Lactobacillus kefiranofaciens subsp. Kefiranofaciens IMAU13289 and application thereof

Abstract

The invention discloses a Lactobacillus kefiraniformis subspecies IMAU13289 and an application thereof, belongs to the technical field of microorganisms, and provides the Lactobacillus kefiraniformis subspecies IMAU13289, which is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms on December 4, 2024, the preservation address is No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.32897. The Lactobacillus kefiraniformis subspecies IMAU13289 has the advantages that the Lactobacillus kefiraniformis subspecies IMAU13289 can be used for preparing the lactobacillus kefiraniformis subspecies IMAU13289; the Lactobacillus kefiranofaciens subsp. Kefiranofaciens IMAU13289 disclosed by the invention has the capability of producing exopolysaccharides at high yield, is high in lactic acid production rate, has drug resistance to neomycin, and can be used for preparing fermented milk with excellent curd characteristics and storage characteristics.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to kumiss-like Lactobacillus kumiss subspecies IMAU13289 and applications thereof. Background Art

[0002] Lactobacillus kumiss subsp. kumiss ( Lactobacillus kefiranofaciens subsp. kefiranofaciens ) has been approved as a new food ingredient, a novel edible probiotic strain. This strain not only serves as a prebiotic functional food, regulates the intestinal microbiome, and improves depression, but also exerts a beneficial anti-osteoarthritis effect by regulating its metabolites and intestinal flora.

[0003] Lactobacillus kumiss subsp. kumiss is a microorganism isolated from kefir grains in the initial stage of camel milk fermentation. It is a starter culture used for Caucasian milk fermentation. The extracellular polysaccharide it produces is called kefir polysaccharide, which is often used as an excellent starter culture. This strain has a very broad application prospect and can be used in different products such as stabilizers, emulsifiers, thickeners, gelling agents and fat substitutes.

[0004] Kefir has long been recognized for its health benefits, including its immunomodulatory effects. However, knowledge regarding the immunomodulatory effects of kefir lactic acid bacteria (LAB) is lacking. Lactobacillus kumiss subsp. kumiss is a key factor in regulating immune function. Studies have found that commercial starter cultures do not colonize and survive well in the human gastrointestinal tract. With technological innovation, more and more companies are committed to finding lactic acid bacteria suitable for fermentation, thereby developing fermented milk with more flavors, functions, and nutrients.

[0005] In summary, this strain will help develop probiotic fermented milk with high probiotic activity and unique metabolic profile, and establish a reference standard for the development of new functional foods in the dairy industry. Therefore, this discovery is of great significance. Summary of the Invention

[0006] To solve the above technical problems, the present invention proposes Kühm-like Lactobacillus kumis subsp. IMAU13289 and its application. The Kühm-like Lactobacillus kumis subsp. IMAU13289 of the present invention has a short coagulation time, a fast lactic acid production rate, good bile salt and alkali resistance, and resistance to neomycin.

[0007] To achieve the above object, the present invention provides a Küsbuccal Lactobacillus subsp. Küsbuccal ( Lactobacillus kefiranofaciens subsp. kefiranofaciens)IMAU13289, the kumiss-like Lactobacillus kumiss-like subspecies IMAU13289 was deposited in the General Microbiology Center of China Culture Collection Administration on December 4, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No.32897.

[0008] The present invention also provides the use of the Kühm-like Lactobacillus subspecies Kühm-like IMAU13289 in preparing a probiotic agent with high exopolysaccharide production.

[0009] The present invention also provides a probiotic agent with high production of extracellular polysaccharides, including Kühm-like Lactobacillus Kühm-like subspecies IMAU13289. The Kühm-like Lactobacillus Kühm-like subspecies IMAU13289 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

[0010] The present invention also provides the use of the Kühm-like Lactobacillus subspecies Kühm-like IMAU13289 in the preparation of a probiotic agent for rapid lactic acid production.

[0011] The present invention also provides a fast-producing lactic acid probiotic, including Kühm-like Lactobacillus Kühm-like subspecies IMAU13289. The Kühm-like Lactobacillus Kühm-like subspecies IMAU13289 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

[0012] The present invention also provides the use of the Kühm-like Lactobacillus subspecies Kühm-like IMAU13289 in preparing a probiotic agent resistant to gastrointestinal fluid digestion.

[0013] The present invention also provides a probiotic agent that is resistant to gastrointestinal fluid digestion, including Kühm-like Lactobacillus Kühm-like subspecies IMAU13289. The Kühm-like Lactobacillus Kühm-like subspecies IMAU13289 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

[0014] The present invention also provides the use of the kumiss-like Lactobacillus kumiss subspecies IMAU13289 in the preparation of a drug-resistant probiotic agent, characterized in that the drug-resistant probiotic agent is resistant to neomycin.

[0015] The present invention also provides a drug-resistant probiotic agent, including Kühm-like Lactobacillus Kühm-like subspecies IMAU13289. The Kühm-like Lactobacillus Kühm-like subspecies IMAU13289 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 32897. The drug-resistant probiotic agent is resistant to neomycin.

[0016] The present invention also provides the use of the kumiss-like Lactobacillus kumiss-like subspecies IMAU13289 in preparing fermented milk with excellent coagulation properties and excellent storage properties, and the fermented milk with excellent coagulation properties and excellent storage properties is prepared, specifically: 1) Preheat milk to 65°C and maintain at 65°C for 15 minutes. Stir continuously and add 6.5% sugar by weight based on the weight of the milk. Mix well and sterilize at 95°C for 40 minutes. Cool to 42°C to obtain a mixture of milk and sugar. 2) mixing Lactobacillus kumiss subsp. kumiss IMAU13289 with a basic starter culture at a volume ratio of 1:1 to obtain a starter culture; 3) inoculating the fermentation agent obtained in step 2) into the mixture of milk and white sugar obtained in step 1), allowing the mixture to ferment at 42° C. until the pH reaches 4.5-4.6, and then stopping the fermentation to obtain fermented milk; The inoculation amount of the fermentation agent in step 3) is calculated based on 4% of the volume fraction of the mixture of milk and white sugar.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention discloses a strain of Lactobacillus kumiss-like subspecies kumiss-like with potential probiotic properties Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, the growth performance, casein hydrolysis, β-galactosidase activity, extracellular polysaccharide content, antioxidant activity, antibacterial activity, carbohydrate utilization ability, drug sensitivity, and cell surface hydrophobicity of the strain were comprehensively evaluated, and it was found that it has good growth performance, a strong adhesion and colonization effect on the intestine, and can adapt to the extreme environment of the gastrointestinal tract, excellent fermentation characteristics, and strong antioxidant capacity. Comprehensive analysis shows that it has potential probiotic properties. In particular, Kühm-like Lactobacillus Kühm-like subspecies IMAU13289 has the ability to produce high extracellular polysaccharides, and has a strong ability to produce lactic acid, and is resistant to gastrointestinal fluid digestion and is resistant to neomycin. It can be used in particular to prepare fermented milk with excellent coagulation properties and excellent storage properties. Therefore, the present invention provides a Kühm-like Lactobacillus Kühm-like subspecies IMAU13289 with new functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is the phylogenetic tree of Lactobacillus kumiss-like subsp. kumiss-like IMAU13289; Figure 2 The colony morphology of Lactobacillus kumiss subsp. kumiss IMAU13289, wherein A is the colony morphology and B is the Gram microscope observation image; Figure 3 This is a growth curve of Lactobacillus kumiss subsp. kumiss IMAU13289; Figure 4 is an acid production curve of Lactobacillus kumiss subsp. kumiss IMAU13289; Figure 5 Effects of different temperatures on the growth of Lactobacillus kumiss subsp. kumiss IMAU13289; Figure 6 Effects of different pH values ​​on the growth of Lactobacillus kumiss subsp. kumiss IMAU13289; Figure 7 This is the standard curve of OPN concentration; Figure 8 is the standard curve of tyrosine; Figure 9 is the glucose standard curve; Figure 10 This is a graph showing the proteolytic activity of Lactobacillus kumiss-like subsp. kumiss-like IMAU13289; Figure 11 This is a graph showing the antioxidant capacity assay results of Lactobacillus kumiss subsp. kumiss IMAU13289; Figure 12 Figure 2 shows the changes in fermentation time and viable bacterial count of milk fermented with Lactobacillus kumiss subsp. kumiss IMAU13289. Uppercase and lowercase letters represent the significance of the same sample at different times, and lowercase letters represent the significance of different samples at the same time. A is significantly higher than B. Figure 13 The figure represents the changes in elasticity factor, viscosity factor, solid-liquid equilibrium value and flow factor of Lactobacillus kumissiformis subsp. kumissiformis IMAU13289 during the fermentation process of preparing fermented milk, where A is the elasticity factor, B is the viscosity factor, C is the solid-liquid equilibrium value, and D is the flow factor. Figure 14The continuous pH and pH during storage of fermented milk prepared by Lactobacillus kumissiformis subsp. kumissiformis IMAU13289, where A is the continuous pH during preparation of fermented milk and B is the pH during storage. Uppercase and lowercase letters in the figure represent the significance of the same sample at different times, and lowercase letters represent the significance of different samples at the same time. Figure 15 Water-holding capacity and viscosity of fermented milk prepared with Lactobacillus kumissiformis subsp. IMAU13289 during storage. A represents the water-holding capacity of the fermented milk during storage, and B represents the viscosity of the fermented milk during storage. Uppercase and lowercase letters in the figure represent the significance of the same sample at different times, and lowercase letters represent the significance of different samples at the same time.

[0020] Certificate of Deposit Lactobacillus kumiss subsp. kumiss IMAU13289, Latin name Lactobacillus kefiranofaciens subsp. kefiranofaciens The strain is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 4, 2024, and the deposit number is: CGMCC No. 32897. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The source of the materials used in the present invention: strain IMAU12319 is a kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens )IMAU12319, from the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCC No.31224; sodium chloride in phosphate buffer was purchased from Tianjin Huihang Chemical Technology Co., Ltd., potassium dihydrogen phosphate was purchased from Tianjin North Tianyi Chemical Reagent Factory, and disodium hydrogen phosphate was purchased from Tianjin Damao Chemical Reagent Factory; API50CHL test kit was purchased from Mérieux (Suzhou) Biological Products Co., Ltd.; LB liquid medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; whole milk was purchased from Inner Mongolia Mengniu Dairy Group Co., Ltd.; sucrose was purchased from Beijing Tangtaizong Technology Co., Ltd.; drug sensitivity paper was purchased from Beekman Biotechnology Co., Ltd.; basic starter culture (PYS-010) (containing Stretpococcus thermophilus and Lactobacillus bulgaricus ) was purchased from Beijing Ketuo Hengtong Biotechnology Co., Ltd.

[0027] The formula of the 11% skim milk culture medium used in the present invention is as follows: 11 g skim milk, 89 mL distilled water, sterilized at 115° C. for 7 min, and rapidly cooled for later use.

[0028] The formula of the MRS liquid culture medium used in the present invention is: 10g soy peptone, 10g beef extract, 5g yeast powder, 20g glucose, 1g Tween 80, 2g dipotassium hydrogen phosphate, 5g anhydrous sodium acetate, 2g sodium citrate, 200mg magnesium sulfate heptahydrate, 54mg manganese sulfate pentahydrate, 1000mL distilled water, the pH is adjusted to 6.3, and sterilized at 121°C for 15min.

[0029] The 0.85% physiological saline used in the present invention comprises 8.5 g of sodium chloride and 1000 mL of distilled water.

[0030] The formula of the simulated gastric fluid used in the present invention is: 40 mL of sterilized PBS + 3.0 g / L pepsin (0.3%), then adjusted to pH 2.5 (adjusted with 0.1 mol / L HCl), filtered through a 0.22 μm filter membrane into a sterilized empty conical flask for later use.

[0031] The formula of the simulated intestinal fluid used in the present invention is: 40 mL of sterilized PBS + 1.0 g / L trypsin (0.1%) + 1.8% bile salt, then adjusted to pH 8.0 (adjusted with 0.1 mol / L NaOH), filtered through a 0.22 μm microfiltration membrane into a sterilized empty conical flask for later use.

[0032] Example 1 Strain isolation and identification process A sample of Mongolian yogurt was serially diluted and inoculated onto MRS medium using the spread plate method. The culture was then anaerobically incubated at 37°C for 36 hours. Single colonies were picked from the plates and purified by streak plate analysis to obtain pure cultures. Purified colonies were inoculated into MRS liquid medium for 24 hours, centrifuged at 12,000 rpm for 5 minutes at 4°C, and washed twice with PBS before collection to obtain bacterial slurry. Genomic DNA was extracted and the 16S rRNA gene (SEQ ID NO. 1) was amplified by PCR. The amplified product was sequenced using universal primers 27F / 1492R. The forward primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2)) and the reverse primer was 1492R (5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO. 3)). Based on the sequencing results, the bioinformatics software MEGA was used to construct a phylogenetic tree to analyze the phylogenetic status of the strains. The phylogenetic tree is shown in the figure below. Figure 1 The strains were preserved using skim milk as a protective agent, packaged in cryopreservation tubes, and stored in a -80°C ultra-low temperature freezer for long-term storage.

[0033]

[0034] Take out the frozen cryovials from the -80℃ freezer, place them in an ice box and thaw them in an ice bath, then inoculate the strains stored in the cryovials into MRS liquid culture medium at a 2% inoculation rate, culture at a constant temperature of 37℃ for 24 hours, and continuously passage for 3 generations to maximize the activity of the strains for use in subsequent experiments.

[0035] Example 2 1. Fermentation characteristics of the strain: 1. Analysis of milk-clotting properties of Lactobacillus kumiss subsp. IMAU13289: The time from inoculation to the formation of curds is recorded as the coagulation time of lactic acid bacteria, with a maximum of 24 hours. Activated Lactobacillus kumiss subsp. kumiss IMAU13289 (strain IMAU13289) was inoculated at a 2% volume fraction into sterilized skim milk culture medium and incubated at 37°C for 12 hours. The coagulation state of the strain was observed and recorded every 3 hours. Activated strain IMAU12319 was inoculated at a 2% volume fraction into sterilized skim milk culture medium and incubated at 37°C for 12 hours. The coagulation state of the strain was observed and recorded every 3 hours.

[0036] Table 1 Coagulation of strain IMAU13289 at different time periods ;

[0037] As shown in Table 1 , the clotting time of strain IMAU13289 was within 12 h, indicating that strain IMAU13289 had the clotting property.

[0038] 2. Analysis of acid production performance of strain IMAU13289: Strain IMAU13289 was inoculated into skim milk at a 2% volume fraction and cultured at 37°C for 24 hours. The pH was measured, the acidity was titrated, and the coagulation time was recorded. Strain IMAU12319 was inoculated into skim milk at a 2% volume fraction and cultured at 37°C for 24 hours. The pH was measured, the acidity was titrated, and the coagulation time was recorded.

[0039] Table 2 Effect of strain IMAU13289 fermentation on the quality of fermented milk ;

[0040] Note: "+++" means the curd is firmer, without whey precipitation, and has a unique aroma; "++" means the curd is firmer, without whey precipitation, and has a fermented taste; "+" means the curd is loose, with less whey precipitation and a weaker fermented taste; " The curds in the "group" were loose, whey precipitation was more serious, and there was a distinct fermented sour smell.

[0041] As shown in Table 2, the strain IMAU13289 reached the fermentation endpoint after 11.3 h of fermentation. At the fermentation endpoint, the pH value of the milk fermented by the strain IMAU13289 was 4.23, and the titratable acidity was 98.7°T, indicating that although the strain IMAU13289 had a slightly longer coagulation time, it had a fast acid production rate.

[0042] Example 3 1. Results of tolerance test on simulated gastrointestinal fluid: 1. pH 3.0 tolerance test: The test bacterial liquid (strain IMAU13289 or strain IMAU12319) was inoculated into MRS liquid medium with a pH of 3.0 at a volume fraction of 2%, and cultured anaerobically at 37°C for 24 hours. The blank control was based on the MRS liquid medium with the corresponding pH without inoculation. The OD value was measured at 600 nm at 0 hour and 24 hours. The fermentation liquid base after culture was dipped with a disposable inoculating loop and streaked on the MRS solid medium to observe the growth on the plate.

[0043] Table 3 Analysis of acid resistance of strains ;

[0044] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0045] As shown in Table 3, strain IMAU13289 can survive at a pH of 3.0.

[0046] 2. 0.3% (w / v) bile salt resistance test: Strain IMAU13289 was inoculated at a 2% volume fraction in MRS liquid medium containing 0.3% bile salts. The inoculated liquid medium was then streaked onto MRS solid medium containing 0.3% bile salts using a disposable inoculating loop. The plate was then incubated upside down at 37°C under anaerobic conditions for 24 hours. Absorbance was measured at 600 nm using a UV spectrophotometer. To ensure accuracy and reproducibility, three replicates were performed in each group, and colony formation was observed. Strain IMAU12319 was inoculated at a 2% volume fraction in MRS liquid medium containing 0.3% bile salts. The inoculated liquid medium was then streaked onto MRS solid medium containing 0.3% bile salts using a disposable inoculating loop. The plate was then incubated upside down at 37°C under anaerobic conditions for 24 hours. The absorbance was measured at 600 nm by UV spectrophotometer. To ensure the accuracy and repeatability of the experiment, three parallel tests were repeated in each group to observe whether colony growth was generated.

[0047] Table 4 Results of 0.3% (w / v) bile salt tolerance test ;

[0048] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0049] As shown in Table 4 , strain IMAU13289 had a certain bile salt tolerance at a bile salt concentration of 0.3% (w / v).

[0050] 3. Analysis of strain tolerance to artificial gastrointestinal fluid: The strains (strain IMAU13289 or strain IMAU12319) resistant to acid stress and bile salt stress were serially subcultured three times to obtain fermentation broth. Subsequently, the supernatant was separated by centrifugation at 4000 r / min for 10 min. The precipitate was washed by centrifugation in phosphate buffered saline (PBS) sterilized in an ultra-high pressure sterilizer, and 5 mL of sterilized PBS solution was added to wash the bacteria by centrifugation. The discarded supernatant was added to sterile PBS until the washing was repeated three times. Then, 40 mL of PBS solution was added to the bacterial slurry and pipetted to mix thoroughly. The concentration of the bacterial suspension was adjusted to 1.0 × 10 8 CFU / mL, which becomes the test bacterial solution.

[0051] 1000 μL of the suspension was thoroughly mixed with 9 mL of simulated gastric fluid (pH 2.5), and fermented in an anaerobic environment at 37°C for 3 hours. The number of viable bacteria was then determined and counted. Another 1000 μL of the bacterial suspension, which had been incubated in simulated gastric fluid for 3 hours, was mixed with 9 mL of simulated intestinal fluid. Samples were collected at 4 and 8 hours under anaerobic conditions at 37°C, and the number of viable bacteria at each time point was counted using the following formula: Artificial gastric fluid survival rate (%) = N1 / N0 × 100% (1); Survival rate in artificial intestinal fluid (%) = N2 / N1×100% (2); Among them, N0 represents the number of viable bacteria in the original bacterial solution at 0 h (CFU / mL); N1 represents the number of viable bacteria in the artificial gastric fluid treatment for 3 h (CFU / mL); and N2 represents the number of viable bacteria in the artificial intestinal fluid treatment for 8 h (CFU / mL).

[0052] Table 5 Analysis results of strain tolerance to artificial gastrointestinal fluid ;

[0053] Note: The data in the table are mean (X) ± standard deviation (SD), and different lowercase letters in the same column indicate significant differences (P<0.05).

[0054] As shown in Table 5, the survival rates of different strains in gastric juice varied. Strain IMAU13289 achieved a high survival rate of 86% after 3 hours of digestion, and 52% remained after 11 hours, surpassing strain IMAU12319. As a probiotic, the presence of a certain number of viable bacteria is a prerequisite for maintaining probiotic function.

[0055] Example 4 1. Growth characteristics of strain IMAU13289: 1. Morphological characteristics of strain IMAU13289: like Figure 2 China A and Figure 2 As shown in middle B, the single colony of strain IMAU13289 is round, with a small diameter, a slightly convex center, neat edges, a smooth surface, and a transparent white color; it was determined to be a Gram-positive bacterium by Gram staining microscopy.

[0056] 2. Growth curve and acid production performance analysis of strain IMAU13289: Take the previously prepared test bacterial suspension and inoculate it into MRS liquid medium at a 2% (v / v) inoculum. Incubate in a 37°C constant temperature incubator. During the growth period, collect 200 μL of the fermentation broth every 2 h (i.e., 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 h) and measure the optical density (OD) of the bacterial suspension. Determine the viable cell count and pH every 4 h (i.e., 0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 h).

[0057] Determination of viable bacteria count: Select a test tube with an appropriate dilution according to the total range of viable lactic acid bacteria, draw 1.0 mL of bacterial suspension onto a sterile plate (set two replicates for each dilution), pour in 15 mL of MRS solid culture medium cooled to 50°C, gently rotate the plate more than 20 times to mix, wait for the agar to solidify, invert the plate, and incubate anaerobically at 37°C for 48 hours. If no colonies grow, the incubation period can be extended to 72 hours. After the incubation period, count the number of colonies on the plate, and the dilution and pouring of the bacterial suspension onto the plate must be completed within 15 minutes.

[0058] OD value determination: Mix the bacterial suspension thoroughly, accurately pipette 200 μL into a 96-well ELISA plate, and measure the absorbance at a wavelength of 600 nm.

[0059] pH measurement: The pH value at each time point was measured using a pH meter.

[0060] The bacterial growth curve reflects the growth pattern of a single microbial population in liquid culture in a specific environment. The growth and acid production characteristics of strain IMAU13289 are shown in Figure 3 and Figure 4 .like Figure 3As shown, the strain IMAU13289 was in the growth retardation period from 0 to 2 hours, and OD 600nm There was no significant change in activity; after a 2-h delay period, it entered the logarithmic growth phase, OD 600nm The value and viable bacteria count increased rapidly from 4 hours onwards; then OD 600nm The growth rate gradually slowed down and stabilized into the stable period; the number of viable bacteria reached its peak at 16 hours, and OD 600nm After 18 hours, the change tends to be stable. Figure 4 As shown, the pH of strain IMAU13289 remained essentially unchanged from 0 to 2 hours. From 2 to 14 hours, it entered the logarithmic growth phase, with increased lactic acid production and a significant decrease in pH as the bacterial population grew. At 20 hours, the pH reached 3.93, then gradually stabilized. After 24 hours, it entered the death phase, with a significant decrease in viable bacterial count and a slight increase in pH. Overall, strain IMAU13289 demonstrated good growth and acid production.

[0061] 3. Growth of strain IMAU13289 at different temperatures: The activated bacterial solution was inoculated into 5 mL of MRS liquid culture medium at a volume fraction of 2%. Five temperature gradients were set. After culturing under constant temperature conditions for 24 h, the absorbance of the fermentation broth at 600 nm was measured to explore the growth temperature range of the strain IMAU13289.

[0062] Depend on Figure 5 It can be seen that the OD of the fermentation broth of strain IMAU13289 600nm The value first increases slowly and then decreases with the increase of temperature. 600nm It reached its maximum value at 37℃. When the culture temperature was ≥45℃, the growth activity of the strain decreased, indicating that high temperature had a certain inhibitory effect on its growth.

[0063] 4. Growth of strain IMAU13289 at different pH values: The bacterial liquid was inoculated into MRS liquid culture medium with initial pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, with an inoculum size of 2% by volume, and cultured at 37°C for 24 hours. The absorbance of the fermentation liquid at 600 nm was then measured to investigate the effect of pH on the growth of the strain.

[0064] Depend on Figure 6The results show that strain IMAU13289 grows well in MRS liquid medium at pH values ​​between 2 and 8. Specifically, growth is slow at pH values ​​below 4.0; growth is robust between 4.0 and 7.0; and bacterial density gradually decreases above pH 7.0. Overall, strain IMAU13289 has an optimal pH of 5.0, indicating that it thrives in a slightly acidic environment.

[0065] Example 5 1. Physiological and biochemical characteristics of strain IMAU13289: 1. Identification of strain IMAU13289 API 50CHL: In this experiment, 50 biochemical assays in the API 50CHL kit were used to identify and test the utilization of 49 sugars and their derivatives by strain IMAU13289.

[0066] Table 6 Determination of carbohydrate types that can be utilized by strain IMAU13289 ;

[0067] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0068] As shown in Table 6, the API 50CHL reaction results showed that the strain IMAU13289 showed positive reactions to 6 carbohydrates, among which the strain IMAU13289 mainly utilized the following carbohydrates: D-galactose, D-glucose (GLU), N-acetyl-glucosamine (NAG), D-maltose (MAL), D-lactose (LAC), and D-trehalose (TRE).

[0069] 2. β-galactosidase activity assay: Strain IMAU13289 or strain IMAU12319 was cultured in MRS liquid medium at 37°C for 24 h. The cells were collected by centrifugation (4°C, 8000 rpm, 15 min) and then sonicated (125 W, 3 s on, 8 s off, 15 min total) to obtain the supernatant as the crude enzyme solution. The total sugar content was determined by UV spectrophotometry.

[0070] When performing the β-galactosidase activity assay, first place a 2.5 mg / mL ONPG solution prepared with pH 6.5 PBS in a 37°C water bath for 5 minutes, then add 1 mL of ONPG solution and 1 mL of crude enzyme solution to the test tube in sequence, mix thoroughly, and react at 37°C for 10 minutes. After the reaction is completed, quickly add 4 mL of 500 mmol sodium carbonate solution, mix well, and let it stand for 5 minutes, then measure the absorbance of the solution at 420 nm. The blank determination operation is basically the same as the above process, except that sodium carbonate solution is added before adding the crude enzyme solution to inactivate the reaction enzyme solution. At the same time, draw a standard curve of o-nitrophenol (ONP) ( Figure 7 ), the β-galactosidase activity in the sample was calculated according to the following formula: Enzyme activity (U / mL) = (c × v × d) / (t × v1) (3); Wherein, c is the concentration of the test ONP; v is the total volume of the reaction solution; d is the sample dilution multiple; t is the reaction time; v1 is the volume of the reaction enzyme solution.

[0071] The amount of o-nitrophenol (ONP) released per mg of dry cell weight per minute was used as the enzyme activity unit (U / 10 4 Cells were collected to measure β-galactosidase activity. Ultrasonication of the test strain at different concentrations was performed, and enzyme activity was then measured using the ONPG method.

[0072] Table 7 β-galactosidase activity ;

[0073] As shown in Table 7, the lactase activity of strain IMAU13289 is 2.93 U / 10 4 cell.

[0074] 3. Determination of proteolytic activity of strain IMAU13289: Using o-phthalaldehyde as substrate, a standard curve for casein hydrolysis activity was constructed ( Figure 8 ), which was used for subsequent proteolytic activity assessment.

[0075] The activated strain IMAU13289 was inoculated into skim milk at a volume fraction of 2%, and cultured at 37°C for 12h. At the same time, skim milk without inoculum was set as a blank control. Accurately draw 2.5mL of sample into a test tube, add 0.5mL of deionized water in turn and mix well, then add 0.5mL of 0.75mol / L trichloroacetic acid (TCA), vortex mix and let stand at room temperature for 10min, filter and obtain the supernatant for use. Take 150μL of supernatant and place it in a test tube, add 3mL of OPA reagent, mix thoroughly, react at the specified temperature for 2min, and then measure its absorbance at 340nm. Since the proteolytic activity is equivalent to the tyrosine content in this system, OD 340nm As the OPA index, comparative analysis was performed based on the standard curve.

[0076] Depend on Figure 10 It can be seen that the OD 340nm Absorbance changes slowly; 6-12h, OD 340nm The absorbance increased rapidly, accelerating between 12 and 18 hours. After 18 hours, the absorbance began to slowly decrease. Overall, the strain exhibited the strongest proteolytic activity during the logarithmic growth phase. After 24 hours of incubation, the final absorbance of strain IMAU13289 reached 0.3812, corresponding to a free amino acid content of 25.39 mg / L.

[0077] 4. Determination of extracellular polysaccharide of strain IMAU13289: This study is based on the glucose standard curve ( Figure 9 ), and use this as a reference for subsequent extracellular polysaccharide determination.

[0078] The activated strain IMAU13289 or strain IMAU12319 was inoculated into 10 mL of MRS medium at a 2% volume fraction and incubated in a 37°C incubator for 24 h. The culture was centrifuged in a refrigerated centrifuge (8000 rpm, 4°C) for 20 min. The supernatant was collected and placed in a boiling water bath for 5 min, cooled to room temperature, and then adjusted to a final concentration of 10% with 80% trichloroacetic acid (TCA). The mixture was stirred thoroughly and allowed to stand overnight. The supernatant was then centrifuged again at 8000 rpm for 20 min to remove protein. The supernatant was then transferred to a conical flask, and three volumes of 95% ethanol were added. The mixture was precipitated overnight at 4°C to induce polysaccharide precipitation as a flocculent precipitate. The precipitate was then centrifuged at 8000 rpm for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in 5 mL of distilled water, placed in a dialysis bag, and stirred continuously with a magnetic stirrer for 48 h. The external solution was replaced every 8 h to ensure thorough dialysis.

[0079] The total sugar content of the dialyzed precipitate was determined using the phenol-sulfuric acid method, with glucose used as the standard to quantify the extracellular polysaccharide content. An uninoculated MRS liquid medium was used as a blank control group. The final extracellular polysaccharide concentration (mg / L) of the strain was determined by subtracting the blank control value from the experimental group's measured value.

[0080] Table 8 Determination of extracellular polysaccharide production of strain IMAU13289 ;

[0081] As shown in Table 8, the extracellular polysaccharide content of strain IMAU13289 reached 151.69 mg / L, indicating that the strain has a high level of extracellular polysaccharide production.

[0082] Example 6 1. Probiotic properties of strain IMAU13289: 1. Determination of antioxidant capacity of strains: (1) Determination of free radical scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH): Take 2 mL of cell suspension, cell-free extract, or fermentation broth and thoroughly mix it with 1 mL of DPPH solution prepared in anhydrous ethanol. After standing in the dark for 30 minutes, centrifuge at 10,000 rpm for 10 minutes. Take the supernatant and zero the instrument. Measure its absorbance at 600 nm using a UV spectrophotometer and record it as B. At the same time, set up a control group by replacing the anhydrous ethanol containing DPPH with anhydrous ethanol. The corresponding absorbance value is recorded as C. Set up a blank group and record the absorbance value as A.

[0083] Cell suspension: Add 40 mL of PBS solution to the bacterial slurry and mix thoroughly, and adjust the concentration of the bacterial suspension to 1.0 × 10 8 CFU / mL.

[0084] Cell-free extract: The cell suspension was disrupted by ultrasonication in an ice bath (200 W power, pulse 30 s / rest 30 s, total 5 min), then centrifuged at 9000 rpm and 4°C for 15 min. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove bacteria.

[0085] Fermentation broth: The strain was inoculated into MRS broth and cultured for 36 h. Centrifuged at 8000 rpm and 4°C for 15 min, the supernatant was collected and sterilized by filtration.

[0086] Given that vitamin C (VC) possesses antioxidant activity and that this activity is positively correlated with its content, a VC aqueous solution was used as a positive control for the DPPH scavenging rate determination in this experiment. The experimental results showed that as the vitamin C content in the VC aqueous solution increased, its DPPH scavenging rate increased accordingly. The subsequent related indicators were calculated based on the following formula: DPPH free radical scavenging rate (%) = [1-(AB) / C] × 100% (4); The absorbance of the blank group was A, the absorbance of the cell suspension, cell-free extract or fermentation broth experimental group at 600 nm was B, and the absorbance of the anhydrous ethanol containing DPPH replaced by anhydrous ethanol was C.

[0087] Depend on Figure 11 The test results showed significant differences in the DPPH radical scavenging rates of different solutions of strain IMAU13289. The VC aqueous solution positive control group achieved a DPPH scavenging efficiency of 87%. For strain IMAU13289, the fermentation broth group exhibited a high DPPH radical scavenging rate of 78%, while the cell suspension group achieved a scavenging rate of 51% and the cell-free extract group achieved a scavenging rate of 37%. This comparison revealed that the fermentation broth group had the highest DPPH scavenging rate of the three solutions and was closest to that of the positive control VC solution. Overall, strain IMAU13289 exhibited strong DPPH radical scavenging ability.

[0088] (2) Superoxide anion (O2 - ) Determination of scavenging ability: Take 0.5 mL of cell suspension, 0.5 mL of cell-free extract, or 0.5 mL of fermentation broth and add 1 mL of 3 mmol / L diethylenetriaminepentaacetic acid, 1 mL of 150 mmol / L tris(hydroxymethyl)methane hydrochloride at pH 8.2, and 1 mL of 1.2 mmol / L pyrogallol in that order. Mix thoroughly and incubate in a 25°C water bath for 10 minutes. After the water bath is complete, measure the absorbance of the system at a wavelength of 325 nm. The calculation formula is: Superoxide anion clearance rate = {1-[(GF) / (ED)]} × 100% (5); Among them, D represents the absorbance of the blank control, E represents the maximum absorbance, F represents the absorbance of the sample group, and G represents the absorbance of the sample blank.

[0089] The color change of the colored intermediate substance before and after adding cell suspension or its cell-free extract or fermentation broth reflects the superoxide anion free radical (O2 - ) generation and reduction.

[0090] like Figure 11 As shown in the figure, the superoxide anion (O2 - ) clearance rate was 100%, among which the superoxide anion (O2 - ) clearance rate was 82%, and the superoxide anion (O2 -) clearance rate was 51%, and the superoxide anion (O2 - ) clearance rate was 21%.

[0091] (3) Determination of hydroxyl radical (-OH) scavenging ability: Measure 2 mL of cell suspension (or an equivalent amount of cell-free extract or fermentation broth) and add, in sequence, 1 mL of a 5 mol / mL salicylic acid ethanol solution, 1 mL of a 5 mmol / L ferric sulfate solution, 1 mL of a 3 mol / mL hydrogen peroxide solution, and 5 mL of double-distilled water. Mix thoroughly and incubate in a 37°C water bath for 15 minutes. Centrifuge at 4°C and 9000 rpm for 10 minutes to obtain the supernatant. Measure its absorbance at 510 nm and record it as H. Replace the sample with ultrapure water as a blank control, and record the corresponding absorbance as I0. The calculation formula is: Hydroxyl radical scavenging rate (%) = [1-(H / I0)] × 100% (6).

[0092] like Figure 11 As shown, the hydroxyl radical scavenging rate of the VC solution control group reached 65%. The hydroxyl radical scavenging rate of strain IMAU13289 in the fermentation broth group was 50%, the hydroxyl radical scavenging rate of strain IMAU13289 in the cell suspension group was 46%, and the hydroxyl radical scavenging rate of strain IMAU13289 in the cell-free extract group was 20%. The fermentation broth had the strongest hydroxyl radical scavenging ability, demonstrating excellent hydroxyl radical scavenging activity.

[0093] 2. Hydrophobicity test of strain IMAU13289: The surface hydrophobicity of strain IMAU13289 was evaluated by hydrocarbon adsorption method. The bacterial solution was centrifuged at 4000 rpm for 5 min to collect the bacterial sludge. After washing twice with PBS buffer, the sludge was resuspended in PBS and the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, and measure the initial absorbance J0 at 600 nm.

[0094] Mix 2 mL of xylene with an equal amount of bacterial suspension, vortex for 10 minutes, and then let it stand at room temperature for 30 minutes. Aspirate the lower aqueous phase and measure the absorbance J at 600 nm. The formula is as follows: Hydrophobicity (%) = (J-J0) / J0×100% (7).

[0095] Table 9 Hydrophobicity test results of strain IMAU13289 ;

[0096] As shown in Table 9, bacterial surface hydrophobicity is positively correlated with bacterial attachment and colonization within organisms; the greater the hydrophobicity, the easier it is to achieve this process. Surface hydrophobicity of strain IMAU13289 was measured, and the strain's hydrophobicity ratio was 30% to 32%.

[0097] 3. Determination of antibacterial activity of strain IMAU13289: The antibacterial activity of the strain was determined using the Oxford cup method. The strain was inoculated into LB solid medium and cultured for 12 hours. The culture was then transferred to LB liquid medium and fermented in a shake flask at 37°C and 180 rpm for 12 hours. The resulting fermentation broth was diluted 10-fold and plated on a plate. The fermentation was continued at 37°C for 24 hours, followed by centrifugation at 4000 rpm for 10 minutes. The supernatant with antibacterial activity was collected for subsequent testing.

[0098] In the antibacterial assay, the experimental strain IMAU13289 was activated by LB solid and fermented in a liquid shake flask. The supernatant was collected after the fermentation liquid was treated. The Oxford cup was placed in a container containing the indicator bacteria Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Listeria monocytogenes ( Listeria monocytogenes ),salmonella( Salmonella For the blank control group, place the Oxford cup on a plate containing indicator bacteria and add MRS broth to the cup. Incubate the cells under the same conditions as the experimental group and observe for the presence of an inhibition zone.

[0099] Table 10 Test results of antibacterial properties of strains ;

[0100] As shown in Table 10, no inhibition zone was observed in the blank control group containing only MRS broth, indicating that the culture medium composition did not interfere with the inhibition assay. Strain IMAU13289 exhibited excellent antibacterial activity against the four common pathogens listed above, with a particularly significant inhibitory effect against Staphylococcus aureus.

[0101] Example 7 Safety evaluation of strain IMAU13289: Using the disc diffusion test method, drug sensitivity tests were conducted on 26 antibiotics. The screened strains were first activated, centrifuged at 4000r / min for 10 minutes, and the supernatant was discarded. The bacteria were resuspended in sterile saline solution, and the turbidity was accurately adjusted to 0.5 McFarland units. 100μL of the bacterial suspension was evenly spread on the surface of MRS solid culture medium. After it was fully dried, the drug-sensitive paper was adhered to the plate coated with lactic acid bacteria using the disc method, and then placed in an anaerobic environment for culture. The sensitivity of the bacteria to each antibiotic was determined based on the size of the inhibition zone around the drug-sensitive sheet.

[0102] Table 11 Drug sensitivity test results ;

[0103] Note: The criteria for judging the diameter of the inhibition zone are sensitive (S), intermediate (I), and resistant (R).

[0104] The test results of 20 commonly used antimicrobial drugs based on the KB susceptibility test are shown in Table 11. The strain IMAU13289 was sensitive to 5 commonly used antimicrobial drugs, with a large number of colonies growing during culture; it was moderately sensitive to 1 of the antimicrobial drugs, with an obvious inhibition ring showing after 4 weeks of culture; it was highly sensitive to 14 antibiotics, with no colonies growing after 4 weeks of culture and a large-caliber inhibition ring forming. These results indicate that the strain has a strong sensitivity to antibiotics.

[0105] Example 8 1. Application of strain IMAU13289 in fermented milk: Commercially available pure milk was preheated to 65°C and stirred for 15 minutes. During the stirring period, 6.5% white sugar was slowly added, mixed and homogenized (low pressure 50 bar, high pressure 190 bar), sterilized at 95°C for 40 minutes, and cooled at 42°C to obtain a milk and white sugar mixture. The strain IMAU13289 and the basic starter culture were mixed in a volume ratio of 1:1 to obtain a fermentation agent. The inoculation amount of the fermentation agent was calculated based on 4% of the volume fraction of the milk and white sugar mixture. The fermentation agent was inoculated into the milk and white sugar mixture and fermented. The mixture was allowed to stand and ferment in a constant temperature box at 42°C until the pH was within the range of 4.5-4.6. Fermentation was stopped and the mixture was refrigerated at 4°C. The relevant indicators were measured after 4 weeks of storage.

[0106] 1. Determination of viable bacterial count in fermented milk: The fermentation time and viable bacteria count of IMAU13289 fermented milk are as follows Figure 12 During the experimental observation period, the number of viable bacteria showed a trend of first decreasing, then increasing and tending to be stable. On the first day, the number of viable bacteria was at a high level, reaching about 1.3×10 9 CFU / mL. By the 7th day, the number of viable bacteria decreased significantly to about 1.2×10 9 CFU / mL, from the 7th to the 14th day, the number of viable bacteria increased to about 1.25×10 9 CFU / mL, the number of viable bacteria on day 21 remained at a level similar to that on day 14, approximately 1.25×10 9 CFU / mL. The International Dairy Federation (IDF) stipulates that during storage, transportation and sales, the number of viable lactic acid bacteria in fermented milk should reach 10 7CFU / mL or above. The total viable bacterial count of the fermented milk sample during storage meets this standard.

[0107] After entering storage, the growth and reproduction of IMAU13289 may have consumed nutrients in the fermented milk, leading to a decrease in viable bacterial counts. For Streptococcus salivarius subspecies thermophilic and Lactobacillus delbrueckii subspecies bulgaricus, in addition to insufficient nutrients in the fermented milk, they also produce large amounts of lactic acid when breaking down lactose. Excessive lactic acid increases the acidity of the fermented milk, which in turn affects microbial activity. Therefore, when acidity is too high, the viable bacterial count tends to decrease.

[0108] 2. Determination of microrheology of strains during fermentation: (1) Determination of strain elasticity factor (EI): The elasticity index (EI) is a parameter used to evaluate and analyze the elastic characteristics of fermented milk. It reflects the degree of elasticity changes during the fermentation process, revealing changes in its structure and texture. The EI value is closely related to the elasticity of the gel structure. An increase in the EI value indicates a tighter gel network within the fermented milk, resulting in enhanced elasticity and a more stable state.

[0109] The changes of EI value of IMAU13289 compound fermented milk during the fermentation process are as follows: Figure 13 As shown in center A, from the time of inoculation to the start of the fermentation phase, the EI values ​​of the fermented milk samples were relatively stable for the initial 0-8 hours, with no significant differences (P>0.05). During this period, the proteins within the fermented milk had not yet established a network structure, resulting in the fermented milk maintaining a liquid state with high fluidity and low viscosity. As fermentation progressed, the 8-hour-old composite fermented milk reached a turning point, the gel point, corresponding to the sharp increase in the viscosity factor in the figure. This indicates that the fermented milk system first transitioned from a liquid to a solid state, with enhanced intermolecular interactions and the initiation of a gel network structure. Subsequently, the viscosity factor rose sharply, similar to the nearly vertical rise of the EI curve. After reaching its peak, the EI value quickly entered a highly elastic phase, then declined slightly, reaching a relatively stable level between 14 and 16 hours. This change reflects that during the acidification process, the original electrostatic repulsion balance of the fermented milk was disrupted, prompting the casein particles to rearrange themselves, forming a three-dimensional network structure, and ultimately forming a stable gel structure.

[0110] (2) Determination of strain viscosity factor (MVI): The viscosity index (MVI) is measured in microns and characterizes the static viscosity of the sample. Under a specified area, the MVI value is positively correlated with the particle movement time and the viscosity of the system. Figure 13 As shown in B, during the fermentation process of IMAU13289 fermented milk, the viscosity factor was 10 -6 ~10-4 Fluctuate frequently, with low and unstable values; the viscosity factor climbs sharply from 9 to 10 h; it tends to be relatively stable and remains at a high level from 10 to 16 h, indicating that the casein micelles aggregate into a stable three-dimensional network structure at this time, the gel system is stable, and the viscosity reaches the peak.

[0111] (3)Determination of the solid-liquid balance value (SLB) of the strain: The solid-liquid balance value is used to characterize the solid-liquid state of the fermented milk sample at different fermentation times under static conditions. When SLB = 0, the sample has solid properties; when 0 < SLB < 0.5, the sample is mainly characterized by solids and initially shows liquid characteristics; when 0.5 < SLB < 1, the liquid characteristics gradually strengthen and the solid characteristics gradually weaken; when SLB = 1, the sample is liquid. As Figure 13 shown in C, the SLB value of IMAU13289 fermented milk is high and fluctuates at the initial stage of fermentation, indicating that the sample has strong fluidity and is in a liquid state. It reaches the gel point at 9 h, and the texture changes to a gel structure. Subsequently, the SLB value rises slightly and then stabilizes, meaning that the gel structure tends to be stable and finally becomes an elastic viscous solid. During this process, the acid production of the strain promotes the dissociation of casein aggregates, making the sample slightly viscous. The SLB value at the end of fermentation is 0.50. Adding IMAU13289 can significantly improve the viscosity and elasticity of the fermented milk, which is beneficial to the stability of the product.

[0112] (4)Determination of the flow factor (FI) of the strain: The flow factor is a key indicator to measure the fluidity of the fermented milk sample, which can directly reveal the movement rate of microscopic particles in the sample. The higher the FI value, the faster the particles move, mainly showing liquid characteristics. On the contrary, it indicates that the particles move slower and tend to be more solid characteristics, and it corresponds to the result of the SLB value. The change of the FI value of IMAU13289 fermented milk during fermentation is as Figure 13 shown in D. At the initial stage of fermentation, the FI value of the fermented milk shows a relatively high level and is accompanied by certain fluctuations, indicating that the particles in the sample move at a relatively fast speed, resulting in an increase in the overall fluidity of the sample. It reaches the gel point at 9 h, and then the FI value drops sharply, indicating that the sample is changing from a liquid state to a solid state. As the fermentation process progresses, the dehydration condensation effect is gradually strengthened, and the FI value of the sample is stably maintained at about 0.01. At this time, the fermented milk samples all exist in a stable solid state. [[ID=十六]] [[ID=十七]]

[0113] 3. Determination of the continuous pH of the strain and the pH during storage: The acid production capacity of lactic acid bacteria is a key indicator for measuring the growth and activity of strains. It not only reflects their efficiency in utilizing nutrients, but also indirectly reflects their adaptability to a specific environment. It is an important parameter for evaluating fermentation characteristics. The acidity of fermented milk is determined by acidic groups such as polypeptides and free amino acid residues. It can determine the activity of microorganisms and affect the quality and taste of the product. Figure 14 China A and Figure 14 During storage (4°C), the activity of lactic acid bacteria is not completely inhibited and they still ferment and produce acid using nutrients in milk as substrates, causing the pH value to drop. Figure 14 The pH value of fermented milk during storage (B) shows changes: 4.5 after 1 day of storage and 4.1 after 21 days. Research shows that the optimal pH value for drinking fermented milk during storage is above 4.0, and the pH value of fermented milk during storage meets the requirements.

[0114] 4. Determination of water holding capacity and viscosity during storage of strains: Water holding capacity reflects the ability of fermented milk to retain water. The higher the water holding capacity, the stronger the gel structure's ability to bind free water, the less whey precipitation, the higher the system stability, and the better the product's sensory and flavor retention. Figure 15 In Figure A, the water-holding capacity of fermented milk increased after 1-7 days of storage, but decreased after 7-21 days. This is due to a continued decrease in pH during the later stages of storage, which resulted in the precipitation of whey and the destruction of the tight gel structure. The water-holding capacity of IMAU13289-fermented milk peaked at 66% after 7 days of storage, indicating that IMAU13289 can enhance the water-holding capacity of fermented milk, improve its quality, and facilitate its storage.

[0115] Viscosity is a core physical parameter that characterizes the viscosity of a fluid. It not only reflects the continuous stability and organizational state of the fermented milk system, but is also a key indicator that affects its storage and transportation performance. High-quality fermented milk usually presents a semi-solid state with uniform organizational structure, good stringiness, and low fluidity. Figure 15 The trend of variation in medium B is similar to the water-holding capacity test results, showing an initial increase followed by a decrease. Initially, exopolysaccharides produced by lactic acid bacteria metabolism, or a decrease in pH, induce casein precipitation and gel formation. Curd particles gradually aggregate and increase viscosity. Later in storage, as the pH continues to decrease, colloidal calcium phosphate dissolves, disrupting protein cross-links and accompanied by whey precipitation, leading to a decrease in system viscosity. The viscosity of this fermented milk reached a peak of 1950 mPa·s after 14 days of storage, demonstrating that IMAU13289 significantly increases fermented milk viscosity, optimizes texture, and improves product quality.

[0116] 5. Changes in texture of strains during storage: Texture characteristics are an important indicator for objectively evaluating the quality of fermented milk. They are not only closely related to taste, but also a key basis for measuring product quality stability. The texture characteristics of fermented milk, such as hardness, consistency, cohesion and viscosity index, are both independent and interrelated. Among them, the milk protein and fat content dominate the hardness of the sample, while the type and amount of starter significantly affect the consistency and cohesion. Hardness is the core characterization parameter of gel structure strength. The higher its value, the more stable the gel network structure and the stronger its deformation resistance. The consistency is quantified by simulating the force-time positive peak area of ​​the stirring process. The cohesion reveals the strength of the internal structural bonding of the sample. The texture test data of IMAU13289 fermented milk during storage are shown in Table 12. Its hardness reached 33.176±3.11 g, reflecting a relatively solid gel structure; the consistency was 246.653±16.27 g·sec, reflecting that a large force was required during stirring; the cohesion was -21.211±4.24 g, the viscosity index was -20.907±5.00 g·sec, and the particle index was 0. These quantitative indicators reveal the texture characteristics of the fermented milk from multiple dimensions and provide a scientific basis for its quality evaluation.

[0117] Table 12 Texture of fermented milk during storage ;

[0118] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Lactobacillus kumiss subsp. kumiss ( Lactobacillus kefiranofaciens subsp. kefiranofaciens )IMAU13289, characterized in that The kumiss-like Lactobacillus kumiss-like subspecies IMAU13289 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

2. Use of the Lactobacillus kumissiformis subsp. IMAU13289 as claimed in claim 1 in preparing a probiotic agent with high exopolysaccharide production.

3. A probiotic agent with high extracellular polysaccharide production, characterized in that: It includes Kühm-like Lactobacillus Kühm-like subspecies IMAU13289, which was deposited in the General Microbiology Center of China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

4. Use of the Kühm-like Lactobacillus subsp. Kühm-like IMAU13289 as claimed in claim 1 in the preparation of a fast-producing lactic acid probiotic.

5. A rapid lactic acid-producing probiotic, characterized in that: It includes Kühm-like Lactobacillus Kühm-like subspecies IMAU13289, which was deposited in the General Microbiology Center of China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

6. Use of the Lactobacillus kumissiformis subsp. IMAU13289 as claimed in claim 1 in the preparation of a probiotic resistant to gastrointestinal digestion.

7. A probiotic resistant to digestion by gastrointestinal fluid, characterized in that: It includes Kühm-like Lactobacillus Kühm-like subspecies IMAU13289, which was deposited in the General Microbiology Center of China Culture Collection Administration on December 4, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 32897.

8. The use of Lactobacillus kumiss subsp. IMAU13289 in the preparation of a drug-resistant probiotic according to claim 1, characterized in that: The drug-resistant probiotic is resistant to neomycin.

9. A drug-resistant probiotic, characterized in that: It includes Kühm-like Lactobacillus Kühm-like subspecies IMAU13289, which was deposited in the General Microbiology Center of China Culture Collection Committee on December 4, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 32897. The drug-resistant probiotic agent is resistant to neomycin.

10. Use of Lactobacillus kumissiformis subsp. IMAU13289 as claimed in claim 1 in preparing fermented milk with excellent coagulation properties and excellent storage properties, characterized in that: The invention provides a fermented milk having excellent coagulation property and excellent storage property, specifically comprising: 1) Preheat milk to 65°C and maintain at 65°C for 15 minutes. Stir continuously and add 6.5% sugar by weight based on the weight of the milk. Mix well and sterilize at 95°C for 40 minutes. Cool to 42°C to obtain a mixture of milk and sugar. 2) mixing Lactobacillus kumiss subsp. kumiss IMAU13289 with a basic starter culture at a volume ratio of 1:1 to obtain a starter culture; 3) inoculating the fermentation agent obtained in step 2) into the mixture of milk and white sugar obtained in step 1), allowing the mixture to ferment at 42° C. until the pH reaches 4.5-4.6, and then stopping the fermentation to obtain fermented milk; The inoculation amount of the fermentation agent in step 3) is calculated based on 4% of the volume fraction of the mixture of milk and white sugar.

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