Propylene glycol-eating Lactobacillus brachyphyllum and application thereof in combined leavening agent and koumiss

Abstract

The invention relates to the technical field of microorganisms, and discloses propylene glycol eating lactobacillus and application thereof in a combined leavening agent and koumiss, the propylene glycol eating lactobacillus MNJ-2 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35857, the preservation number is CGMCC No.35857, and the preservation number is CGMCC No.35857. The invention relates to a lactobacillus plantarum strain, which has good acid resistance, cholate resistance and intestinal epithelial cell adhesion capacity, shows the potential of serving as a probiotic and has no obvious antagonism with other strains, and further relates to a leavening agent prepared by combining the strain with lactobacillus plantarum ZKLp l 800, lactobacillus paracasei ZKL200 and saccharomyces cerevisiae ZKM011 in proportion. Compared with traditional koumiss, the koumiss produced by applying the combined leavening agent is more stable in state, richer in fragrance and richer in taste, meanwhile, the traditional sour taste and wine fragrance are reserved, and the koumiss has wide application prospects in the aspect of industrial production.

Description

Lactobacillus propylene glycol and its application in combined starter cultures and fermented mare's milk Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Lactobacillus propylene glycol and its application in combined fermentation agents and fermented mare's milk. Background Technology

[0002] Fermented mare's milk, a traditional beverage made by fermenting fresh mare's milk with specific microorganisms, is a traditional fermented food of herders with a long history, dating back to the Spring and Autumn Period according to historical records. Besides being a daily drink, fermented mare's milk also holds an important place in Mongolian medicine, being one of the earliest dietary drinks used in therapy and as an adjunct treatment for various diseases.

[0003] Traditional methods of making fermented mare's milk are typically quite primitive. They usually begin by fermenting a small amount of fresh mare's milk using fermented cow's milk as a primary starter. This fermented mare's milk is then used as a secondary starter in new fresh mare's milk to expand the fermentation process, requiring periodic stirring until fermentation is complete. However, because the starter cultures (i.e., the source of the microbial culture) used vary across regions and batches, fermented mare's milk produced in different areas exhibits significant differences in taste and flavor, resulting in inconsistent product quality and hindering standardized and large-scale industrial production.

[0004] Modern microbiological research has found that the unique flavor and efficacy of fermented mare's milk mainly originate from its complex microbial community. Analysis of the bacterial composition of fermented mare's milk samples from different pastoral areas revealed that *Lentilactobacillus diolivorans* is one of the dominant lactobacilli. Further research showed that the *Lentilactobacillus diolivorans* strain MNJ-2, isolated from the samples, not only possesses excellent acid and bile salt tolerance but also exhibits good adhesion to Caco-2 small intestinal epithelial mimic cells.

[0005] Therefore, how to solve the problems of inconsistent flavor and unstable quality of traditional fermented mare's milk, and realize the industrial production of fermented mare's milk, is an urgent problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides Lactobacillus propylene glycol and its application in combined fermentation agents and fermented mare's milk, solving the problems of inconsistent quality (such as stability, aroma, and taste), large flavor differences, and difficulty in achieving industrialized production in traditional fermented mare's milk production.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: Propylene glycol-treated Lactobacillus and its application in combined fermentation agents and fermented mare's milk.

[0008] The purpose of this invention is to provide a combined fermentation agent composed of Lactobacillus MNJ-2, Lactobacillus plantarum ZKLpl800, Lactobacillus paracasei ZKL200, and Saccharomyces cerevisiae ZKM011.

[0009] Another object of the present invention is to provide a method for preparing fermented mare's milk.

[0010] The objective of this invention and the technical problem it solves are achieved through the following technical solution. According to this invention, a *Lactobacillus propylene glycol-based* MNJ-2 strain is provided. This strain has the accession number CGMCC No. 35857 and the accession date is September 10, 2025. The depositary institution is the China General Microbiological Culture Collection Center (CGMCC).

[0011] The objectives of this invention and the technical problems it addresses can be further achieved by the following technical measures.

[0012] In a first aspect of the present invention, the survival rate of the lactobacillus MNJ-2, which is resistant to acidic environment at pH 2.5, can reach 34.4% after 3 hours; and the survival rate of the lactobacillus MNJ-2, which is resistant to bile salts at 3‰, can reach 58.1% after 3 hours.

[0013] In a second aspect of the invention, the propylene glycol-treated Lactobacillus MNJ-2 achieves an adhesion rate of up to 37.5% to Caco-2 cells, which are mimics of small intestinal epithelial cells.

[0014] In a third aspect of the present invention, the propylene glycol-treated Lactobacillus MNJ-2 does not antagonize any of the various strains preserved in this laboratory and has good compatibility.

[0015] In a fourth aspect of the present invention, the combined fermentation agent composed of *Lactobacillus propylene glycol-produced* MNJ-2, *Lactobacillus plantarum* ZKLpl 800, *Lactobacillus paracasei* ZKL200, and *Saccharomyces cerevisiae* ZKM011 has a live bacteria ratio of 0.1-10.0% for *Lactobacillus propylene glycol-produced* MNJ-2, 0.01-0.5% for *Lactobacillus plantarum* ZKLpl 800, 0.01-0.5% for *Lactobacillus paracasei* ZKL200, and 0.01-0.5% for *Saccharomyces cerevisiae* ZKM011.

[0016] In a fifth aspect of the present invention, the method for preparing the fermented mare's milk is as follows:

[0017] (1) Preprocessing

[0018] Fresh mare's milk is filtered through an 80-100 mesh filter to remove impurities;

[0019] (2) Ingredients

[0020] Add 5-8% (w / v) of white sugar to the fresh mare's milk after removing impurities and stir until dissolved;

[0021] (3) Preheating

[0022] Heat the pre-treated fresh mare's milk to 45-55℃;

[0023] (4) Homogeneous

[0024] The pretreated fresh mare's milk was homogenized under a pressure of 15-25 MPa.

[0025] (5) Sterilization

[0026] The homogenized fresh mare's milk was sterilized at 65-75℃ for 30-50 minutes.

[0027] (6) Cooling

[0028] The sterilized fresh mare's milk was quickly placed in cold water at -5 to -15°C to cool it down to 35 to 45°C.

[0029] (7) Vaccination

[0030] The combined starter culture of *Lactobacillus propylene glycol-treated* MNJ-2, *Lactobacillus plantarum* ZKLpl 800, *Lactobacillus paracasei* ZKL200, and *Saccharomyces cerevisiae* ZKM011 was prepared at a concentration of 1.0–5.0 × 10⁻⁶. 9 Inoculate fresh mare's milk after it has been cooled down with a dose of CFU / 100ml.

[0031] (8) Fermentation

[0032] The inoculated fresh mare milk was placed in a shaker at 35-42℃ for constant temperature fermentation. The shaker speed was set to 50-150 rpm / min, and the fermentation time was 24-48 hours. The acidity during the initial fermentation was 70-90°T.

[0033] (9) Sterilization after fermentation

[0034] Sterilize the fermented mare's milk at 65-75℃ for 3-5 minutes.

[0035] (10) Cooling after fermentation

[0036] After fermentation, the fermented mare's milk is placed in cold water at -5 to -15°C to cool it down to 2 to 8°C.

[0037] Biological Preservation Instructions

[0038] Classification and nomenclature: Lentilactobaci l lus diolivorans MNJ-2 was deposited on September 10, 2025 at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35857.

[0039] This invention provides Lactobacillus propylene glycol and its application in combined fermentation agents and fermented mare's milk.

[0040] It has the following beneficial effects:

[0041] 1. This invention provides a *Lactobacillus malaccensis* strain MNJ-2 (accession number CGMCC No. 35857) with excellent probiotic potential. Through examples, it has been verified that the survival rate of this strain can reach 34.4% in an acidic environment of pH 2.5 and 58.1% in an environment with 3% bile salts. Furthermore, the adhesion rate to small intestinal epithelial mimic cells Caco-2 reaches 37.5%, indicating that this strain can effectively resist the harsh environment of the human digestive tract and has the ability to colonize the intestine, laying a solid foundation for exerting its potential probiotic function.

[0042] 2. The propylene glycol-treated Lactobacillus MNJ-2 screened in this invention has good compatibility, which provides the possibility for developing efficient and stable compound fermentation agents. The antagonistic experiment results show that this strain has no antagonistic effect with a variety of common Lactobacillus, Bifidobacterium, Streptococcus and yeast, and can grow synergistically. This characteristic solves the problem of mutual inhibition that may exist in the mixed fermentation of different functional strains, and ensures the normal functioning of each strain in the subsequent combined fermentation agent of this invention.

[0043] 3. This invention provides a combined fermentation agent composed of specific strains (Lactobacillus propylene glycol MNJ-2, Lactobacillus plantarum ZKLpl800, Lactobacillus paracasei ZKL200, and Saccharomyces cerevisiae ZKM011) and its application method, which effectively improves the quality and standardization of fermented mare's milk. Compared with fermented mare's milk produced by traditional natural fermentation, the product made using the fermentation agent of this invention has the characteristics of moderate sweetness and sourness, mellow alcohol taste, and rich fermentation aroma, and the product is stable. It solves the problems of inconsistent flavor and unstable quality in traditional processes, and provides a reliable technical solution for the industrial production of fermented mare's milk. Attached Figure Description

[0044] Figure 1 shows the cell morphology of Lactobacillus MNJ-2 under an optical microscope.

[0045] Figure 2 shows the colony morphology of Lactobacillus MNJ-2, which is fed with propylene glycol. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Refer to Figures 1 and 2:

[0048] Example 1: Isolation and Identification of Propylene Glycol-Consuming Lactobacillus MNJ-2

[0049] Strain isolation:

[0050] Sample collection: 25 mL of fermented mare's milk collected from Xinjiang was added to 225 mL of sterile physiological saline and thoroughly shaken to obtain the sample solution.

[0051] For cell enrichment, pipette 2-5 mL of (1) sample solution and add it to a 300 mL Erlenmeyer flask containing 100 mL of sterile MRS broth medium. After sealing with tissue culture membrane, place in a constant temperature incubator and incubate at 36-38℃ for 24-48 h to obtain the enriched culture solution.

[0052] To isolate the strain, pipette 1 mL of enrichment culture and transfer it to a test tube containing 9 mL of sterile physiological saline (w / v concentration 0.85%). After mixing by pipetting, 10... -1 Diluent; pipette 10 -1 1 mL of diluent was transferred to a test tube containing 9 mL of sterile physiological saline. After mixing by pipetting, 10 mL of diluent was obtained. -2 Diluent; and so on, 10 can be obtained sequentially. -3 10 -4 10 -5 Dilute the medium to a specific concentration. Take sterile MRS solid medium, melt it, pour it into a petri dish, and after it has completely cooled and solidified, pipette 0.1–0.2 mL of each graded dilution into the medium and spread it evenly using a spreader. Then place the spread plates in an incubator at 36–38°C and incubate anaerobically for 72–96 hours, observing the colony growth. After clearly visible colonies appear on the plates, select single colonies that meet the requirements for further strain purification.

[0053] For strain purification, selected single colonies were picked and streaked onto pre-cooled Petri dishes containing sterile MRS solid medium. The cultures were then incubated aerobically at 36–38°C for 72–96 hours. Subsequently, single colonies grown on the Petri dishes were picked and streaked onto Petri dishes containing MRS solid medium, again incubated aerobically at 36–38°C for 72–96 hours. After three consecutive streaking incubations, pure cultures were obtained. The pure cultures were transferred to cryovials containing 15–20% sterile glycerol and stored in liquid nitrogen. Simultaneously, the pure cultures were inoculated onto MRS solid medium slant tubes, and the cultured slant tubes were temporarily stored at 2–8°C. The composition of the MRS broth medium used for isolation and purification is as follows: 10.0 g casein peptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 5.0 g sodium acetate, 2.0 g diammonium citrate, 1.0 g Tween-80, 2.0 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, 0.05 g MnSO₄·H₂O, and 1000 mL distilled water. 15% agar was added to the MRS broth medium to prepare the MRS solid medium.

[0054] Test results:

[0055] Physiological and biochemical characteristics and morphological features:

[0056]

[0057]

[0058] Sequencing of 16S rRNA and PheS gene of strain MNJ-2:

[0059] The 16S rRNA gene sequence is shown in SEQ ID NO.1, and the pheS sequence is shown in SEQ ID NO.2.

[0060] Test results:

[0061] Based on a comprehensive analysis of experimental data including cell and colony morphology, physiological and biochemical characteristics, 16S rRNA gene, and pheS gene of this strain, and with reference to Bergey's Manual of Systematic Bacteriology and relevant research papers in the International Journal of Systematic and Evolutionary Microbiology, the MNJ-2 strain of this invention has been identified as *Lentilactobacil lus diolivorans*.

[0062] Example 2: Study on the relevant characteristics of Lactobacillus MNJ-2 (propylene glycol-fed)

[0063] Acid resistance properties:

[0064] Strain activation:

[0065] Pick a single colony from the above-mentioned prepared Lactobacillus MNJ-2 slant test tube and inoculate it into a 10ml sterile container;

[0066] The culture medium was placed in a 50 ml Erlenmeyer flask containing MRS broth, and then placed in a constant temperature shaker at 36–38 °C and 100–150 rpm / min for 36–48 h of activation culture. 200 μL of the activated solution was then taken and activated twice more using the same method to obtain the activated bacterial culture.

[0067] Centrifugation: Centrifuge the activated bacterial solution at 4℃ and 8000r / min for 10min, discard the supernatant, and collect the bacterial cells.

[0068] Inoculation and culture: The bacterial cells were suspended in an equal volume of MRS broth medium with a pH of 2.5 and incubated at a constant temperature of 36-38°C.

[0069] Survival rate determination: Samples were taken at 0h and 3h, and viable bacteria were counted using the plate count method. The viable bacteria count at 0h was used as a control. The survival rate was calculated using the following formula:

[0070]

[0071] In the formula: A1 is the number of viable bacteria of MNJ-2 after culturing in MRS liquid medium with pH 2.5 for 0 h, CFU / mL; A2 is the number of viable bacteria of MNJ-2 after culturing in MRS liquid medium with pH 2.5 for 3 h, CFU / mL.

[0072] Acid resistance test results: The survival rate of MNJ-2 after culturing in MRS liquid medium with a pH of 2.5 for 3 h is shown in Table 2 below.

[0073] Table 2: Survival rate of MNJ-2 after 3 h of culture in MRS liquid medium at pH 2.5.

[0074]

[0075] Bile salt tolerance:

[0076] Activation of the bacterial strain: Pick a single colony from the prepared slant tube of *Lactobacillus propylene glycol-treated* MNJ-2 and inoculate it into a 50ml Erlenmeyer flask containing 10ml of sterile MRS broth. Then place the flask in a constant-temperature shaker at 36–38℃ and 100–150 rpm / min for 36–48 hours. After activating, take 200μL of the above activation solution and repeat the activation process twice more to obtain the activated bacterial culture.

[0077] Centrifugation: Centrifuge the activated bacterial solution at 4℃ and 8000r / min for 10min, discard the supernatant, and collect the bacterial cells.

[0078] Inoculation and culture: The bacterial cells were suspended in an equal volume of MRS broth medium with a bile salt concentration of 3% and cultured at a constant temperature of 36-38℃.

[0079] Survival rate determination: Samples were taken at 0h and 3h, and viable bacteria were counted using the plate count method. The viable bacteria count at 0h was used as a control. The survival rate was calculated using the following formula:

[0080]

[0081] In the formula: B1 is the number of viable bacteria of MNJ-2 cultured in MRS liquid medium with a bile salt concentration of 3% for 0 h, CFU / mL;

[0082] B2 represents the viable count (CFU / mL) of MNJ-2 cells cultured in 3% bile salt MRS liquid medium for 3 hours.

[0083] Results of bile salt tolerance test: The survival rate of MNJ-2 after culturing in MRS liquid medium with a bile salt concentration of 3% for 3 hours is shown in Table 3 below.

[0084] Table 3: Survival rate of MNJ-2 cells after 3 h of culture in MRS liquid medium with 3% bile salt concentration.

[0085]

[0086] Intestinal epithelial cell adhesion characteristics:

[0087] Strain activation:

[0088] A single colony from the prepared Lactobacillus MNJ-2 slant test tube was inoculated into a 50ml Erlenmeyer flask containing 10ml of sterile MRS broth. The flask was then placed in a constant-temperature shaker at 36–38°C and 100–150 rpm / min for 36–48 hours of activation. 200μL of the activation solution was then taken and activated twice more using the same method to obtain the activated bacterial culture.

[0089] Centrifugation: Centrifuge the activated bacterial solution at 4℃ and 8000r / min for 10min, discard the supernatant, and collect the bacterial cells.

[0090] Preparation of bacterial suspension: The bacterial cells described in 3.2 above were resuspended in an appropriate amount of 0.85% (w / v) sterile physiological saline to ensure a viable count of 102. 8 ~10 9 CFU / mL, for later use.

[0091] Adhesive cell preparation: Caco-2 small intestinal epithelial mimic cells were cultured to a near-complete monolayer.

[0092] Lactic acid bacteria adhesion experiment: The prepared MNJ-2 bacterial suspension was co-cultured with Caco-2 cells at 36-38℃ for 36-48h to allow the lactic acid bacteria and Caco-2 cells to come into full contact.

[0093] Washing: Pour out the mixed culture medium and gently wash the adherent cells with an appropriate amount of sterile 0.85% (w / v) physiological saline to remove unadhered lactic acid bacteria.

[0094] Cell lysis: Add an appropriate amount of cell lysis buffer to the Caco-2 adherent cells to completely lyse the cells and release the lactic acid bacteria.

[0095] Prepare an appropriate amount of diluent: Dilute the lysate appropriately so that the concentration of lactic acid bacteria is within the countable range.

[0096] Plate counting: Spread the diluted solution evenly on a plate containing MRS solid medium and count the colonies according to the plate counting method.

[0097] Adhesion rate determination: The number of viable bacteria in Caco-2 cells before and after adhesion was measured, and the Caco-2 cell adhesion rate was calculated according to the following formula.

[0098]

[0099] In the formula: C I C1 represents the initial number of MNJ-2 cells, CFU / mL; C2 represents the number of MNJ-2 cells adhering to Caco-2 cells, CFU / mL.

[0100] Results of MNJ-2 adhesion rate to Caco-2 in small intestinal epithelial cells:

[0101] The adhesion rate of MNJ-2 to Caco-2 cells in small intestinal epithelial cells is shown in Table 4.

[0102] Table 4: Adhesion rate of MNJ-2 to Caco-2 cells in small intestinal epithelial mimics

[0103]

[0104] Antagonism:

[0105] Strain activation: The glycerol seed culture preserved in liquid nitrogen in our laboratory was activated according to the following methods.

[0106] Activation of Lactobacillus and Bifidobacterium:

[0107] The glycerol seeds of each Lactobacillus and Bifidobacterium strain, which were placed in liquid nitrogen, were thawed in a water bath at 40–50°C. Under aseptic conditions, 200 μL of the nutrient solution was taken and inoculated into a 50 ml Erlenmeyer flask containing 10 ml of sterile MRS broth medium. The flask was then placed in an anaerobic incubator at a temperature of 36–38°C and activated for 12–24 h. The above steps were repeated to subculture and activate the strains twice to obtain activated bacterial solutions.

[0108] Streptococcal activation:

[0109] Glyceryl spores of various thermophilic streptococci and lactic acid streptococci strains, placed in liquid nitrogen, were thawed in a water bath at 40–50°C. Under aseptic conditions, 200 μL of each spore was inoculated into a 50 ml Erlenmeyer flask containing 10 ml of sterile IRIE liquid medium. The flask was then placed in an anaerobic incubator at 36–38°C for 12–24 hours of activation culture. The above steps were then repeated.

[0110] The bacterial strain was subcultured and activated twice to obtain an activated bacterial solution.

[0111] Yeast activation:

[0112] The glycerol seed culture of each yeast strain, which was placed in liquid nitrogen, was thawed in a water bath at 40–50°C. Under aseptic conditions, 200 μL of the culture was taken and inoculated into a 50 ml Erlenmeyer flask containing 10 ml of sterile YPD liquid culture medium. The flask was then placed in a constant temperature shaker at 28–30°C and 100–150 rpm / min. After activating the culture for 12–24 h, the culture was removed and the above steps were repeated. The strain was subcultured and activated twice to obtain the activated bacterial solution.

[0113] Propylene glycol-treated Lactobacillus MNJ-2 activation:

[0114] A single colony from the prepared Lactobacillus MNJ-2 slant test tube was inoculated into a 50ml Erlenmeyer flask containing 10ml of sterile MRS broth. The flask was then placed in a constant-temperature shaker at 36–38°C and 100–150 rpm / min for 36–48 hours of activation. 200μL of the activation solution was then taken and activated twice more using the same method to obtain the activated bacterial culture.

[0115] Pour culture of each test strain:

[0116] The activated lactobacillus, bifidobacteria, streptococci, and yeast were diluted separately with 0.8% (w / v) sterile physiological saline until the viable count was 10. 5 ~10 6 CFU / mL. Then, 200 μL of each bacterial dilution was pipetted into a sterile Petri dish, and 20 ml of the corresponding solid culture medium for the activation of each strain was added to the Petri dish. After shaking well, the Petri dishes were placed in a clean bench to air dry for 30 min.

[0117] Preparation of MNJ-2 supernatant:

[0118] The activated Lactobacillus MNJ-2 bacterial culture was centrifuged to prepare the fermentation supernatant. The centrifugation conditions were: 4℃, 8000r / min for 10min.

[0119] Antagonism experiment:

[0120] Antagonism experiments were performed using the Oxford cup method. Sterile Oxford cups (6 mm inner diameter) were placed in each dry solid Petri dish containing the respective bacterial culture, and slightly pressed down to ensure no gaps between the cup and the culture medium. Then, 200 μL of *Lactobacillus montmorillonite* MNJ-2 fermentation supernatant was added to each Oxford cup. The Petri dishes were then placed in the same culture conditions used for the activation of each strain. After 24–36 h, the size and diameter of the inhibition zone were observed and measured. Each sample was tested in triplicate, and the average value was taken. The control group consisted of Oxford cups containing 200 μL of the corresponding liquid culture medium used for the activation of each strain, under identical culture conditions.

[0121] Antagonistic experiment results: The antagonistic experiment results of Lactobacillus MNJ-2 with propylene glycol and various strains preserved in this experiment are shown in Table 5 below.

[0122] Table 5: Antagonistic Experiment Results of MNJ-2 with Various Strains

[0123]

[0124]

[0125] The acid resistance results above show that MNJ-2 has a survival rate of 34.4% in an environment with a pH of 2.5 and a survival rate of 58.1% in an environment with a bile salt concentration of 3%. This good acid and bile salt resistance is the basis for the strain to exert its probiotic function after entering the intestine. Meanwhile, MNJ-2 achieved an adhesion rate of 37.5% to Caco-2 cells, a mimicking small intestinal epithelial cells. This high adhesion rate ensures that the strain can continue to exert its probiotic function after entering the intestine.

[0126] Further antagonistic experiments showed that the Lactobacillus MNJ-2, to which this invention pertains, can grow synergistically with most strains, indicating that strain MNJ-2 can be used as a fermenting agent in combination with other strains.

[0127] Example 3: Formulation of the fermentation agent

[0128] Preparation of bacterial suspension:

[0129] Preparation of propylene glycol-treated Lactobacillus MNJ-2 suspension:

[0130] Preparation of first-generation seed culture: Pick a single colony from the prepared Lactobacillus MNJ-2 slant test tube, inoculate it into a 50ml Erlenmeyer flask containing 10ml MRS broth medium, and then place it in a constant temperature shaker. Set the shaker temperature to 36-38℃ and the rotation speed to 100-150rpm / min. After activation culture for 36-48h, take it out. This is the first-generation seed culture.

[0131] Preparation of second-generation seed culture: Take 0.2-0.5 ml of first-generation seed culture and inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of MRS broth medium. Continue to activate it using the same culture method as above. The activated seed culture is used as the second-generation seed culture.

[0132] Preparation of third-generation seed solution: Third-generation seed solution was obtained using the same inoculation and culture methods.

[0133] Centrifugation: Place the obtained third-generation seed liquid in a centrifuge, set the centrifugation temperature to 2-8℃, the centrifugation speed to 6000-8000 rpm / min, and centrifuge for 5-10 minutes. Remove the liquid and discard the supernatant to obtain MNJ-2 centrifuged bacterial sludge.

[0134] Resuspension of bacterial sludge: The obtained centrifuged bacterial sludge of MNJ-2 was resuspended in 10 ml of sterilized fresh mare's milk and used as one of the components of the combined fermentation agent. It was then refrigerated at 2-8℃ for later use.

[0135] Preparation of *Lactobacillus plantarum* ZKLpl 800 bacterial suspension (isolated and preserved by our company):

[0136] Preparation of first-generation seed culture: Take 0.2-0.5 ml of liquid nitrogen-preserved Lactobacillus plantarum ZKLpl 800 glycerol seed and inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of MRS broth medium. Then place it in a constant temperature shaker, set the shaker temperature to 36-38℃ and the rotation speed to 100-150 rpm / min, and take it out after activating culture for 12-24 h. This is the first-generation seed culture.

[0137] Preparation of second-generation seed culture: Take 0.2-0.5 ml of first-generation seed culture and inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of MRS broth medium. Continue to activate it using the same culture method as above. The activated seed culture is used as the second-generation seed culture.

[0138] Preparation of third-generation seed solution: Third-generation seed solution was obtained using the same inoculation and culture methods.

[0139] Centrifugation: Place the obtained third-generation seed liquid in a centrifuge, set the centrifugation temperature to 2-8℃, the centrifugation speed to 6000-8000 rpm / min, and centrifuge for 5-10 minutes. Remove the liquid and discard the supernatant to obtain ZKLpl 800 centrifuged bacterial sludge.

[0140] Resuspension of bacterial sludge: The obtained ZKLpl 800 centrifuged bacterial sludge was resuspended in 10 ml of sterilized fresh mare's milk and used as one of the components of the combined fermentation agent. It was then refrigerated at 2-8℃ for later use.

[0141] Preparation of Lactobacillus paracasei ZKL200 (isolated and preserved by our company) bacterial suspension:

[0142] Preparation of first-generation seed culture: Take 0.2-0.5 ml of Lactobacillus paracasei ZKL200 glycerol seed culture preserved in liquid nitrogen, inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of MRS broth medium, and then place it in a constant temperature shaker. Set the shaker temperature to 36-38℃ and the rotation speed to 100-150 rpm / min. After activation culture for 12-24 h, take it out. This is the first-generation seed culture.

[0143] Preparation of second-generation seed culture: Take 0.2-0.5 ml of first-generation seed culture and inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of MRS broth medium. Continue to activate it using the same culture method as above. The activated seed culture is used as the second-generation seed culture.

[0144] Preparation of third-generation seed solution: Third-generation seed solution was obtained using the same inoculation and culture methods.

[0145] Centrifugation: Place the obtained third-generation seed liquid in a centrifuge, set the centrifugation temperature to 2-8℃, the centrifugation speed to 6000-8000 rpm / min, and centrifuge for 5-10 minutes. Remove the liquid and discard the supernatant to obtain ZKL200 centrifuged bacterial sludge.

[0146] Resuspension of bacterial sludge: The obtained ZKL200 centrifuged bacterial sludge was resuspended in 10 ml of sterilized fresh mare's milk and used as one of the components of the combined fermentation agent. It was then refrigerated at 2-8℃ for later use.

[0147] Preparation of a suspension of Saccharomyces cerevisiae ZKM011 (isolated and preserved by our company):

[0148] Preparation of first-generation seed culture: Take 0.2-0.5 ml of glycerol seed of Saccharomyces cerevisiae ZKM011 preserved in liquid nitrogen, inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of YPD medium, and then place it in a constant temperature shaker. Set the shaker temperature to 28℃ and the rotation speed to 100-150 rpm / min. After activation culture for 12-24 h, take it out. This is the first-generation seed culture.

[0149] Preparation of second-generation seed culture: Take 0.2-0.5 ml of first-generation seed culture and inoculate it into a 50 ml Erlenmeyer flask containing 10 ml of YPD medium. Continue to activate it using the same culture method as above. The activated seed culture is used as the second-generation seed culture.

[0150] Preparation of third-generation seed solution: Third-generation seed solution was obtained using the same inoculation and culture methods.

[0151] Centrifugation: Place the obtained third-generation seed liquid in a centrifuge, set the centrifugation temperature to 2-8℃, the centrifugation speed to 6000-8000 rpm / min, and centrifuge for 5-10 minutes. Remove the liquid and discard the supernatant to obtain ZKM011 centrifuged bacterial sludge.

[0152] Resuspension of bacterial sludge: The obtained ZKL200 centrifuged bacterial sludge was resuspended in 10 ml of sterilized fresh mare's milk and used as one of the components of the combined fermentation agent. It was then refrigerated at 2-8℃ for later use.

[0153] The YPD medium used above is prepared as follows: Dissolve 10g of yeast extract and 20g of peptone in 900ml of water, sterilize at 121℃ for 20min, and then add 100ml of 20g glucose solution sterilized at 115℃ for 15min. If preparing plates, add 20g of agar powder.

[0154] Fermentation agent preparation: Prepare the bacterial suspensions of the above strains according to the preparation ratios in the table below.

[0155] Table 6: Fermentation Agent Composition Ratio

[0156]

[0157] Example 4: Preparation of the fermented mare's milk of the present invention

[0158] Steps for making fermented mare's milk:

[0159] Pre-treatment: Filter the freshly collected mare's milk through an 80-100 mesh filter to remove impurities.

[0160] Ingredients: Add 5-8% (w / v) of white sugar to fresh mare's milk after removing impurities and stir until dissolved.

[0161] Preheating: Place the filtered fresh mare's milk in a constant temperature water bath and heat it to 45-55℃.

[0162] Homogenization: The fresh mare's milk, which has been heated to 45-55°C in a water bath, is homogenized under a pressure of 15-25 MPa.

[0163] Sterilization: After homogenization, the fresh mare's milk is dispensed into sterilized 300ml Erlenmeyer flasks and sealed. Each Erlenmeyer flask contains 100ml of fresh mare's milk. Then, the Erlenmeyer flasks containing the fresh mare's milk are placed in a water bath and sterilized at 65-75℃ for 30-50 minutes.

[0164] Cooling: Quickly place the sterilized fresh mare's milk in cold water at -5 to -15°C to cool it down to 35 to 45°C.

[0165] Inoculation: Inoculate each of the fermentation agents prepared in Table 2 into Erlenmeyer flasks containing cooled fresh mare's milk. The number of inoculated bacteria per 100 ml of fresh mare's milk is shown in Table 2.

[0166] Fermentation: Place the inoculated fresh mare's milk in a shaker at 35-42℃ for constant temperature fermentation. Set the shaker speed to 50-150 rpm / min and the fermentation time to 24-48 hours.

[0167] Sterilization after fermentation: Sterilize the fermented mare's milk at 65-75℃ for 3-5 minutes.

[0168] Cooling after fermentation: Place the fermented mare's milk in cold water at -5 to -15°C to cool it down to 2 to 8°C.

[0169] Determination of indicators in fermented mare's milk:

[0170] Fermentation acidity determination:

[0171] Reagent preparation:

[0172] Sodium hydroxide standard solution (0.1000 mol / L): Weigh 0.75 g of potassium hydrogen phthalate, a working standard reagent, dried to constant weight in an electric oven at 105℃~110℃. Dissolve it in 50 mL of carbon dioxide-free water, add 2 drops of phenolphthalein indicator (10 g / L), and titrate with the prepared sodium hydroxide solution until the solution turns pink and remains so for 30 seconds. Perform a blank test simultaneously.

[0173] Reference solution: Dissolve 3g of cobalt sulfate heptahydrate in water and bring the volume to 100mL.

[0174] Phenolphthalein indicator solution: Weigh 0.5g of phenolphthalein and dissolve it in 75mL of 95% ethanol, add 20mL of water, then add sodium hydroxide solution dropwise until a light pink color appears, and then add water to make up to 100mL.

[0175] Distilled water without carbon dioxide: Boil water for 15 minutes to remove carbon dioxide, cool, and seal.

[0176] Preparation of the reference solution: Add 2.0 mL of the reference solution to an Erlenmeyer flask containing an equal volume of the corresponding solution, gently rotate to mix, and obtain the standard reference color. If multiple similar products are to be measured, this reference solution can be used throughout the entire measurement process, but the time should not exceed 2 hours.

[0177] Determination: Weigh 10g (accurate to 0.001g) of the well-mixed sample and place it in a 150mL Erlenmeyer flask. Add 20mL of freshly boiled water cooled to room temperature, mix well, add 2.0mL of phenolphthalein indicator solution, mix well, and then titrate with sodium hydroxide standard solution, rotating the flask while adding the solution, until the color is similar to that of the reference solution and does not fade within 5 seconds. The entire titration process should be completed within 45 seconds. During the titration, purge the Erlenmeyer flask with nitrogen gas to prevent the solution from absorbing carbon dioxide from the air. Record the volume of sodium hydroxide standard titration solution consumed (V1) and substitute it into the following formula for calculation.

[0178] Blank titration: Perform a blank experiment using an equal volume of water and read the volume (V0) of sodium hydroxide standard solution consumed. The volume of sodium hydroxide consumed in the blank should not be less than zero; otherwise, distilled water that meets the requirements should be prepared and used again.

[0179] Presentation of analysis results:

[0180]

[0181] In the formula: X1 is the acidity of the sample, in degrees (°T) [calculated as the number of milliliters of 0.1 mol / L sodium hydroxide consumed per 100g sample, in milliliters per 100g (mL / 100g)]; c1 is the molar concentration of the sodium hydroxide standard solution, in moles per liter (mol / L); V1 is the volume of sodium hydroxide standard solution consumed during titration, in milliliters (mL); V0 is the volume of sodium hydroxide standard solution consumed in the blank experiment, in milliliters (mL); 100 is 100g of sample; m1 is the mass of the sample, in grams (g); 0.1 is the theoretical definition of the molar concentration of sodium hydroxide, in moles per liter (mol / L).

[0182] The result is expressed as the arithmetic mean of two independent measurements obtained under repeatability conditions, and is retained to three significant figures.

[0183] Titration results:

[0184] Table 7: Titration Results of Acidity of Various Fermented Mare's Milk

[0185]

[0186]

[0187] Alcohol content measurement:

[0188] Sample preparation:

[0189] Removal of carbon dioxide from the sample:

[0190] Pour approximately 300 mL of the wine sample, which has returned to room temperature, into a 1000 mL Erlenmeyer flask. Stopper the flask with a rubber stopper, gently shake, release the gas, and then stopper it again. Repeat this process until no more bubbles escape. Filter the flask using a single layer of medium-speed dry filter paper (with a glass watch glass placed over the funnel).

[0191] After removing carbon dioxide, the sample is collected in a stoppered conical flask and prepared fresh before use.

[0192] Sample distillation: After the clean, dry 100mL volumetric flask and sample have been kept at a constant temperature of 20℃, accurately measure 100mL of the sample into a 500mL distillation flask. Rinse the volumetric flask three times with 50mL of water, and combine the washings with the 500mL distillation flask. Add a few boiling stones (or glass beads), connect a serpentine condenser, and use the original volumetric flask used for sampling as the receiver (with an ice bath). Turn on the cooling water (the cooling water temperature should be below 15℃) and slowly heat for distillation, collecting the distillate. When it is close to the mark, remove the volumetric flask, stopper it, and keep it in a 20℃ water bath for 30 minutes. Then add water (20℃) to the mark, mix well, and set aside.

[0193] Determination of the sample solution: Wash and dry the density bottle, and weigh it with a thermometer and a side-hole cover. Repeat drying and weighing until the difference between two consecutive weighings does not exceed 2 mg, i.e., constant weight (m0).

[0194] Remove the stopper with the thermometer, fill the pre-weighed density bottle with boiled water cooled to 15°C, insert the stopper with the thermometer (there should be no air bubbles in the bottle), and immediately immerse it in a constant temperature water bath at (20±0.1)°C. Maintain this temperature for 20 minutes when the contents reach 20°C. Quickly absorb any liquid overflowing from the side tube with filter paper, ensuring the liquid level in the side tube is flush with the tube opening. Immediately cover the side tube with the vent cap, remove the density bottle, wipe the liquid off the outer wall of the bottle with filter paper, and then weigh (m1).

[0195] Pour out the water, rinse the density bottle with anhydrous ethanol and then with ether, blow it dry, rinse the density bottle repeatedly with the sample distillate 3 to 5 times, and then fill it.

[0196] Statement of analytical results: The density of the sample at 20℃ (ρ) 20 The air buoyancy correction value (A) is calculated using the following formula.

[0197]

[0198] In the formula: ρ 20 ρ is the density of the sample at 20℃, in grams per liter (g / L); 998.20 is the density of distilled water at 20℃, in grams per liter (g / L); m2 is the mass of the density bottle and sample at 20℃, in grams (g); m0 is the mass of the density bottle, in grams (g); A is the air buoyancy correction value; m1 is the mass of the density bottle and water under buoyancy correction, in grams (g); ρ u 1.2 g / L is the density of dry air at 20℃ and 1013.25 hPa; 997.0 is the difference between the density of distilled water and dry air at 20℃, in grams per liter (g / L).

[0199] Based on the density ρ of the sample 20 Consult the table of alcohol-water solution density and ethanol concentration (alcohol content) to obtain the alcohol content, expressed as volume fraction %vol.

[0200] The result is expressed as the arithmetic mean of two independent measurements obtained under repeatability conditions, and is rounded to one decimal place.

[0201] Alcohol content test results:

[0202] Table 8: Results of alcohol content determination for various fermented mare's milk products

[0203]

[0204]

[0205] Determination of total lactic acid bacteria count:

[0206] Sample preparation: After thoroughly shaking each fermented mare's milk sample, use a sterile pipette to draw 25 mL of the sample and place it into a sterile conical flask containing 225 mL of diluent (with an appropriate number of sterile glass beads pre-placed in the flask). Shake thoroughly to prepare a 1:10 sample homogenate.

[0207] Dilution and incubation: Use a 1mL pipette to draw 1mL of the 1:10 sample homogenate and slowly pour it along the tube wall into a sterile test tube containing 9mL of diluent (be careful not to let the tip of the pipette tip touch the diluent). Shake the test tube to mix it thoroughly to prepare a 1:100 sample homogenate.

[0208] Take another 1mL pipette tip and, following the above procedure, homogenize the sample in 10-fold increments. Use a 1mL sterile pipette tip for each increment of dilution.

[0209] Lactic acid bacteria count: Based on the estimated total number of viable bacteria in each mare's milk wine sample, select... For each consecutive appropriate dilution, pipette 1 mL of sample homogenate into a sterile plate, and prepare two plates for each dilution.

[0210] After transferring the diluted solution into a petri dish, cool it to... Pour MRS agar medium into Petri dishes Rotate the pan to mix thoroughly.

[0211] After the culture medium solidifies, invert it and anaerobically incubate at 36℃±1℃ for a period of time.

[0212] The process from sample dilution to plate pouring must be completed within 15 minutes.

[0213] Colony count: Can be observed with the naked eye, or if necessary, with a magnifying glass or colony counter. Record the dilution factor and the corresponding number of colonies. Colony count is expressed in colony-forming units (CFU).

[0214] Select colony count in Count the total number of colonies on plates with no spreading bacterial growth. For plates with less than 30 CFU, record the specific colony count; for plates with more than 300 CFU, record as overcountable. The colony count for each dilution should be the average of two plates.

[0215] Methods for calculating total bacterial count:

[0216] If only one dilution plate has a colony count within the appropriate range, calculate the average colony count of the two plates, and then multiply the average by the corresponding dilution factor to obtain the total colony count per mL of sample.

[0217] If the colony counts on plates of two consecutive dilutions are within the appropriate counting range, calculate according to formula (7):

[0218]

[0219] In the formula: N is the number of colonies in the sample; ∑C is the sum of colony counts on plates (including plates with colony counts within the appropriate range); n1 is the number of plates at the first dilution (low dilution factor); n2 is the number of plates at the second dilution (high dilution factor); d is the dilution factor (first dilution factor).

[0220] Report on total bacterial count:

[0221] If the colony count is less than 100 CFU, round it off according to the rounding principle and report it as an integer.

[0222] When the colony count is greater than or equal to 100 CFU, the third digit is rounded according to the rounding principle, taking the first two digits and replacing the remaining digits with 0; alternatively, it can be expressed in exponential form of 10, rounded according to the rounding principle, and then used with two significant figures. The result is reported in CFU / mL.

[0223] Lactic acid bacteria test results:

[0224] Table 9: Results of Lactic Acid Bacteria Determination in Fermented Mare's Milk

[0225]

[0226]

[0227] Sensory evaluation of fermented mare's milk: Ten volunteers were randomly selected to conduct sensory evaluations of nine groups of fermented mare's milk. The evaluation was conducted according to the standards shown in Table 10.

[0228] Table 10: Sensory Evaluation Standards for Fermented Mare's Milk

[0229]

[0230] The sensory evaluation results of each fermented mare's milk are shown in Table 11 below.

[0231] Table 11: Sensory evaluation results of each fermented mare's milk sample

[0232]

[0233]

[0234] The sensory evaluation results show that experimental group 3 achieved the highest sensory score, reaching 91 points. The fermented mare's milk produced using this group of starter cultures had a moderate sweet and sour taste, a wine-like aroma, and a rich fermented fragrance, which was favored by most volunteers. The combination with the highest sensory score is consistent with the experimental results; that is, the optimal combination is achieved when the ratio of *Lactobacillus propylene glycol-modified* MNJ-2: *Lactobacillus plantarum* ZKLpl800: *Lactobacillus paracasei* ZKL200: *Saccharomyces cerevisiae* ZKM011 = 10:0.2:0.2:0.2, with an initial inoculation viable count of 1.06 × 10⁻⁶. 9 CFU / 100ml, fermented mare's milk under these conditions has an acidity of 82.6°T, an alcohol content of 3.1% vol, and a viable cell count of up to 1.0 × 10⁻⁶ CFU / 100ml. 9 CFU / ml.

[0235] In summary, this invention discloses a *Lactobacillus MNJ-2* strain and its applications. This strain exhibits good acid and bile salt tolerance and good intestinal epithelial cell adhesion ability, showing potential as a probiotic. Antagonistic experiments with various laboratory-preserved strains did not show significant antagonism, indicating its suitability as a fermentation agent. Furthermore, a fermentation agent was prepared by combining this strain with *Lactobacillus plantarum* ZKLpl800, *Lactobacillus paracasei* ZKL200, and *Saccharomyces cerevisiae* ZKM011 isolated and preserved by our company in a specific ratio. This combined fermentation agent was inoculated into fresh mare's milk after pretreatment, ingredient mixing, preheating, homogenization, sterilization, and cooling. After fermentation, sterilization, and cooling, fermented mare's milk was obtained. Fermented mare's milk produced by this method not only retains the original sour and alcoholic flavor but also exhibits greater stability, richer aroma, and a more complex taste, showing broad application prospects in the industrial production of fermented mare's milk. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The *Lactobacillus MNJ-2* oxidase described in this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. Propylene glycol-fed Lactobacillus MNJ-2, characterized in that, The Lactobacillus MNJ-2, which is propylene glycol-treated, was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35857.

2. The propylene glycol-treated Lactobacillus MNJ-2 according to claim 1, characterized in that, The 16S rRNA gene sequence of the propylene glycol-treated Lactobacillus is shown in SEQ ID NO.1, and the pheS sequence is shown in SEQ ID NO.

2.

3. A combined fermentation agent, characterized in that, The combined fermentation agent comprises Lactobacillus MNJ-2, Lactobacillus plantarum ZKLpl 800, Lactobacillus paracasei ZKL200, and Saccharomyces cerevisiae ZKM011 as described in claims 1-2.

4. The combined fermentation agent according to claim 3, characterized in that, The combined fermentation agent is composed of the following components in the following proportions of live bacteria: Lactobacillus MNJ-2 (propanediol-treated) 0.1-10.0 parts; Lactobacillus plantarum ZKLpl 800 0.01-0.5 parts; Lactobacillus paracasei ZKL200 0.01-0.5 parts; and Saccharomyces cerevisiae ZKM011 0.01-0.5 parts.

5. The use of the combined fermentation agent according to claim 3 or 4 in the preparation of fermented mare's milk.

6. The application according to claim 5, characterized in that, The preparation of fermented mare's milk includes the following steps: a) pre-treating fresh mare's milk, the pre-treating including filtration, ingredient mixing, preheating, homogenization and sterilization; b) cooling the pre-treated mare's milk raw material to an inoculation temperature of 35-45°C; c) inoculating the mare's milk raw material with one of the combined fermentation agents for fermentation; d) sterilizing and cooling the fermented mare's milk after fermentation.

7. The method for preparing fermented mare's milk according to claim 6, characterized in that, The specific parameters of the pretreatment step are as follows: a) Filter the fresh mare's milk through an 80-100 mesh filter to remove impurities, and then add 5-8% w / v of white sugar to the filtered fresh mare's milk; b) Heat the liquid to 45-55°C and homogenize it under a pressure of 15-25 MPa; c) Sterilize the homogenized liquid at 65-75°C for 30-50 minutes.

8. The method for preparing fermented mare's milk according to claim 6, characterized in that, The specific parameters for the fermentation step are: 1.0~5.0×10 9 Inoculate with a total inoculum of CFU / 100ml and ferment at 35-42℃ for 24-48 hours. Stop fermentation when the acidity reaches 70-90°T at the end of fermentation.

9. The method for preparing fermented mare's milk according to claim 6, characterized in that, The combined fermentation agent used in step c) is a combined fermentation agent.

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