Domestication method for improving acid tolerance and fermentation density of lactobacillus plantarum

Abstract

The invention belongs to the field of improvement of acid tolerance and high-density fermentation of probiotics, and particularly relates to a domestication method for improving acid tolerance and fermentation density of lactobacillus plantarum and an obtained strain. The invention provides a domestication method for improving acid tolerance and fermentation density of lactobacillus plantarum, which is characterized in that Lactiplantarum ZJ316 (CCTCC NO: M 208077) is used as a wild strain, the wild strain is domesticated, and the obtained domesticated strain ZJ316PV (Lactiplantarum ZJ316PV) has the preservation number of CGMCC NO.33082. The acclimatization method for improving acid tolerance and fermentation density of lactobacillus plantarum has the advantages that the acclimatization method comprises the following steps: taking Lactiplantarum ZJ316 (CCTCC NO: M 208077) as the wild strain; the acid tolerance of the domesticated strain ZJ316PV is superior to that of the wild strain ZJ316. The invention also provides a method for improving the PV fermentation density of the wild strain ZJ316 and the domesticated strain ZJ316.

Description

Technical Field

[0001] This invention belongs to the field of improving the acid tolerance of probiotics and their high-density fermentation, specifically involving a domestication method and the resulting strain for improving the acid tolerance and fermentation density of Lactobacillus plantarum. Background Technology

[0002] Lactobacillus plantarum is widely found in traditionally fermented vegetables. It is a facultative anaerobic bacterium belonging to the genus Lactobacillus in the family Lactobacillusceae of the phylum Firmicutes in the kingdom Bacteria, and is commonly used in the fermentation industry. Compared to other similar probiotics, Lactobacillus plantarum has a stronger ability to produce lactic acid during fermentation. Fermenting yogurt with Lactobacillus plantarum not only gives it a unique flavor, but also allows it to metabolize and produce various short-chain fatty acids and beneficial components during fermentation, which are conducive to promoting human intestinal health. Lactobacillus plantarum is also considered a potential probiotic that resists the invasion of pathogenic microorganisms into the intestines, as it can produce antimicrobial peptides encoded by genes such as plnJ and plnK, which can effectively inhibit the growth of foodborne pathogens. Improving the acid resistance of lactic acid bacteria helps enhance their survival rate and activity in gastric acid or acidic environments, thereby improving the effectiveness of probiotics, especially their function after yogurt fermentation or digestion by gastric acid.

[0003] Currently, methods for improving the acid tolerance of lactic acid bacteria, both domestically and internationally, mainly focus on screening acid-resistant strains. In recent years, this has gradually extended to methods such as gene regulation, gene editing, and domestication, including screening for strongly acid-resistant strains from the gastrointestinal tract or reorganizing genes to highly express acid-resistant genes. Due to the complexity of the basic metabolic network related to the acid tolerance of lactic acid bacteria, it is difficult to obtain positive mutant strains through rational genetic engineering design. However, adaptive laboratory domestication techniques can achieve rapid diversity evolution of strains by artificially simulating evolutionary environments, thereby obtaining beneficial mutant strains with genetic stability. Previous studies have shown numerous cases where adaptive domestication techniques have successfully improved the resistance of strains to different stress environments, such as pH, osmotic pressure, temperature, UV irradiation, and metabolic byproducts, indicating that using this technology to improve the tolerance of microorganisms to environmental stress is a very effective solution.

[0004] *Lactobacillus plantarum* typically possesses a certain degree of acid tolerance, maintaining stable growth even at pH 4.0. Studies have shown that it primarily maintains its cytoplasmic pH around 7.0 through glutamate decarboxylase and F0F1-ATPase. When cultured in normal MRS medium, *Lactobacillus plantarum* accumulates large amounts of lactic acid during the late logarithmic growth phase, causing a rapid pH drop. At this point, the bacterial community reaches a plateau where growth and death are intertwined. Long-term laboratory nutrient-enrichment and acid-adaptation acclimatization of strains can enhance the bacteria's ability to utilize environmental substances and effectively improve their survival rate during post-fermentation processes. Therefore, research on the laboratory nutrient-enrichment and acid-adaptation acclimatization of probiotics is of significant importance in the food industry's fermentation field.

[0005] Invention CN113355271A, entitled "Methods for Improving the Acid Stress Resistance of Lactic Acid Bacteria and Their Applications," discloses the use of chemical inducers, such as β-glucosides including salicin, to induce the regulation of the bg1 operon gene expression in *Lactobacillus*, *Bifidobacterium*, and *Streptococcus*, thereby improving the distribution of lactic acid bacteria subpopulations and their acid tolerance. This method can be applied to extend the shelf life of food, pharmaceuticals, feed, and chemicals.

[0006] The invention CN113355271A, entitled "Method for Improving the Acid Stress Resistance of Lactic Acid Bacteria and Its Application", states that adding weak acids or weak acid salts, including but not limited to acetic acid, propionic acid, n-butyric acid and potassium acetate, to the fermentation culture medium of Lactobacillus plantarum can improve the acid resistance of Lactobacillus plantarum cells.

[0007] Invention CN115948316A, entitled "A Method for Improving the Acid Tolerance of Lactic Acid Bacteria," discloses that by using gene recombination technology to transform the special functional genes carbamate kinase or arginine deiminase into the target genome, the acid tolerance of lactic acid bacteria NZ9000 is improved. Under the same acid stress conditions, the survival rate is increased by 1.25 to 1.5 times compared to the original strain.

[0008] Invention CN115261298A, entitled "A Method for Domesticating Thermoresistant Lactobacillus plantarum and a Method for Preparing Lactobacillus plantarum Powder," discloses that the invention employs a gradient temperature increase method, including but not limited to multi-stage high-temperature cultivation, until a water bath survival rate of ≥80% is achieved at 69–71°C. Furthermore, the invention incorporates a protective agent made from sodium alginate, calcium chloride, chitosan, yeast cell wall, glucono-δ-lactone, skim milk powder, and sodium stearate, which is applied to the spray drying method for preparing the bacterial powder from this thermoresistant strain.

[0009] The invention CN114044750B, "Method for Purifying Indole-3-Lactic Acid from Fermentation Supernatant of *Lactobacillus plantarum* ZJ316 (accession number CCTCC NO: M208077)," utilizes the fermentation supernatant of *Lactobacillus plantarum* ZJ316 to purify indole-3-lactic acid. *Lactobacillus plantarum* strain ZJ316 was initially isolated from healthy infant fecal samples and possesses many probiotic characteristics, such as significantly improving pig growth and pork quality, and exhibiting antibacterial activity against various in vitro pathogens, including *Micrococcus luteus*, *Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli*, *Salmonella enterica*, and *Listeria monocytogenes*. Whole-genome sequencing of *Lactobacillus plantarum* strain ZJ316 (BGI-Shenzhen, Shenzhen, China) was performed using shotgun sequencing. Detailed wild-type genome assembly and annotation can be found in the following published article (https: / / doi.org / 10.1128 / genomea.00094-13).

[0010] Li X, Gu Q, Lou X, Zhang Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a method for domesticating a strain of Lactobacillus plantarum with high fermentation characteristics and acid tolerance, and the resulting strain.

[0012] To address the aforementioned problems, this invention provides a domestication method for improving the acid tolerance and fermentation density of Lactiplantibacillus plantarum, using Lactiplantibacillus plantarum ZJ316 (CCTCC NO:M 208077) as a wild strain for domestication.

[0013] As an improvement to the domestication method of the present invention for increasing the acid tolerance and fermentation density of Lactobacillus plantarum, the domestication process is divided into the following four stages:

[0014] Phase 1: Acclimation culture was carried out using MRS liquid medium with a pH of 5.5 (±0.02) for 10±0.5 days;

[0015] Second stage: Acclimation culture was carried out using MRS liquid medium with pH 4.5 (±0.01) for 20±0.5 days;

[0016] Phase 3: Acclimation culture was carried out using MRS liquid medium with a pH of 4.0 (±0.01) for 30±0.5 days;

[0017] Phase 4: The strain was acclimatized using MRS liquid medium at pH 3.5 (±0.01) until the following condition was met: the amount of cells obtained by the cultured strain in MRS medium changed significantly compared with the amount of cells obtained by the wild strain in MRS medium; this phase 4 ended; the resulting strain was named the acclimatized strain ZJ316PV.

[0018] As a further improvement to the domestication method of improving the acid tolerance and fermentation density of Lactobacillus plantarum of the present invention: the "significant change" refers to the fact that the amount of cells obtained by the cultured strain in MRS medium is ≥1.2 times the amount of cells obtained by the wild strain in MRS medium, which is judged as a significant change.

[0019] As a further improvement to the domestication method of the present invention for improving the acid tolerance and fermentation density of Lactobacillus plantarum:

[0020] Each stage involved inoculating the bacterial culture into the culture medium every 12 hours, with each inoculation amount being 1% (v / v), and the culture temperature being 37℃.

[0021] The first, second, and third stages are for shaking culture at a speed of 180±20 rpm; the fourth stage is for static culture.

[0022] Note: In this invention, the fourth stage of cultivation takes approximately 40 days.

[0023] The present invention also provides a domesticated strain ZJ316PV (Lactiplantibacillus plantarum ZJ316PV), with accession number CGMCC NO.33082.

[0024] The domesticated strain ZJ316PV exhibits better acid tolerance than the wild strain ZJ316 (CCTCC NO:M 208077).

[0025] The domesticated strain ZJ316PV exhibits better acid tolerance within the pH range of 3.0–4.0, and its two enzymes, which play a major role in acid resistance, show higher activity. The domesticated strain also demonstrates greater survival ability and strategies at the fermentation endpoint.

[0026] This invention also provides a culture medium for both the domesticated strain ZJ316PV and the wild strain ZJ316, which may be any of the following:

[0027] MRS modified culture medium: Dissolve 20g trehalose, 15g peptone, 2.5g yeast extract, 5g beef extract, 15g disodium glycerophosphate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.2g ferric sulfate, 0.05g manganese sulfate monohydrate, and 1g T-80 in 1000ml distilled water, and autoclave at 121℃ for 15min for later use.

[0028] The optimal MRS medium is as follows: glucose 20 g / L, peptone 15 g / L, beef extract 5 g / L, yeast extract 2.5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.05 g / L, and Tween 80 1 mL / L. Weigh the above ingredients according to the above proportions, dissolve them in 1 L of distilled water, and autoclave at 121 °C for 15 min for later use.

[0029] Note: Solid MRS is mixed with 20 g / L agar.

[0030] This invention also provides a method for increasing the fermentation density of wild strain ZJ316 and domesticated strain ZJ316PV: using MRS modified medium and high-level MRS medium;

[0031] The inoculation rate was 2%.

[0032] Fermentation conditions were: 34℃, 180rpm, 16h.

[0033] In this invention, the tested strain was *Lactiplantibacillus plantarum* ZJ316 (CCTCC NO: M208077). The whole genome data has been submitted to the National Center for Biotechnology Information (NCBI), NCBI accession numbers: NC_020229 (chromosome), NC_021903 (plasmid pLP-ZJ101), NC_021904 (plasmid pLP-ZJ102), and NC_021912 (plasmid pLP-ZJ103).

[0034] This invention explores the acid tolerance mechanism of Lactobacillus plantarum through adaptive domestication and endows it with the ability to resist adverse environments such as gastric acid.

[0035] This invention provides a method for acclimatizing a strain of *Lactobacillus plantarum* with high fermentation characteristics and acid tolerance; it also provides an inoculation treatment method to reduce the influence of other ineffective components in the seed culture on secondary fermentation; and it further provides an improved culture medium suitable for high-density fermentation of the acclimatized strain ZJ316. This invention provides a theoretical basis for the laboratory acclimatization of *Lactobacillus plantarum* strains to nutrient enrichment and acid tolerance through multi-omics analysis and physiological and biochemical tests on wild-type and acclimatized strains. The results of this invention provide a theoretical basis for the principles of probiotic acid tolerance and industrial fermentation of probiotics.

[0036] As an improvement to the physiological growth level testing of the strains of this invention: Traditional inoculation methods cannot guarantee consistent initial viable cell counts between wild-type and domesticated strains, and cannot guarantee whether metabolic waste from the seed culture and residual culture medium will affect the next stage of fermentation. Therefore, the inoculation method was improved. Wild-type and domesticated strains preserved using the method of this invention were used, and the viable cell counts in multiple centrifuge tubes were tested using a 10-fold serial dilution method and plate count method. The experiment was repeated three times. The counted viable cell count of the wild-type strain was recorded as X1 CFU / ml, and the viable cell count of the domesticated strain was recorded as X2 CFU / ml. During testing, the bacterial culture from the original preservation tube was used for inoculation, and the inoculation volume of the domesticated or wild-type strain was changed to ensure that the initial viable cell counts were essentially consistent. The OD values ​​of wild-type and domesticated strains grown on normal MRS medium were tested and compared. 600 Growth kinetics curves, viable cell count growth curves, and biomass mass after 24 hours of fermentation were obtained. This invention also measured the OD (Organic Demand) of the domesticated strain at different inoculum amounts.600 Growth curves were generated to verify the effect of the initial viable cell count on growth and to support subsequent culture medium optimization.

[0037] This invention conducted a strong acid stress challenge test on wild-type and domesticated strains of *Lactobacillus plantarum* ZJ316. The MRS (microbial slurry) was pre-adjusted to different pH values ​​(2.00, 3.00, 4.02) with concentrated hydrochloric acid, and the pH stabilized at 2.00, 3.00, and 4.00 after high-temperature and high-pressure sterilization, respectively. This ensured a consistent initial viable cell count per milliliter of bacterial solution in each tube. Control and replicate wells were set up. After inoculation, the bacterial solution was swirled for 30 seconds, and 200 μL of the solution was transferred to a 96-well plate for growth kinetic testing. This invention also tested acid survival rate; that is, the fermented sludge obtained by inoculation in the above manner was washed once with PBS buffer and then directly exposed to hydrochloric acid-phosphate buffer systems of different pH values. Subsequently, the viable cell count at each time point was tested using a tenfold serial dilution method and plate count method.

[0038] This invention presents comparative genomic and reference transcriptomic analyses of wild-type and domesticated strains of *Lactobacillus plantarum* ZJ316 and ZJ316PV. Specifically, the analysis involves steps such as sample preparation, DNA library construction, RNA-seq library construction, sequencing methods, data quality control, genome assembly, general database annotation, sequence alignment and gene set enrichment analysis, and gene structure analysis.

[0039] This invention optimizes the culture medium for *Lactobacillus plantarum* ZJ316PV. The medium includes several buffer salts (disodium glycerol phosphate, trisodium phosphate, monosodium glutamate, sodium acetate), carbon sources (glucose, sorbitol, galactose, galactitol, trehalose, lactose, fructose, xylose, mannose, sucrose, oligodextrose, fructooligosaccharides, maltose, arabinose, cellobiose), nitrogen sources (peptone, yeast extract, beef extract), and trace metals (ferrous sulfate). The optimized scheme involves inoculating the culture medium with glycerol preservation tubes of the domesticated strain, and preparing a modified medium by subtracting or supplementing the aforementioned buffer salts, carbon sources, and nitrogen sources from the MRS basal medium. Growth kinetics testing methods are used to initially screen suitable factor levels. Static fermentation is then used to further refine the fermentation results using a single-factor design, with the viable cell count per milliliter of bacterial culture as the test index. Orthogonal optimization design is then used to further refine the static fermentation results using a composite design. The predicted optimal scheme is then replicated and validated. Attached Figure Description

[0040] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0041] Figure 1 The 24-hour viable cell count growth curve, 24-hour biomass mass, and A of wild-type Lactobacillus plantarum strain ZJ316 and its domesticated strain PV (ZJ316PV) were compared.600 Growth curves, 24-hour growth phenotypes, strong acid stress challenge test, and comparison of survival rates under strong acid stress.

[0042] Figure 1 middle:

[0043] (A); Comparison of viable cell count growth curves of Lactobacillus plantarum ZJ316 and its domesticated strain PV after 24 hours of fermentation.

[0044] (B); Comparison of biomass quality after 24 hours of fermentation by Lactobacillus plantarum ZJ316 and its domesticated strain PV.

[0045] (C); A fermentation of Lactobacillus plantarum ZJ316 and its domesticated strain PV for 16 h 600 Comparison of growth curves

[0046] (D); Phenotypic comparison of Lactobacillus plantarum ZJ316 and its domesticated strain PV. The left test tube contains the domesticated strain, and the right test tube contains the wild-type strain.

[0047] (E); A strain (ZJ316PV) controlling different initial viable cell counts. 600 Growth curve (1),

[0048] (F); A strain (ZJ316PV) controlled for different initial viable cell counts. 600 Growth curve (2),

[0049] (G); Survival rate test of Lactobacillus plantarum ZJ316 under strong acid stress

[0050] (H); Survival rate test of domesticated strain (ZJ316PV) under strong acid stress.

[0051] Figure 2 The diagram shows the global chromosome alignment of Lactobacillus plantarum ZJ316 and its domesticated strains, the comparison of 16S rDNA, the alignment of 16S rDNA operons, the bubble chart of differentially functional metabolic gene enrichment analysis, the volcano chart of gene differential expression, and the whole genome map of Lactobacillus plantarum ZJ316PV. Figure 2 middle:

[0052] (A); Schematic diagram of global chromosome alignment between Lactobacillus plantarum ZJ316 and its domesticated strain PV (first 1–45000 bp).

[0053] (B); Complete genome map of Lactobacillus plantarum ZJ316PV

[0054] (C); Global comparison of 16S rDNA of Lactobacillus plantarum ZJ316 and its domesticated strain PV

[0055] (D); Comparison of 16S rDNA operons between Lactobacillus plantarum ZJ316 and its domesticated strain PV (1 of the five 16S rDNA operons),

[0056] (E); Bubble chart showing the differential functional metabolic gene enrichment analysis between Lactobacillus plantarum ZJ316 and its domesticated strain PV.

[0057] (F): Volcano diagram of differential gene expression between Lactobacillus plantarum ZJ316 and its domesticated strain PV.

[0058] Figure 3 The ATPase activity test and glutamate decarboxylase activity test were performed on Lactobacillus plantarum ZJ316 and its domesticated strain.

[0059] Figure 3 middle:

[0060] (A); ATPase enzyme activity standard curve

[0061] (B); Standard curve of glutamate decarboxylase activity

[0062] (C): Comparison of ATPase activity between Lactobacillus plantarum ZJ316 and its domesticated strain PV

[0063] (D): Comparison of glutamate decarboxylase activity between Lactobacillus plantarum ZJ316 and its domesticated strain PV.

[0064] Figure 4 The following are the test diagrams for sugar fermentation, buffer salt fermentation optimization, and micronutrient fermentation optimization of Lactobacillus plantarum ZJ316PV.

[0065] Figure 4 middle:

[0066] (A); Sugar fermentation test of Lactobacillus plantarum ZJ316PV Figure 1 (8 types, including a glucose control group)

[0067] (B); Sugar fermentation test of Lactobacillus plantarum ZJ316PV Figure 2 (8 types)

[0068] (C); Nitrogen source fermentation test diagrams of Lactobacillus plantarum ZJ316PV (3 types)

[0069] (D); Optimization test results for Lactobacillus plantarum ZJ316PV buffer salt fermentation (4 types)

[0070] (E); Micronutrient fermentation optimization test diagram of Lactobacillus plantarum ZJ316PV (1 species)

[0071] Figure 5 To verify the viable cell count and fermentation quality of Lactobacillus plantarum ZJ316PV;

[0072] Figure 5 middle:

[0073] (A) Verification of viable cell count in fermentation of Lactobacillus plantarum ZJ316PV (4 sugars, 2 organic salts)

[0074] (B) Biomass quality verification of Lactobacillus plantarum ZJ316PV (4 sugars, 2 organic salts);

[0075] Figure 6 See Appendix 1.

[0076] Figure 7 This is a sequence list. Detailed Implementation

[0077] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0078] 0.1M PBS, i.e., 0.1M phosphate buffer (pH 7.2).

[0079] MRS medium: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 0.5 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1 mL / L Tween 80. Weigh the above ingredients according to the proportions and dissolve them in 1 L of water. Dispense according to the experimental procedure. Add 20 g / L agar to the solid MRS medium and autoclave at 121 °C for 15 min for later use.

[0080] Example 1:

[0081] S1. The selected test strain was *Lactiplantibacillus plantarum* ZJ316 (CCTCC NO: M208077), which was used as the wild-type strain. Basic physiological and biochemical tests showed that when this strain was cultured in MRS medium (37℃, 180 rpm), the lag phase was 0–2 h, the logarithmic growth phase was 2–14 h, the late logarithmic growth phase was reached 12–15 h, and the plateau phase was reached 15–16 h. Therefore, 12 h was chosen as the subculture time point, at which time the pH of the fermentation broth was 3.9 (±0.05).

[0082] The laboratory domestication process is divided into four stages:

[0083] The first stage involved a 10-day culture process using MRS liquid medium with a pH of 5.5 (±0.02).

[0084] The second stage involved a 20-day culture process using MRS liquid medium with a pH of 4.5 (±0.01).

[0085] The third stage involved a 30-day culture process using MRS liquid medium at pH 4.0 (±0.01).

[0086] The fourth stage involved a 40-day incubation period using MRS liquid medium at pH 3.5 (±0.01) until the following condition was met: the cell count of the cultured strain in MRS medium showed a significant difference compared to the cell count of the wild-type strain in MRS medium. This fourth stage was then concluded, and the resulting strain was named the domesticated strain ZJ316PV. A significant change was defined as a cell count of the cultured strain in MRS medium ≥ 1.2 times that of the wild-type strain in MRS medium.

[0087] Each stage consisted of the following: the bacterial culture was inoculated into the corresponding culture medium every 12 hours, with an inoculation amount of 1% (v / v) each time, and the culture temperature was 37℃. The first three stages of the acclimatization process were shake culture (180 rpm), and the fourth stage was static culture.

[0088] Note: In each of the above stages, the pH of the MRS liquid culture medium was adjusted using 9M HCl, based on the standard MRS culture medium.

[0089] The cell count of the domesticated strains in MRS medium was compared with that of the wild-type strains in MRS medium using the following method:

[0090] The test strains obtained from the fourth stage of domestication and the wild strain ZJ316 (CCTCC NO: M 208077) were subjected to the following operations:

[0091] Subsequent physicochemical tests on all tested strains were conducted using the following steps to establish the original strain library and the working strain library:

[0092] The fermentation broth obtained on day 40 was streaked, and single colonies were picked for activation culture (10 ml, MRS, 24 h, 37℃, 180 rpm), followed by subculturing (20 ml, 2%, MRS, 12 h, 37℃, 180 rpm), and then scaled up (2%, MRS, 15 h, 37℃, 80 rpm). A sterile, clean centrifuge bottle was used to enrich the bacterial slurry by low-temperature centrifugation (4℃, 2000 g, 10 min). 100 mL of sterile PBS was added, vortexed, and centrifuged once more under the same conditions. The supernatant was discarded, and another 100 mL of sterile PBS was added, vortexing for 1 min. 400 μL of the bacterial suspension was then aliquoted into 1.5 mL centrifuge tubes pre-filled with 600 μL of 50% glycerol, mixed thoroughly, and stored at -80℃ for later use. 1 mL of the remaining bacterial suspension was used for additional 16S rDNA sequencing identification. As an improvement to this method, sterile 25% skim milk powder emulsion can be used to replace the above-mentioned "50% glycerin".

[0093] Random samples were taken from the above-mentioned working bacterial culture library, vortexed, and diluted with 100 μL of the culture medium from the preservation tube and 900 μL of 0.1M (pH 7.2–7.4) PBS buffer in a 1.5 ml centrifuge tube. This dilution process was repeated several times until a suitable gradient was achieved (empirical conditions suggest a colony count of 30–300 on the plate is ideal). The viable cell count in the diluted culture medium was then calculated using the plate count method. Based on the number of dilutions, the corresponding order of magnitude was estimated to calculate the viable cell count per milliliter of enriched bacterial suspension in the original wild-type and domesticated strain preservation tubes. The inoculation volume was adjusted according to the difference in viable cell count to ensure that the initial viable cell count after inoculation was as consistent as possible.

[0094] Take other preservation tubes and inoculate them into conventional MRS medium at an inoculation rate of 1% for 12 hours; the primary fermentation broth is obtained.

[0095] Preparation of secondary seed culture: Take the above primary seed culture and culture it in test tubes (2%, 200 ml, MRS, 12 h, 37 ℃, 180 rpm).

[0096] Preparation of the third-level seed culture: Take the above-mentioned second-level seed culture and add it to an Erlenmeyer flask containing liquid culture medium (1L, 2%, MRS, 12h, 37℃, 180rpm).

[0097] Unless otherwise specified, the primary seed culture is generally left to stand for incubation. Other seed cultures are incubated for 12 hours before inoculation. If incubation continues for 15-16 hours, the culture can be used for physicochemical analysis of the fermentation endpoint. Furthermore, before inoculation, the culture medium to be inoculated should be pre-placed in a 37°C oven for 30 minutes to reduce the impact of sudden temperature changes during inoculation on the lag phase of the strain.

[0098] The number of dilutions of the test strains obtained from the fourth stage of domestication is equal to the number of dilutions of the wild strain ZJ316.

[0099] When the number of viable bacteria in the diluted bacterial solution of the test strain obtained in the fourth stage of domestication is ≥1.2 times that of the wild strain ZJ316, it is determined that the amount of bacteria obtained by the test strain (i.e., the strain obtained by domestication culture) in MRS medium has changed significantly compared with the amount of bacteria obtained by the wild strain in MRS medium; only then is this fourth stage ended (in this invention, the fourth stage lasts for about 40 days), and the obtained strain is named Lactiplantibacillusplantarum ZJ316PV (abbreviated as: domesticated strain PV or domesticated strain ZJ316PV).

[0100] That is, the domesticated strain finally obtained by this invention is: Lactiplantibacillus plantarum ZJ316PV, accession number CGMCC No: 33082, accession date: January 15, 2025.

[0101] S2. The traditional inoculation method involves inoculating the seed culture with the secondary fermentation broth. This can lead to a significant drop in the initial pH of fermentation when the inoculation rate is too high, and the impact of an excessively high inoculation rate on the fermentation results is unknown. This experiment used the initial viable cell count per milliliter as the unit of inoculation rate to investigate the effect of different initial viable cell counts of the domesticated strain on the fermentation results, in order to verify the influence of the initial viable cell count on growth and provide support for subsequent culture medium optimization.

[0102] Wild-type and domesticated strains stored at -80℃ were used to test the viable cell count in multiple centrifuge tubes using a 10-fold serial dilution method and a plate count method. The experiment was repeated three times. The 10-fold serial dilution method involved transferring 100 μL of the mixed fermentation broth into a 1.5 ml centrifuge tube pre-filled with 900 μL of 0.1 M PBS, pipetting thoroughly 20–30 times, and repeating the dilution process until the appropriate gradient was reached. The plate count method involved transferring 100 μL of the final dilution to the center of a solid agar plate, spreading it evenly with a sterile disposable spreader, repeating this process three times, and incubating upside down at 37℃ for 48 h. The number of colonies on each plate was counted, and the average value was recorded as the viable cell count per 100 μL of the final dilution.

[0103] The viable count of wild-type strains was recorded as X1 CFU / ml, and the viable count of domesticated strains was recorded as X2 CFU / ml. During testing, the bacterial culture from the preservation tube was inoculated, and the inoculation volume for either the domesticated or wild-type strain was varied to ensure a consistent initial viable count. In this Example 1 design, the viable counts measured in all wild-type and domesticated strain preservation tubes were 5 (±1) × 10⁻⁶. 10After being stored at -80℃ for 3 months, the viable bacterial count still ranged from 3 to 5 × 10⁻⁶ CFU / ml. 10 Between CFU / ml. The culture tube was repeatedly frozen once (specifically, after being removed from the culture tube and brought to room temperature, it was frozen again at -80°C for 1 hour, then thawed at room temperature and the viable count was tested). The final viable count decreased to 1–2 × 10⁻⁶. 10 CFU / ml.

[0104] Viable cell count growth curve test: 1 ml of bacterial culture was taken from the stock tube and added to 100 ml of MRS liquid medium for shake-flask fermentation (37℃, 180 rpm). The bacterial culture was taken every 2 hours and diluted to an appropriate gradient. Viable cell count was performed using the plate count method. Before the experiment, the medium was preheated to 37℃, and the stock tubes were thawed at -4℃ and then brought to room temperature. The experiment was repeated three times. The results are as follows: Figure 1 As shown in (A), the number of viable bacteria in the domesticated strain is relatively high in the later stage of fermentation.

[0105] Biomass quality testing: 1 ml of bacterial culture was taken from the preservation tube and added to 100 ml of MRS liquid medium for shake-flask fermentation (37℃, 180 rpm). 1 ml of the bacterial culture was collected every 2 hours, centrifuged, and the supernatant was discarded. The wet weight was recorded, ensuring no water droplets remained on the tube wall. The experiment was repeated three times. Results are as follows: Figure 1 As shown in (B), the domesticated strain has a higher biomass mass in the later stage of fermentation.

[0106] OD 600 Growth test: Take 100 μL of bacterial culture from the preservation tube and add it to a 10 ml MRS liquid culture medium test tube. Take 200 μL and put it into a 96-well plate, setting up as many replicates as possible. Pour 300 μL of pure water around the outer ring, cover the plate, and test at 34℃ for 16 h. Measure the absorbance at 600 nm every 30 min. The results are as follows: Figure 1 As shown in (C), the fermentation characteristics of the domesticated strain are superior to those of the wild strain.

[0107] Biological phenotypic test: 100 μL of bacterial culture was taken from the preservation tube and added to a 10 ml MRS liquid culture medium test tube. The culture was incubated at 37°C for 16 hours, followed by static incubation. Results are as follows: Figure 1 As shown in (D), the amount of bacteria in the test tube of the domesticated strain (left) is significantly greater than that in the test tube of the wild strain (right), thus indicating that the fermentation characteristics of the domesticated strain are superior to those of the wild strain.

[0108] Domesticated strain inoculum testing: The specific method is as follows: A glycerol storage tube of the domesticated strain from a working strain library with known viable bacterial concentration was used. Kinetic growth curves were tested at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% inoculum concentrations, using 1% (2.5 × 10^7 CFU / ml) as the standard. Furthermore, kinetic growth curves of secondary fermentation broths with inoculum rates of 1%, 2%, and 3% (v / v) using traditional inoculum methods were tested. Several multiples of bacterial culture were taken from the storage tube and added to 10 ml MRS liquid culture medium test tubes, followed by vortexing for 10 minutes. The OD values ​​were then measured using the above-mentioned method. 600 The growth test method was used for the experiment. The results are as follows: Figure 1 As shown in (E and F), the initial viable cell count of the domesticated strain in the fermentation range of 5.0 × 10^7 to 1.0 × 10^8 CFU / ml is acceptable. Figure 1 (E) is the growth curve without subtracting the initial reading of the microplate reader. Figure 1 (F) is the growth curve after subtracting the initial fermentation readings.

[0109] The specific analysis process is as follows:

[0110] 1. Inoculation with different concentrations of live bacteria increases OD 600 The presence of different initial values, along with small error bars and relatively small systematic errors, indicates that the experimental design was successful.

[0111] 2. Other ineffective components of the seed culture were removed during inoculation, resulting in a high initial viable count (10% initial inoculation rate, i.e., 2.5 × 10⁻⁶). 8 (CFU / ml) is beneficial for shortening the fermentation cycle (especially the lag phase), reaching the plateau phase in 8–10 hours; at a lower initial viable count (1% initial inoculum, i.e., 2.5 × 10⁻⁶), it can achieve the desired results. 7 (CFU / ml), the plateau phase can be reached in 12-16 hours;

[0112] 3. Figure F shows that the lower the initial viable cell count, the lower the OD before and after fermentation. 600 The larger the difference, the higher the initial viable cell count and the higher the OD value before and after fermentation. 600 The smaller the difference, the higher the growth potential of the lower concentration of viable cells. Furthermore, according to Figure E, different initial viable cell counts have little effect on the optical density 600 at the fermentation endpoint; they all tend to converge. This indicates that within the experimental design range (2.5 × 10⁻⁶), the optical density 600 is within acceptable limits. 7 ~2.5×10 8The initial viable cell count (CFU / ml) had little effect on the fermentation endpoint, only shortening the fermentation cycle. Further analysis of the final fermentation pH showed that the final pH at all 10 inoculation levels was 3.78 (±0.01). Since in actual practice, inoculation is done by transferring the bacterial culture to the culture medium, it is impossible to avoid the influence of other metabolic waste products, metabolic signaling molecules, and residual culture medium in the seed culture on secondary fermentation. Therefore, this invention evaluated the optimal inoculation rate. The above analytical results indicate that at 5.0 × 10⁻⁶ CFU / ml… 7 ~1.0×10 8 Inoculation within the initial viable cell count range of CFU / ml is highly recommended, as it ensures high fermentation efficiency and reduces the impact of ineffective components from the seed culture on secondary fermentation.

[0113] S3. This step compared survival rates under acid stress.

[0114] MRS culture medium was pre-adjusted to different pH values ​​(2.00, 3.00, 4.01) with concentrated hydrochloric acid, and then autoclaved at high temperature and pressure until the pH stabilized at 2.00, 3.00, and 4.00, respectively. The culture was then inoculated into MRS medium at each pH value as described in step S2. Control wells (using MRS medium with unchanged pH as a control) and replicate wells (to reduce systematic error, samples from the same tube were repeatedly added to blank wells, and the average value was taken) were set up. The inoculated culture was swirled for 30 seconds, and 200 μL of the culture was transferred to a 96-well plate for growth kinetic testing. The conditions were set at 34℃ for 16 h, A = 600, and the plate was shaken for 1 minute before each reading. The survival rate test involved adding 100 μL of bacterial suspension from the preservation tube to a 10 ml MRS liquid culture medium tube, incubating for 16 h, centrifuging to obtain bacterial sludge, resuspending the sludge in 0.1 M phosphate buffer, centrifuging again, and resuspending it in 4 ml of 0.1 M phosphate buffer. The sludge was then aliquoted into 1.5 ml centrifuge tubes, and centrifuged again to remove the buffer. The sludge was then mixed with 1 ml of hydrochloric acid-phosphate buffer at different pH values ​​and resuspended to fully expose the bacteria to a strongly acidic environment. After each sampling test, the tubes were returned to a 37°C biochemical incubator. 100 μL samples were taken at 0 h, 1 h, 2 h, 3 h, and 4 h for serial dilution and plate colony counting. The experiment was repeated three times. Survival rate was calculated starting with an initial viable count of 100%, and each subsequent count was calculated as a percentage of the average remaining viable count. Results are shown below. Figure 1 As shown in (G and H), the domesticated strain has better acid tolerance than the wild strain.

[0115] S4. This step involves comparative genomic and transcriptomic analysis of *Lactiplantibacillus plantarum* ZJ316 (wild-type strain) and *Lactiplantibacillus plantarum* ZJ316PV (domesticated strain). This includes total DNA / RNA extraction, sequencing, assembly, annotation, and statistical analysis. Furthermore, the activities of glutamate decarboxylase (GAD) and the F0F1-proton pump (ATPase) in the wild-type and domesticated strains were investigated.

[0116] The initial procedure involved taking 100 μL of bacterial culture from the domesticated strain preservation tube and adding it to 10 ml of MRS liquid medium for static incubation for 12 h. This was followed by scale-up culture at 2% (v / v) at 37°C for 16 h at 180 rpm. Before the experiment, the medium was preheated to 37°C, and the preservation tubes were thawed at 4°C and then brought to room temperature. After 16 h of fermentation, the cells were collected by centrifugation (6000 g, 4°C, 10 min), washed 1-2 times with sterile PBS buffer, and treated in liquid nitrogen for 1 min to terminate cell metabolism. The cells were then stored at -80°C for later use.

[0117] The genome sequencing assembly process was performed according to the standard protocol provided by PacBio, including sample quality testing, library construction, library quality testing, and library sequencing.

[0118] Library construction includes the following steps: 1. Disrupting the DNA sample using g-TUBE; 2. Repairing the damage to the disrupted DNA sample; 3. End repair of the DNA; 4. Connecting dumbbell adapters; 5. Digesting with exonucleases; 6. Screening for target fragments using BluePippin to obtain the sequencing library.

[0119] The information analysis process mainly includes the following steps: 1. Raw data quality control, filtering out CCS reads that are too short; 2. Genome assembly, assembling the filtered CCS reads de novo and correcting errors in the assembled genome; 3. Genome component analysis, mainly including: repetitive sequences, coding genes, non-coding RNA, prophages, gene islands, CRISPR, etc.; 4. Functional annotation, mainly including general databases such as Nr, Uniprot, COG, KEGG, etc.; 5. Comparative genomic analysis, including chromosome genome alignment, plasmid genome alignment, functional gene variation analysis, and 16S rDNA alignment.

[0120] A schematic diagram of comparative genomics is shown below. Figure 2 (A) shows the preliminary results (first 1-45000bp), as shown in Table 1. Detailed comparison results are provided in Appendix 1. Figure 6Among these mutated CDS genes, genes encoding transposases were found to be more active, providing potential for the strains to adapt to acidic, nutrient-rich laboratory environments. Simultaneously, mutations were observed in genes related to cell membrane proteins, cell starvation / quiescent protection proteins, glycoside hydrolase family proteins, ribosomal proteins, and histidine kinases, which are membrane receptors in the TCS system. The mutated cell membrane protein genes can be divided into two categories based on their function: one primarily responsible for the exchange of substances across the membrane, and the other responsible for bacterial adhesion, anchoring, and aggregation. In addition, some genes that may not be related to acid-adaptive evolution were mutated, such as alkaline phosphatase, Ig-like proteins, 3-dehydroquinic acid synthase, and DNA transposases.

[0121] The results of genome assembly are as follows Figure 2 As shown in (B), Circos visualizes the genome, allowing for a clearer exploration of the positional relationships between genomic components on the genome. Figure 2 (B) shows a circle diagram of the assembled domesticated strain's chromosomes. The diagram reveals a relatively even distribution of transcriptional regulatory elements and ribosomal DNA genes, with genes exhibiting a roughly 50 / 50 orientation. However, their biological significance may require comparative analysis across multiple strains. Typically, genes and other functional elements in a genome are not randomly distributed; they may exhibit certain patterns due to factors such as chromosomal topology.

[0122] 16S rDNA alignment results are as follows Figure 2 As shown in (C) and (D), the results indicate that the wild-type strain has five copies of 16S rDNA in its chromosomal genome, and all sequences are identical. However, the 16S rDNA at the same location in the whole genome of the domesticated strain and the wild-type strain are all different, with the domesticated strain sequence being 1567 bp in length, identical to that of the wild-type strain. The 16S rDNA of the wild-type strain is shown below. Figure 7 As shown in sequence 1, the 16S rDNA of the domesticated strains are as follows: Figure 7 The sequences 2 to 6 are shown.

[0123] 16S rDNA plays a significant role in ribosome assembly, biological system classification, and biological evolution mechanisms. It consists of ten conserved regions and nine variable regions (V1-V9). The numerous point mutations found in domesticated strains are mainly concentrated in the V2 and P3 regions. Figure 2In (C), the seventh sequence is a partitioning diagram of the variable and conserved regions obtained from the ZJ316 16S rDNA analysis, for comparative reference. Furthermore, the number of tRNAs and 5S rDNAs in the domesticated strain ZJ316PV changed; the number of tRNA genes increased by 6 (2 Ile-tRNAs, 2 Ala-tRNAs, 1 Lys-tRNA, and 1 Val-tRNA), and the number of 5S rDNAs increased by one. Both the newly inserted tRNAs and 5S rDNAs are located within the rDNA operon. Figure 2 (D) shows a comparison of a segment of rDNA operon before and after mutation. This mutation trend may indicate that in a nutrient-rich environment, the strain needs more of these special tRNAs to participate in the mRNA translation process to synthesize proteins.

[0124] Table 1 Comparison of basic genome information between wild and domesticated strains

[0125]

[0126] Transcriptome analysis began with the extraction of total RNA from bacterial cells using the Trizol assay, followed by quality control using a Thermo NanoDrop One and an Agilent 4200 Tape Station. After passing quality control, ribosomal RNA was removed using the Epicentre Ribo-Zero rRNA Removal Kit, and strand-specific libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. The specific steps for constructing the library include: 1. Fragmenting mRNA into short fragments; 2. Using mRNA as a template, synthesizing one-stranded cDNA using six-base random hexamers; 3. Adding buffer, dNTPs (replacing dUTP with dTTP in dNTPs), DNAPolymerase I, and RNase H to synthesize two-stranded cDNA; 4. Purifying the double-stranded cDNA using AMPure XP beads; 5. End-repairing the purified double-stranded cDNA, adding an A tail, and ligating sequencing adapters; 6. Degrading the second strand of cDNA containing U using USER enzyme; 7. Selecting fragment sizes using AMPure XP beads; 8. Finally, performing PCR amplification and purifying the PCR product using AMPure XP beads to obtain the final library. Libraries that pass quality control will be sequenced using the Illumina NovaSeq high-throughput sequencing platform with PE150 sequencing. The raw image data files obtained from sequencing will be converted into raw sequencing reads through base calling analysis. The results will be stored in FASTQ (.fq) file format, containing sequence information for each read and its corresponding sequencing quality information. The information analysis workflow mainly includes the following steps: 1. Sequencing data quality control; 2. Sequence alignment analysis; 3. Transcriptome quality assessment; 4. Expression level statistics; 5. Differential gene analysis statistics; 6. Gene Set Enrichment Analysis (GSEA); 7. Gene structure analysis.

[0127] After acclimatization to nutrient and acid conditions, the transcriptome of domesticated strains underwent certain changes. Preliminary analysis using basic transcriptomics tools, with the wild-type whole genome as a reference genome, showed that under the same single-batch fermentation conditions, the expression of at least 328 genes differed significantly between domesticated and wild-type strains (|log2 FC|>0.4, P value<0.05). However, due to gene variation and duplicative transposable genes causing some gene transcriptomes not to match the reference genome, the count values ​​of some genes are not particularly valuable for analysis. To address this issue, data reanalysis is typically performed on individual genes using the domesticated strain reference genome, followed by further correction of the expression data of those genes with erroneous analyses. Figure 2 (E) shows the corrected gene expression differential volcano plot. Based on the background gene proportion and p. adjust, 19 sets of data with biological or statistical significance were selected, and KEGG functional enrichment analysis was performed using Prism 9 software to plot the results. The results are as follows... Figure 2 As shown in (F), analysis of the figure reveals that the differential expression of pyruvate metabolism, phosphotransferase system, and fatty acid synthesis pathway is mainly a result of laboratory adaptive evolution. Furthermore, enrichment analysis of other differentially expressed fatty acids also primarily points to carbon and nitrogen sources, indicating that the laboratory domestication process has enhanced the strain's ability to convert environmental nutrients, primarily carbon sources.

[0128] GadB and ATPase enzyme activity assay:

[0129] The level of glutamate decarboxylase (GAD) in bacterial cells was determined using a double-antibody sandwich method. A microplate was coated with purified GAD antibody to prepare a solid-phase antibody. Glutamate decarboxylase (GAD) was added sequentially to the wells coated with the monoclonal antibody, followed by binding with HRP-labeled GAD antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The color intensity was positively correlated with the amount of GAD in the sample. The absorbance was measured at 450 nm using a microplate reader, and the concentration of GAD activity in the sample was calculated using a standard curve. ATPase was analyzed similarly. The specific procedures are as follows: The bacterial culture was cultured overnight to the late logarithmic phase (16 h). Then, 1 ml of the bacterial culture was centrifuged to collect the bacterial sludge (12000 g, 1 min). The sludge was resuspended twice with 1 ml of PBS buffer, followed by resuspending with 2 ml of PBS buffer. Empty wells served as blank controls. 200 μL of the bacterial suspension was used to measure the OD value at 600 nm. All bacterial suspension samples were diluted to OD = 0.5 ± (0.01). All cell samples were disrupted using an ultrasonic cell disruptor (150 W, 3 s on, 6 s off, 4.5 mins). Before each sonication, the probe was cleaned with ddH2O. During sonication, the probe was immersed to a depth of 1 cm below the liquid surface. Centrifuge tubes were secured with ice and a fixative. The supernatant was collected by centrifugation (6000 g, 3 min). Enzyme activity was further measured according to the kit instructions. The experiment was repeated three times, with three replicates for each level. Data were plotted and analyzed using Prism 9 for macOS V 9.4.1 software.

[0130] The standard curves and results of each enzyme activity are as follows: Figure 3 As shown in (A~D), the ATPase standard curve is "Y=0.01034*X-0.06671R". 2 =0.9953P<0.0001", GadB standard curve "Y=0.01069*X-0.001701R" 2 =0.9862P<0.0001". The standard curves for each enzyme showed good linearity and can be used for further analysis.

[0131] Enzyme activity tests showed that the ATPase and GadB enzyme activities of the domesticated strain (169.51 U / L, 146.31 U / L) were higher than those of the wild strain (157.11 U / L, 133.06 U / L), indicating that the strain's acid resistance has been improved during the long-term acid-adaptive evolution process.

[0132] S5. The culture medium for Lactobacillus plantarum ZJ316PV was optimized.

[0133] Preliminary screening of carbon source levels: MRS medium with glucose removed was supplemented with glucose at a ratio of 5.0 (±0.05)% (m / v), and then simultaneously inoculated with MRS medium without added carbon source at an inoculation ratio of 1% (v / v). The MRS medium with added glucose was diluted in wells with MRS medium without added carbon source to different multiples, with a total bacterial volume of 200 μL in each well, and at least two replicates were set up. 300 μL of pure water was added to the outer ring, and the plates were tested with the lids on at 34℃ for 16 h. The absorbance at 600 nm was measured every 30 min, and the plate was shaken for 1 minute before each test. Results are as follows: Figure 4 (A) Glucose portion shown.

[0134] Preliminary screening of nitrogen source levels: MRS medium excluding peptone, yeast extract, or beef extract was supplemented with nitrogen source at ratios of 2.5 (±0.025)% (m / v), 1.25 (±0.01)% (m / v), and 2.5 (±0.025)% (m / v), respectively. These were then simultaneously inoculated with MRS medium without nitrogen source at an inoculation ratio of 1% (v / v). MRS medium supplemented with the desired nitrogen source was diluted in wells with MRS medium without the desired nitrogen source to different concentrations, with a total bacterial volume of 200 μL in each well. At least two replicates were set up. 300 μL of pure water was added to the outer ring, and the plate was capped and incubated at 34°C for 16 h. The absorbance at 600 nm was measured every 30 min, with the plate shaken for 1 minute before each test. Results are as follows: Figure 4 (C) Nitrogen source section shown.

[0135] First, the five factors most significantly affecting viable cell count were identified through single-factor subtraction of the culture medium. The levels of each factor were then screened based on growth kinetics tests. An orthogonal design was used to optimize the carbon and nitrogen sources and buffer salts in the MRS medium. The test indicators were CFU(b) after eight dilutions and the final pH(a) of fermentation. This experiment was repeated three times. The experimental design and results are shown in Table 2. Ignoring factor B, which had the least impact, the established model was significant, as shown in Table 3. This result indicates that yeast extract has the least impact on viable cell count.

[0136] Table 2L16(4) 5 Orthogonal experimental factor level design and optimal design prediction verification results L16(4) 5 Orthogonal table and results

[0137]

[0138]

[0139] Table 3L16(4) 5ANOVA results for the selected factorial model predicting viable cell count.

[0140] Source Sum of Squares df Mean Square F-value p-value Model 27889 12 2324.08 26.95 0.0101 AA 5572.25 3 1857.42 21.54 0.0157 CC 7397.25 3 2465.75 28.59 0.0104 DD 12160.25 3 4053.42 47 0.0051 EE 2759.25 3 919.75 10.66 0.0415 Residual 258.75 3 86.25 Cor Total 28147.75 15

[0141] Based on the model predictions, three combinations with viable cell counts greater than 220 were selected for repeated validation. The validation results are shown in Table 2 below. Considering cost-effectiveness, Experiment 17 was selected as the optimal level, namely: 2% (m / v) glucose as the basic carbon source, 1.5% (m / v) peptone, 0.25% (m / v) yeast extract, and 0.5% (m / v) beef extract as the basic nitrogen source, and 0.5% (m / v) sodium acetate as the buffer salt, with other components of the culture medium remaining unchanged. Analysis of the final pH of the fermentation response showed that factors A, D, and E had a significant impact on the final pH of fermentation, with E (buffer salt) playing a dominant role and D (glucose) playing a secondary dominant role. Therefore, a reasonable buffer salt system can be further optimized to maintain the final pH of fermentation within a suitable range, thereby enhancing the survival rate of viable cells under moderately strong acid conditions (the relevant experimental design process is described in Example 2).

[0142] That is, the optimal level of MRS medium (Experiment 17) is: glucose 20 g / L, peptone 15 g / L, beef extract 5 g / L, yeast extract 2.5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL / L. Weigh the above proportions and dissolve them in 1 L of water. Dispense according to the experimental procedure. Add 20 g / L agar to the solid MRS and autoclave at 121 °C for 15 min for later use.

[0143] In summary, this embodiment represents an improvement on the basic MRS medium. The medium formulation obtained in Experiment 17 was used for secondary shake-flask fermentation to verify the viable cell count, and the final viable cell count reached 3.95 (±0.45) × 10⁻⁶. 10 CFU / mL. Compared to general MRS medium, it can increase the viable cell count by 1.5 to 2 times. The optimized medium in this example was optimized without changing the basic composition of MRS medium; only the ratio of the main carbon and nitrogen sources in the medium was changed during the optimization process. Since MRS medium is generally used for fermentation in industry, the above scheme is a conservative optimization scheme. This scheme explores the optimal nutrient supply strategy for domesticated strains while also meeting the nutrient supply strategy of industrial design, achieving dual benefits in terms of cost and manpower.

[0144] Example 2:

[0145] This invention represents a variation of the culture medium optimization for *Lactobacillus plantarum* ZJ316PV. The optimization scheme disregards cost-effectiveness, aiming to maximize the number of viable fermentation cells. The results are based on genomic and reference transcriptome studies. Selected sources include: carbon (glucose, sorbitol, galactose, mannose, mannitol, galactooligosaccharides, arabinose, trehalose, fructose, sucrose, oligodextrose, fructooligosaccharides, maltose, cellobiose, inulin), nitrogen (peptone, yeast extract, beef extract), organic salts (monosodium glutamate, disodium glycerophosphate, sodium acetate, trisodium phosphate), and trace inorganic salts (ferrous sulfate).

[0146] Wild-type and domesticated strains, preserved at -80℃ with the fermentation supernatant removed, were used to test the viable cell count in multiple centrifuge tubes using a 10-fold serial dilution method and a plate count method. The experiment was repeated three times. The 10-fold serial dilution method involved transferring 100 μL of the mixed fermentation broth into a 1.5 ml centrifuge tube pre-filled with 900 μL of 0.1 M PBS, and then pipetting thoroughly 20–30 times. This dilution process was repeated until the appropriate gradient was reached. The plate count method involved transferring 100 μL of the final dilution to the center of a solid agar plate, spreading it evenly with a sterile disposable spreader, and repeating this process three times. The plates were incubated upside down at 37℃ for 48 h. The number of colonies on each plate was counted, and the average value was recorded as the viable cell count per 100 μL of the final dilution. The viable cell count of the wild-type strain was recorded as X1 CFU / ml, and the viable cell count of the domesticated strain was recorded as X2 CFU / ml. During testing, the bacterial culture from the preservation tube was inoculated, and the inoculation volume of the domesticated or wild-type strain was varied to ensure that the initial viable cell count was essentially the same. In this Example 2, the viable cell counts measured in all wild-type and domesticated strain preservation tubes were 7 (±1) × 10⁻⁶. 10 CFU / ml, viable bacterial count still ranged from 5 to 7 × 10⁻⁶ after 3 months of storage. 10 Between CFU / ml. After one repeated freezing and storage, the viable bacterial count decreased to 1–2 × 10⁻⁶. 10 CFU / ml, the lag period of primary fermentation was extended by 1 (±0.5) h.

[0147] Preliminary screening of carbon source factors and levels: MRS medium with glucose removed was supplemented with various carbon sources at a ratio of 5.0 (±0.05)% (m / v), and then simultaneously inoculated with MRS medium without added carbon sources at an inoculation ratio of 1% (v / v). The MRS medium with added carbon sources was diluted in wells with MRS medium without added carbon sources to different multiples, with a total bacterial volume of 200 μL in each well, and at least two replicates were set up. 300 μL of pure water was added to the outer ring, and the plates were tested with the lid on at 34℃ for 16 h. The absorbance at 600 nm was measured every 30 min, and the plate was shaken for 1 minute before each test. All preliminary screening results for carbon sources are as follows: Figure 4As shown in (A and B). The results indicate that glucose dimers (D-cellobiose, D-trehalose), isomers (D-mannose), and its sugar alcohol (mannitol) can be well utilized. D-cellobiose, D-trehalose, D-mannose, and D-glucose were selected for further verification of biomass quality and viable cell count during fermentation, and the results are shown below. Figure 4 (As shown in A and B). This study found that trehalose and cellobiose had better utilization rates. However, due to the high price of cellobiose, it was not suitable as a culture medium. Therefore, the carbon source of the MRS medium was replaced, and the MRS medium using trehalose as the carbon source further improved fermentation characteristics. In the following medium optimizations, trehalose (2%, mass / volume) was used as a substitute for glucose.

[0148] Preliminary screening of nitrogen source levels: MRS medium excluding peptone, yeast extract, or beef extract was supplemented with nitrogen source at ratios of 2.5 (±0.025)% (m / v), 1.25 (±0.01)% (m / v), and 2.5 (±0.025)% (m / v), respectively. These were then simultaneously inoculated with MRS medium without nitrogen source at an inoculation ratio of 1% (v / v). MRS medium supplemented with the desired nitrogen source was diluted in wells with MRS medium without the desired nitrogen source to different concentrations, with a total bacterial volume of 200 μL in each well. At least two replicates were set up. 300 μL of pure water was added to the outer ring, and the plate was capped and incubated at 34°C for 16 h. The absorbance at 600 nm was measured every 30 min, with the plate shaken for 1 minute before each test. Results are as follows: Figure 4 As shown in (C), an appropriate level was selected to verify the number of viable cells during fermentation, but the results did not meet expectations; the number of viable cells per milliliter did not increase with the increase of different nitrogen source concentrations. Since the nitrogen source utilization properties of the cells remain basically unchanged during fermentation, the nitrogen source optimization results in Example 1 were used as the nitrogen source optimization in this example, without changing the nitrogen source ratio.

[0149] As part of the screening and optimization of organic salt factors and levels in this example: the tested organic salts included disodium glycerophosphate polyhydrate, trisodium phosphate, monosodium glutamate (MSG), and sodium acetate. Sodium acetate was added to the MRS medium after removing it, following the preliminary screening method for carbon source factors and levels described above. The added sodium acetate concentration was 1.25 (±0.01)% (m / v). Disodium glycerophosphate polyhydrate, trisodium phosphate, and MSG were added additionally to the MRS medium. The added disodium glycerophosphate polyhydrate concentration was 5.0 (±0.05)% (m / v), the added trisodium phosphate concentration was 1.25 (±0.01)% (m / v), and the added MSG concentration was 5.0 (±0.05)% (m / v). The MRS medium containing one of the additional tested organic salts was diluted to different factors in each well with MRS medium without that tested organic salt. The total volume of bacterial culture in each well was 200 μL, with at least two replicate wells. Finally, 300 μL of pure water was added to the outer ring, and the plate was tested at 34°C for 16 hours. The absorbance at 600 nm was measured every 30 minutes, with the plate shaken for 1 minute before each test. Results are as follows: Figure 4 As shown in (D). According to... Figure 4 (D) Results: Appropriate factors and levels were selected for validation of fermentation viable cell count and biomass quality. Results are as follows: Figure 5 (As shown in A and B). The results indicate that 1% monosodium glutamate or 1% disodium glycerophosphate has better fermentation characteristics.

[0150] For the optimization of trace metal elements in this example: the tested trace metal salt solutions contained ferrous sulfate. The added ferrous sulfate was an additional addition to the MRS medium, at a ratio of 0.125 (±0.001)% (m / v). The MRS medium containing the additional trace element was diluted to different factors in each well with MRS medium without the added trace element. The total volume of bacterial culture in each well was 200 μL, with at least two replicates. Finally, 300 μL of pure water was added to the outer ring, and the plate was tested at 34°C for 16 h, with absorbance measured at 600 nm every 30 min. The plate was shaken for 1 minute before each test. The results are as follows: Figure 4 As shown in (E).

[0151] The above optimization results were summarized, and the viable cell count was verified by shake-flask fermentation as the MRS improved medium. The final optimized medium composition was confirmed to be: trehalose (2%), peptone (1.5%), yeast extract (0.25%), beef extract (0.5%), disodium glycerophosphate (1.5%), sodium acetate (0.5%), dipotassium hydrogen phosphate (0.2%), magnesium sulfate heptahydrate (0.05%), ferric sulfate (0.02%), manganese sulfate monohydrate (0.005%), and T-80 (0.1%).

[0152] The preparation method of the modified MRS medium (i.e., the optimized MRS medium) is as follows: Dissolve 20g trehalose, 15g peptone, 2.5g yeast extract, 5g beef extract, 15g disodium glycerophosphate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.2g ferric sulfate, 0.05g manganese sulfate monohydrate, and 1g T-80 in 1000ml distilled water, and autoclave at 121℃ for 15min for later use.

[0153] Wild-type strain ZJ316 (CCTCC NO: M 208077) was inoculated at a rate of 2% onto conventional MRS medium. Fermentation conditions were 34℃, 180 rpm, and 16 h. The viable cell count of the resulting fermentation broth was 2.14 (±0.25) × 10⁻⁶. 10 CFU / ml;

[0154] Wild-type strain ZJ316 (CCTCC NO: M 208077) was inoculated onto modified MRS medium at a 2% inoculum size. Fermentation conditions were the same as above, and the resulting fermentation broth showed a viable cell count of 2.42 (±0.30) × 10⁻⁶. 10 CFU / ml.

[0155] The domesticated strain ZJ316PV was inoculated at a rate of 2% onto conventional MRS medium, and fermentation conditions were the same as above. The viable cell count of the resulting fermentation broth was 2.73 (±0.35) × 10⁻⁶. 10 CFU / ml;

[0156] The domesticated strain ZJ316PV was inoculated at a rate of 2% onto the optimized medium No. 17 from Example 1, and fermentation conditions were the same as above. The viable cell count of the resulting fermentation broth was 3.95 (±0.45) × 10⁻⁶. 10 CFU / ml, initial fermentation pH was 5.91 (±0.02), final pH was 3.82 (±0.01);

[0157] The domesticated strain ZJ316PV was inoculated at a rate of 2% onto the modified MRS medium from Example 2. The fermentation conditions were: 34°C, 180 rpm, 16 h. The resulting fermentation broth had a viable cell count of 7.2 (±1.0) × 10⁻⁶. 10 CFU / ml, initial fermentation pH was 7.02 (±0.02), final pH was 4.04 (±0.01).

[0158] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for domestication that improves the acid tolerance and fermentation density of *Lactobacillus plantarum*, characterized in that: Using Lactiplantibacillus plantarum ZJ316 (CCTCC NO:M 208077) as a wild strain, the wild strain was domesticated.

2. The domestication method for improving the acid tolerance and fermentation density of *Lactobacillus plantarum* according to claim 1, characterized in that: The domestication process is divided into the following four stages: Phase 1: Acclimation culture was carried out using MRS liquid medium with a pH of 5.5 (±0.02) for 10±0.5 days; Second stage: Acclimation culture was carried out using MRS liquid medium with pH 4.5 (±0.01) for 20±0.5 days; Phase 3: Acclimation culture was carried out using MRS liquid medium with a pH of 4.0 (±0.01) for 30±0.5 days; Phase 4: The strain was acclimatized using MRS liquid medium at pH 3.5 (±0.01) until the following condition was met: the amount of cells obtained by the cultured strain in MRS medium changed significantly compared with the amount of cells obtained by the wild strain in MRS medium; this phase 4 ended; the resulting strain was named the acclimatized strain ZJ316PV.

3. The domestication method for improving the acid tolerance and fermentation density of *Lactobacillus plantarum* according to claim 2, characterized in that... The term "significant change" refers to a situation where the bacterial cell count of the cultured strain in MRS medium is ≥1.2 times that of the wild-type strain in MRS medium, which is considered a significant change.

4. The domestication method for improving the acid tolerance and fermentation density of *Lactobacillus plantarum* according to claim 2 or 3, characterized in that: Each stage involved inoculating the bacterial culture into the culture medium every 12 hours, with each inoculation amount being 1% (v / v), and the culture temperature being 37℃. The first, second, and third stages are for shaking culture at a speed of 180±20 rpm; the fourth stage is for static culture.

5. The domesticated strain ZJ316PV (Lactiplantibacillus plantarum ZJ316PV), characterized by: The accession number is CGMCCNO.33082.

6. The domesticated strain ZJ316PV according to claim 5, characterized in that: The domesticated strain ZJ316PV exhibits better acid tolerance than the wild strain ZJ316.

7. A culture medium for domesticated strain ZJ316PV and wild strain ZJ316, characterized in that... For any of the following: MRS Modified Culture Medium: Dissolve 20g trehalose, 15g peptone, 2.5g yeast extract, 5g beef extract, 15g disodium glycerophosphate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.2g ferric sulfate, 0.05g manganese sulfate monohydrate, and 1g T-80 in 1000ml distilled water. The optimal MRS medium is as follows: glucose 20 g / L, peptone 15 g / L, beef extract 5 g / L, yeast extract 2.5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.05 g / L, and Tween 80 1 mL / L. Weigh out the above proportions and dissolve them in 1 L of distilled water.

8. A method for increasing the fermentation density of wild strain ZJ316 and domesticated strain ZJ316PV, characterized in that: MRS modified medium and high-quality MRS medium were used. The inoculation rate was 2%. Fermentation conditions were: 34℃, 180rpm, 16h.

Images