0.25 g / L manganese sulfate. The amount of glycerophosphate choline added is 0.02% to 0.05% of the weight of the modified MRS medium.
[0007] Furthermore, in step one, the activation culture conditions are: culture at 30–32°C under anaerobic conditions for 12–16 hours, until the bacterial culture OD... 600 It reaches 0.8 to 1.0.
[0008] Furthermore, in step two, the stress adaptation culture medium consists of: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 10–30 g / L lactic acid, and 1–3 g / L acetic acid.
[0009] Furthermore, in step two, the method of gradually reducing pH is as follows: initial pH 5.0, adjusted to pH 4.0 after 8 hours of culture, and then adjusted to pH 2.5–3.5 after another 8 hours of culture; then lysophosphatidylcholine is added and cultured at 30°C under anaerobic conditions for 24–36 hours; the amount of lysophosphatidylcholine added is 0.01%–0.03% of the weight of the stress adaptation medium; the pH adjuster is a mixed solution of lactic acid and acetic acid.
[0010] Furthermore, in step three, the inoculation amount of the acclimatization bacterial solution is 0.1% to 0.5% of the dry weight of the fermentation substrate; the supplementation amount of glycerophosphate choline or its hydrochloride is 0.01%; the fermentation conditions are: initial pH 2.5 to 3.5, temperature 28 to 32°C, and moisture content 50% to 70%.
[0011] Furthermore, in step three, the target fermentation system is either a liquid fermentation system or a solid fermentation system; the solid fermentation system is preferably a spirit fermentation mash or a vinegar fermentation mash.
[0012] This application also seeks protection for the application of the above method in improving the tolerance of Lactobacillus acetic acid to extremely acidic and / or high osmotic pressure environments.
[0013] This application also seeks protection for the application of the above method in the brewing of liquor or vinegar.
[0014] This application also seeks protection for the application of the above method in improving the flavor consistency between batches of fermented products.
[0015] Beneficial effects 1. This invention combines choline cycle metabolism enhancement with stress adaptation training to specifically activate the inherent acid resistance mechanism of Lactobacillus acetic acid in Jinshan. By adding precursor substances such as glycerophosphate choline and lysophosphatidylcholine, the strain is provided with sufficient metabolic substrates, which significantly improves its abundance (accounting for more than 80% of the total number of 16sRNA bacteria), survival rate (increased by more than 70%), and metabolic stability in an extremely acidic environment of pH 2.5 to 3.5.
[0016] 2. This invention employs a gradual pH reduction stress acclimatization method to simulate the stress environment changes in industrial fermentation, allowing the strains to gradually adapt to extreme conditions and avoiding cell damage caused by sudden stress. The acclimatized strains are more likely to colonize quickly and form an advantage in actual fermentation.
[0017] 3. The method of the present invention is simple to operate, the choline precursors used are inexpensive and easy to obtain, and the amount added is low, which will not have a negative impact on the flavor and quality of the fermented product. It is suitable for traditional solid-state fermentation processes such as liquor and vinegar, and does not require modification of existing production equipment.
[0018] 4. After applying the method of the present invention, the relative abundance of 16S rRNA of Lactobacillus acetic acid in the fermentation system can be maintained at more than 80%, the survival rate can be increased to more than 70%, the growth of miscellaneous bacteria can be effectively inhibited, the flavor consistency deviation between fermentation batches is less than 20%, and the problem of large quality fluctuation in traditional fermentation can be significantly improved, which has important industrial application value. Attached Figure Description
[0019] Figure 1 This diagram illustrates the enhanced tolerance of Lactobacillus acetic acid to extreme acid stress.
[0020] Figure 1 In Chinese: GPC: glycerophosphocholine. LPC: lysophosphatidylcholine. Ach: Acetylcholine; Lactic acid; Betaine: betaine; Choline: choline; GlpQ: glycerophosphodiesterphosphodiesterase. BCT: betaine / carnitine / choline transporter. H+: Hydrogen ion; Phosphate: Phosphate group; Glycerol: Glycerin; OH: hydroxyl group; R1: Receptor / transfer protein site Acyl-COA: Acyl-CoA. Detailed Implementation
[0021] The following detailed description illustrates the specific implementation method: A method (liquid system) for improving the tolerance of Lactobacillus acetic acid to extreme acid stress includes the following steps: (1) Activation of the strain: Glycerol phosphate choline (GPC) was added to modified MRS medium (10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate and 0.25 g / L manganese sulfate), pH 6.2, and sterilized at 121℃ for 20 min; inoculated Acet ilactobacillus jinshanensis The L214 strain was cultured at 30°C under anaerobic conditions for 14 hours to obtain an activated bacterial solution.
[0022] (2) Stress adaptation culture: The activated bacterial solution was transferred to the stress adaptation medium (20 g / L lactic acid, 2 g / L acetic acid, and the other components were the same as the modified MRS medium) at an inoculation rate of 8%. The initial pH was 5.0. After culturing for 8 h, the pH was adjusted to 4.0 with lactic acid-acetic acid solution. After culturing for another 8 h, the pH was adjusted to 3.0. At the same time, lysophosphatidylcholine (LPC) was added. The culture was carried out at 30 °C under anaerobic conditions for 30 h. The amount of lysophosphatidylcholine added was 0.01% to 0.03% of the weight of the stress adaptation medium.
[0023] (3) Fermentation system enhancement: The bacterial culture after stress adaptation and the mixed bacterial culture of the mash system were inoculated into liquid modified MRS medium (the initial pH was adjusted to 2.5, 3.0 and 3.5 respectively using lactic acid-acetic acid mixture) and fermented for 7 days; the inoculation amount of the bacterial culture after stress adaptation was 0.3% of the weight of liquid modified MRS medium, and the inoculation amount of the mixed bacterial culture of the mash system was 1% of the weight of liquid modified MRS medium.
[0024] In this application, the mixed microbial system of the fermented mash system is: 10g of fermented mash is added to 100ml of sterile distilled water and thoroughly shaken and mixed. After standing, the supernatant is taken as the mixed microbial system of the fermented mash system.
[0025] Examples 1-6 refer to the "A method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress (liquid system)" in the specific implementation. The specific parameter differences are detailed in Table 1. In Table 1, the fermentation temperature of Examples 1-6 refers to the fermentation temperature of the enhanced fermentation stage of the fermentation system.
[0026] Control group 1: The mixed microbial culture of the mash system was inoculated into liquid modified MRS medium (the initial pH was adjusted to 3.0 using a mixture of lactic acid and acetic acid) and fermented for 7 days at a fermentation temperature of 30℃; the inoculation amount of the mixed microbial culture of the mash system was 1% of the weight of the liquid modified MRS medium.
[0027] Control group 2: Lactobacillus acetic acid bacteria that had not undergone strain activation and stress adaptation culture ( Acetilactobac illus jinshanensis L214 strain and the mixed bacterial strain of the fermentation mash system were inoculated into liquid modified MRS medium (the initial pH was adjusted to 2.5 using a lactic acid-acetic acid mixture) and fermented for 7 days at a fermentation temperature of 30℃. The inoculation amount of Lactobacillus jinshanensis was 0.3% of the weight of the liquid modified MRS medium, and the inoculation amount of the mixed bacterial strain of the fermentation mash system was 1% of the weight of the liquid modified MRS medium.
[0028] The only difference between control group 3 and control group 2 was that the initial pH of the liquid modified MRS medium was 3.5.
[0029] The only difference between control group 4 and control group 2 was that the initial pH of the liquid modified MRS medium was 3 and the fermentation temperature was 32℃.
[0030] The only difference between control group 5 and example 3 is that it does not include step (2) stress adaptation culture and step (3) fermentation system enhancement stage, and the activated bacterial solution is used to replace the bacterial solution after stress adaptation culture.
[0031] The only difference between control group 6 and example 3 is that: in step (1) the strain activation stage, glycerophosphate choline is not added.
[0032] After fermentation, the relative abundance and survival rate of Lactobacillus acetic acid were measured.
[0033] (1) The relative abundance of Lactobacillus aceticus in Jinshan was obtained by high-throughput sequencing of the 16S rRNA amplicon of the target microorganism in the sample and bioinformatics analysis. The entire 16S rRNA gene segment (V1-V9) was amplified using the Illumina MiSeq sequencing platform. Bioinformatics analysis was performed using QIIME2 software with parameters set as quality filtering (Trimmomatic, Q20 threshold), OTU clustering (97% similarity), and species annotation (comparison with SILVA138 database). Finally, the relative abundance percentage of Lactobacillus aceticus in the total bacterial community was calculated.
[0034] (2) The survival rate was determined based on heavy water (D2O) labeled culture combined with Raman spectroscopy single-cell sorting analysis. The specific procedure was as follows: fermentation samples were taken, inoculated into modified MRS medium containing 2% D2O, and cultured anaerobicly at 32℃ for 24 h. Subsequently, a confocal Raman spectrometer (excitation wavelength 532 nm, scanning range 200-3000 cm⁻¹) was used. - ¹) The detection was carried out by sorting single cells by flow cytometry and analyzing Raman peaks using OPUS software (a ratio of CD peak to CH peak ≥ 0.1 was used to determine the labeling positive, i.e., the surviving strain). The final survival rate was calculated using the formula: survival rate = number of positive strains / total number of strains × 100%.
[0035] The specific parameters and test results of Examples 1-6 and Control Groups 1-6 are shown in Table 1 below: Table 1
[0036] As shown in Table 1, when the simulated liquid fermentation system was left untreated (control group 1), the relative abundance of *Lactobacillus jinshanensis* after fermentation was only 12.06%, and the survival rate was 20.70%. Even with direct addition of *Lactobacillus jinshanensis* through exogenous enhancement (control groups 2, 3, and 4), its abundance and survival rate remained low after fermentation. This is because the system was not suitable for the growth of *Lactobacillus jinshanensis*, thus direct addition was ineffective. When *Lactobacillus jinshanensis* was first subjected to stress and certain substances were added (control groups 5 and 6), its abundance and survival rate improved significantly, but remained below 60%. However, the effect was very significant when two substances were added for stress culture (Examples 1-6).
[0037] A method for improving the tolerance of Lactobacillus acetic acid in Jinshan and its application in solid-state fermentation systems includes the following steps: (1) Activation of the strain: Glycerol phosphate choline (GPC) was added to modified MRS medium (10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate and 0.25 g / L manganese sulfate), pH 6.2, and sterilized at 121℃ for 20 min; inoculated Acet ilactobacillus jinshanensis The L214 strain was cultured at 30°C under anaerobic conditions for 14 hours to obtain an activated bacterial solution.
[0038] (2) Stress adaptation culture: The activated bacterial solution was transferred to the stress adaptation medium (20 g / L lactic acid, 2 g / L acetic acid, and the other components were the same as the modified MRS medium) at an inoculation rate of 8%. The initial pH was 5.0. After culturing for 8 h, the pH was adjusted to 4.0 with lactic acid-acetic acid solution. After culturing for another 8 h, the pH was adjusted to 3.5. At the same time, lysophosphatidylcholine (LPC) was added. The culture was carried out at 30 °C under anaerobic conditions for 30 h. The amount of lysophosphatidylcholine added was 0.01% to 0.03% of the weight of the stress adaptation medium.
[0039] (3) Fermentation system enhancement: The cultured bacterial solution after stress adaptation was inoculated into baijiu mash and vinegar mash (moisture content 50%-60%) for fermentation. The initial fermentation temperature was 30℃, and the initial pH was adjusted to 2.5-3.5 using a lactic acid-acetic acid mixture. On the third day of fermentation, anhydrous glycerophosphate choline or its hydrochloride form was added by spraying (the amount added was 0.01% of the weight of baijiu mash or vinegar mash), and then fermentation continued for 30 days. The inoculation amount of the cultured bacterial solution after stress adaptation was 0.3% of the weight of baijiu mash or vinegar mash.
[0040] Examples 7-15 refer to the "A method for improving the tolerance of Lactobacillus acetic acid in Jinshan and its application in solid-state fermentation system" in the specific implementation method. The specific parameter differences are detailed in Table 2.
[0041] Control group 7: Baijiu mash (moisture content 50%-60%) was fermented at an initial fermentation temperature of 30℃ and the initial pH was adjusted to 3.0 using a lactic acid-acetic acid mixture; fermentation lasted for 30 days.
[0042] Control group 8: Vinegar mash (moisture content 50%-60%) was fermented at an initial fermentation temperature of 30℃ and the initial pH was adjusted to 3.0 using a lactic acid-acetic acid mixture; fermentation lasted for 30 days.
[0043] Control group 9: Lactobacillus acetic acid bacteria that have not undergone strain activation and stress adaptation culture ( Acetilactobac illus jinshanensis The L214 strain was inoculated into the baijiu mash (moisture content 50%-60%) for fermentation. The initial fermentation temperature was 30℃, and the initial pH was adjusted to 2.5 using a lactic acid-acetic acid mixture. Fermentation lasted for 30 days, and the inoculation amount of Lactobacillus acetic acid was 0.3% of the weight of the baijiu mash.
[0044] Control group 10: Lactobacillus acetic acid bacteria that have not undergone strain activation and stress adaptation culture ( Acetilactoba cillus jinshanensisL214 strain was inoculated into vinegar mash (moisture content 50%-60%) for fermentation. The initial fermentation temperature was 30℃, and the initial pH was adjusted to 2.5 using a lactic acid-acetic acid mixture. Fermentation lasted for 30 days, and the inoculation amount of Lactobacillus jinshanensis was 0.3% of the weight of vinegar mash.
[0045] The only difference between control group 11 and control group 9 was that the initial pH was 3.
[0046] The only difference between control group 12 and control group 10 was that the initial pH was 3.
[0047] After fermentation, the relative abundance and survival rate of Lactobacillus acetic acid in Jinshan were tested, as well as the relative deviation of flavor substances (such as skeletal components and trace components such as ethyl lactate and ethyl acetate) between fermentation batches of vinegar mash / wine mash.
[0048] (1) The relative abundance of Lactobacillus aceticus in Jinshan was obtained by high-throughput sequencing of the 16S rRNA amplicon of the target microorganism in the sample and bioinformatics analysis. The entire 16S rRNA gene segment (V1-V9) was amplified using the Illumina MiSeq sequencing platform. Bioinformatics analysis was performed using QIIME2 software with parameters set as quality filtering (Trimmomatic, Q20 threshold), OTU clustering (97% similarity), and species annotation (comparison with SILVA138 database). Finally, the relative abundance percentage of Lactobacillus aceticus in the total bacterial community was calculated.
[0049] (2) The survival rate was determined based on heavy water (D2O) labeled culture combined with Raman spectroscopy single-cell sorting analysis. The specific procedure was as follows: fermentation samples were taken, inoculated into modified MRS medium containing 2% D2O, and cultured anaerobicly at 32℃ for 24 h. Subsequently, a confocal Raman spectrometer (excitation wavelength 532 nm, scanning range 200-3000 cm⁻¹) was used. - ¹) The detection was carried out by sorting single cells by flow cytometry and analyzing Raman peaks using OPUS software (a ratio of CD peak to CH peak ≥ 0.1 was used to determine the labeling positive, i.e., the surviving strain). The final survival rate was calculated using the formula: survival rate = number of positive strains / total number of strains × 100%.
[0050] (3) Flavor consistency was determined using gas chromatography-mass spectrometry (GC-MS) to qualitatively and quantitatively detect volatile flavor compounds in the fermentation system, and the results were evaluated through multivariate statistical analysis. A gas chromatography (Agilent 7890A) and mass spectrometry (Agilent 5975C) platform was used with an HP-5MS column (30m × 0.25mm × 0.25 μm). The temperature was programmed (initial 40℃, held for 3 min, then increased to 250℃ at 5℃ / min, held for 5 min). The injection volume was 1 μL, and the split ratio was 10:1. Samples were pretreated using solid-phase microextraction (SPME). Qualitative analysis was performed using NIST library comparison, and quantification was performed using n-hexanol as an internal standard. Finally, SPSS software was used to calculate the relative deviation and similarity of the target flavor compounds from three or more batches.
[0051] The specific parameters and test results of Examples 7-15 and Control Groups 7-12 are shown in Table 2 below: Table 2
[0052] As shown in Table 2, when abnormalities occurred in the actual food fermentation system without any intervention (control groups 7 and 8), the relative abundance of *Lactobacillus aceticus* after fermentation was only 30-36%, and the survival rate was 35-38%. Similarly, even when *Lactobacillus aceticus* was directly added to the baijiu or vinegar fermentation system through exogenous enhancement (control groups 9, 10, 11, and 12), its abundance and survival rate were very low after fermentation. This is because the system was no longer suitable for the growth of *Lactobacillus aceticus*, so direct addition was ineffective. However, when *Lactobacillus aceticus* was first subjected to stress and supplemented with substances before direct addition (Examples 7-15), its abundance and survival rate were significantly improved, demonstrating a very significant effect.
[0053] Mechanism explanation: A schematic diagram illustrating the enhanced tolerance of Lactobacillus acetic acid to extreme acid stress is attached. Figure 1 As shown.
[0054] Betaine is a typical compatible solute that stabilizes proteins and membrane structures under hyperosmolar or acid stress, helping cells maintain osmotic homeostasis. Phosphatidylcholine (PC) is the main structural membrane lipid, determining the physicochemical properties and adaptability of the membrane. *Lactobacillus jinshanensis* carries a betaine / carnitine / choline transporter (BCT), which can take up choline with high affinity. The ingested choline can be further converted into the compatible solute betaine, enhancing the bacterium's stress tolerance.
[0055] Meanwhile, lysophosphatidylcholine (LPC) is a surface-active molecule that can insert into bacterial membranes, depolarize membranes, and increase the membrane permeability of Gram-positive bacteria, while its hydrolysis product, glycerophosphocholine (GPC), and long-chain fatty acids may support bacterial survival.
[0056] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. A method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress, characterized in that: Includes the following steps: Step 1, Strain activation: Add glycerophosphate choline to the modified MRS medium, then inoculate with Lactobacillus acetic acid for activation culture to obtain activated bacterial solution; Step 2, stress adaptation culture: The activated bacterial solution obtained in Step 1 is inoculated into the stress adaptation culture medium, and the pH is gradually reduced to carry out stress acclimatization. Lysophosphatidylcholine is added during the acclimatization process, and culture is carried out to obtain the acclimatized bacterial solution. Step 3: Fermentation system enhancement: Inoculate the domesticated bacterial culture obtained in Step 2 into the target fermentation system, and supplement the fermentation system with glycerophosphate choline or its hydrochloride to carry out fermentation.
2. The method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress according to claim 1, characterized in that: In step one, the modified MRS medium consists of: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate, and 0.25 g / L manganese sulfate. The amount of glycerophosphate choline added is 0.02% to 0.05% of the weight of the modified MRS medium.
3. The method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress according to claim 2, characterized in that: In step one, the activation culture conditions are: culturing at 30–32℃ under anaerobic conditions for 12–16 hours, until the bacterial culture OD... 600 It reaches 0.8 to 1.
0.
4. The method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress according to claim 3, characterized in that: In step two, the stress adaptation culture medium consists of: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 10–30 g / L lactic acid, and 1–3 g / L acetic acid.
5. The method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress according to claim 4, characterized in that: In step two, the method of gradually reducing pH is as follows: initial pH 5.0, after culturing for 8 hours, adjust to pH 4.0, continue culturing for another 8 hours, and then adjust to pH 2.5-3.5; then add lysophosphatidylcholine and culture at 30°C under anaerobic conditions for 24-36 hours; the amount of lysophosphatidylcholine added is 0.01%-0.03% of the weight of the stress adaptation medium; the pH adjuster is a mixed solution of lactic acid and acetic acid.
6. The method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress according to claim 5, characterized in that: In step three, the inoculation amount of the acclimatization bacterial solution is 0.1% to 0.5% of the dry weight of the fermentation substrate; the supplementation amount of glycerophosphate choline or its hydrochloride is 0.01%; the fermentation conditions are: initial pH 2.5 to 3.5, temperature 28 to 32°C, and moisture content 50% to 70%.
7. A method for improving the tolerance of Lactobacillus acetic acid to extreme acid stress according to claim 6, characterized in that: In step three, the target fermentation system is either a liquid fermentation system or a solid fermentation system.
8. The application of the method according to any one of claims 1 to 7 in improving the tolerance of Lactobacillus acetic acid to extreme acidic and / or high osmotic pressure environments.
9. The application of the method according to any one of claims 1 to 7 in the brewing of baijiu or vinegar.
10. The application of the method according to any one of claims 1 to 7 in improving the flavor consistency between batches of fermented products.