Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 and application thereof

Abstract

The application discloses a lactobacillus kefiri subsp. kefiri IMAU13289 and application, belongs to the technical field of microorganisms, provides a lactobacillus kefiri subsp. Lactobacillus Lactobacillus kefiranofaciens subsp. kefiranofaciens ) IMAU13289, which is preserved in the China General Microbiological Culture Collection Center on December 4, 2024, and the address is No. 1, Xibaixili, Chaoyang District, Beijing, and the preservation number is CGMCC No. 32897. The lactobacillus kefiri subsp. kefiri IMAU13289 has the ability of high yield of extracellular polysaccharide, a fast lactic acid production rate, and neomycin resistance, and can be used for preparing fermented milk with excellent coagulation characteristics and excellent storage characteristics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 and application thereof. BACKGROUND

[0002] Lactobacillus kefiranofaciens subsp. kefiranofaciens has been approved as a new food material, that is, a new type of edible probiotic strain. Lactobacillus kefiranofaciens subsp. kefiranofaciens The strain can not only be used as a probiotic functional food, regulate intestinal microecology and improve melancholia, but also play a good anti-osteoarthritis effect through regulation of its metabolites and intestinal flora.

[0003] Lactobacillus kefiranofaciens subsp. kefiranofaciens is a microorganism isolated from kefir grains in the initial stage of camel milk fermentation, and is a starter for the fermentation of Caucasian cow milk.

[0004] Kefir has long been considered beneficial to health, and its health benefits include immune regulation. However, there is a lack of knowledge about the immune regulation induced by kefir lactic acid bacteria (LAB), and Lactobacillus kefiranofaciens subsp. kefiranofaciens plays an important role in regulating immune function. Studies have found that commercial starters cannot well colonize and survive in the human gastrointestinal tract, and with the innovation of technology, more and more enterprises are committed to finding suitable lactic acid bacteria for fermentation, so as to develop fermented milk with more flavors, more functions and more nutrients.

[0005] In summary, the strain helps to develop probiotic fermented milk with high probiotic activity and unique metabolic profile, and establishes a reference standard for the development of new functional foods in the dairy industry, so the discovery is of great significance. SUMMARY

[0006] To solve the above technical problems, the present application provides Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 and application thereof.

[0007] To achieve the above purpose, the present application provides Lactobacillus kefiranofaciens subsp. kefiranofaciens (ATCC 15697) Lactobacillus kefiranofaciens subsp. kefiranofaciensLactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 was preserved in China General Microbiological Culture Collection Center on December 04, 2024, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is: CGMCC No. 32897.

[0008] The application also provides an application of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in preparing a probiotic agent with high yield of exopolysaccharide.

[0009] The application also provides a probiotic agent with high yield of exopolysaccharide, which comprises Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, and the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 was preserved in China General Microbiological Culture Collection Center on December 04, 2024, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is: CGMCC No. 32897.

[0010] The application also provides an application of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in preparing a probiotic agent with rapid lactic acid production.

[0011] The application also provides a probiotic agent with rapid lactic acid production, which comprises Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, and the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 was preserved in China General Microbiological Culture Collection Center on December 04, 2024, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is: CGMCC No. 32897.

[0012] The application also provides an application of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in preparing a probiotic agent with gastrointestinal fluid digestion resistance.

[0013] The application also provides a probiotic agent with gastrointestinal fluid digestion resistance, which comprises Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, and the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 was preserved in China General Microbiological Culture Collection Center on December 04, 2024, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is: CGMCC No. 32897.

[0014] The application also provides an application of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in preparing a drug-resistant probiotic agent, and the drug-resistant probiotic agent has drug resistance to neomycin.

[0015] The application further provides a drug-resistant probiotic agent, comprising Lactobacillus kefiri subsp. kefiri IMAU13289, which is preserved in the China General Microbiological Culture Collection Center on December 4, 2024, has a preservation address of No. 1, Beichen West Road, Haidian District, Beijing, a preservation number of CGMCC No. 32897, and drug resistance to neomycin.

[0016] The application further provides application of the Lactobacillus kefiri subsp. kefiri IMAU13289 in preparation of fermented milk with excellent curd properties and excellent storage properties, and preparation of fermented milk with excellent curd properties and excellent storage properties, specifically:

[0017] 1) preheat milk to 65 DEG C, keep 65 DEG C for 15 min, continuously stir during the period and add white sugar with a mass fraction of 6.5% according to the mass of the milk, mix and homogenize, sterilize at 95 DEG C for 40 min, cool to 42 DEG C, and obtain a mixture of milk and white sugar;

[0018] 2) mix Lactobacillus kefiri subsp. kefiri IMAU13289 and a basic starter according to a volume ratio of 1:1, and obtain a fermentation starter;

[0019] 3) inoculate the fermentation starter obtained in step 2) into the mixture of milk and white sugar obtained in step 1), and perform fermentation at 42 DEG C until the pH is 4.5-4.6, stop the fermentation, and obtain fermented milk;

[0020] The inoculation amount of the fermentation starter in step 3) is calculated according to 4% of the volume fraction of the mixture of milk and white sugar.

[0021] Compared with the prior art, the application has the following advantages and technical effects:

[0022] The application discloses Lactobacillus kefiri subsp. kefiri with potential probiotic properties Lactobacillus kefiranofaciens subsp. kefiranofaciensIMAU13289, comprehensive evaluation of the growth performance of the strain, casein hydrolysis, beta-galactosidase activity, exopolysaccharide content, antioxidant activity, antibacterial activity, carbohydrate utilization ability, drug sensitivity, cell surface hydrophobicity, and found that its growth performance is good, the intestinal adhesion and colonization effect is strong, and can adapt to the extreme environment of gastrointestinal tract, fermentation characteristics are excellent, and has strong antioxidant capacity, and comprehensive analysis has potential probiotic characteristics. Especially wine-like lactobacillus wine-like subspecies IMAU13289 has the ability of high yield of exopolysaccharide, and strong lactic acid production capacity, and tolerance to gastrointestinal juice digestion, and has drug resistance to neomycin, especially can be used for preparing fermented milk with excellent coagulation characteristics and excellent storage characteristics. Therefore, the present application provides a wine-like lactobacillus wine-like subspecies IMAU13289 with new functions. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0024] Figure 1 The phylogenetic tree of wine-like lactobacillus wine-like subspecies IMAU13289 is shown in the figure;

[0025] Figure 2 The colony morphology of wine-like lactobacillus wine-like subspecies IMAU13289 is shown in the figure, wherein A is the colony morphology, and B is the gram microscope observation figure;

[0026] Figure 3 The growth curve of wine-like lactobacillus wine-like subspecies IMAU13289 is shown in the figure;

[0027] Figure 4 The acid production curve of wine-like lactobacillus wine-like subspecies IMAU13289 is shown in the figure;

[0028] Figure 5 The effect of different temperatures on the growth of wine-like lactobacillus wine-like subspecies IMAU13289 is shown in the figure;

[0029] Figure 6 The effect of different pH values on the growth of wine-like lactobacillus wine-like subspecies IMAU13289 is shown in the figure;

[0030] Figure 7 The OPN concentration standard curve is shown in the figure;

[0031] Figure 8 The tyrosine standard curve is shown in the figure;

[0032] Figure 9 Figure 7 is a standard curve of glucose;

[0033] Figure 10 Figure 8 is a graph of the proteolytic activity of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289;

[0034] Figure 11 Figure 9 is a graph of the antioxidant capacity determination results of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289;

[0035] Figure 12 Figure 10 is a graph of the fermentation time and viable cell count changes of the fermented milk prepared by Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, where the capital letters and small letters in the graph represent the significance of different times of the same sample and the same time of different samples, respectively, and A is significantly higher than B;

[0036] Figure 13 Figure 11 is a graph of the changes of the elastic factor, viscosity factor, solid-liquid balance value and flow factor of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in the process of preparing fermented milk by fermentation, where A is the elastic factor, B is the viscosity factor, C is the solid-liquid balance value, and D is the flow factor;

[0037] Figure 14 Figure 12 is a graph of the continuous pH of the fermented milk prepared by Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 and the pH during storage, where A is the continuous pH of the fermented milk prepared, and B is the pH during storage, and the capital letters and small letters in the graph represent the significance of different times of the same sample and the same time of different samples, respectively;

[0038] Figure 15 Figure 13 is a graph of the water holding capacity and viscosity of the fermented milk prepared by Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 during storage, where A is the water holding capacity of the fermented milk during storage, and B is the viscosity of the fermented milk during storage, and the capital letters and small letters in the graph represent the significance of different times of the same sample and the same time of different samples, respectively.

[0039] Deposit certificate

[0040] Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, in Latin is Lactobacillus kefiranofaciens subsp. kefiranofaciens The strain is preserved in the General Microbiological Center of China Microbial Culture Collection and Preservation Management Committee, located at No. 3, Beichen West Road, Chaoyang District, Beijing, on December 04, 2024, with the preservation number of CGMCC No. 32897. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be merely illustrative of certain aspects, features and embodiments of the present application, not a limitation thereof.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only exemplary of the various preferred embodiments and are not intended to be limiting on the scope of the application. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. Various embodiments of the present application will be described in detail, with reference to the drawings, wherein like reference numerals represent like parts, and each reference to technical literature in the specification is intended to include all patents, patent applications, scientific articles, and other technical literature cited therein.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the specification and any document incorporated herein by reference, the specification will control.

[0044] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the application disclosed herein. Each of the references recited in the specification is incorporated by reference herein in its entirety.

[0045] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0046] Sources of materials used in the present application: strain IMAU12319 is Lactobacillus kefiranofaciens (Lactobacillus kefiri) Lactobacillus kefiranofaciens IMAU12319, is from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No.31224; sodium chloride in phosphate buffer is purchased from Tianjin Huirong Chemical Technology Co., Ltd., potassium dihydrogen phosphate is purchased from Tianjin Beifang Tianyi Chemical Reagent Factory, sodium phosphate dibasic is purchased from Tianjin Damao Chemical Reagent Factory; API50CHL kit is purchased from Merieux (Suzhou) Bioproducts Co., Ltd.; LB liquid medium is purchased from Qingdao Gaosi Technology Park Haibo Biotechnology Co., Ltd.; whole milk is purchased from Inner Mongolia Mengniu Dairy Group Co., Ltd.; sucrose is purchased from Beijing Tangtai Technology Co., Ltd.; drug sensitivity paper is purchased from Bikeman Biotechnology Co., Ltd.; basic starter (PYS-010) (containingStretpococcus thermophilus and Lactobacillus bulgaricus ) purchased from Beijing Kedao Hengtong Biotechnology Co., Ltd.

[0047] The formula of the 11% skim milk medium used in the application is: 11 g of skim milk and 89 mL of distilled water. Sterilize at 115°C for 7 min, and cool down for standby.

[0048] The formula of the MRS liquid medium used in the application is: 10 g of soybean peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 1 g of Tween 80, 2 g of potassium phosphate dibasic, 5 g of anhydrous sodium acetate, 2 g of sodium citrate, 200 mg of magnesium sulfate heptahydrate, 54 mg of manganese sulfate pentahydrate, 1000 mL of distilled water, pH adjusted to 6.3, sterilized at 121°C for 15 min.

[0049] The 0.85% physiological saline used in the application is: 8.5 g of sodium chloride and 1000 mL of distilled water.

[0050] The formula of the simulated gastric juice used in the application is: 40 mL of sterilized PBS + 3.0 g / L pepsin (0.3%), then adjust pH to 2.5 (adjust with 0.1 mol / L HCL), filter with 0.22 μm filter membrane into a sterilized empty conical flask, and wait for use.

[0051] The formula of the simulated intestinal juice used in the application is: 40 mL of sterilized PBS + 1.0 g / L trypsin (0.1%) + 1.8% bile salt, then adjust pH to 8.0 (adjust with 0.1 mol / L NaOH), filter with 0.22 μm filter membrane into a sterilized empty conical flask, and wait for use.

[0052] Example 1

[0053] Strain isolation and identification process

[0054] Mongolia acid camel milk samples were gradient diluted and inoculated on MRS medium by spread plate method, and then cultured anaerobically at 37°C for 36h. Single colonies were picked from the plate and purified by plate streaking method to obtain pure culture. The purified single colonies were inoculated in MRS liquid medium for 24h culture, and then centrifuged at 12000r / min at 4°C for 5min. The bacterial cells were collected after washing twice with PBS to obtain bacterial slurry. The genomic DNA of the bacterial cells was extracted, and the 16S rRNA gene (SEQ ID NO. 1) was amplified by PCR technology. The amplified product was sequenced by using universal primers 27F / 1492R, wherein the forward primer was 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 phylogenetic tree was constructed by using bioinformatics software MEGA to analyze the phylogenetic position of the strain, and the phylogenetic tree diagram was as shown in Figure 1 The strain was preserved by using skimmed milk as a protective agent, and then divided into cryogenic tubes and stored in a -80°C ultra-low temperature refrigerator for long-term preservation.

[0055]

[0056] The frozen cryovials were taken out from the -80 °C freezer and placed in an ice box for thawing. The cryovials were inoculated with the preserved strain at a 2% inoculation volume in MRS liquid medium, and incubated at 37 °C for 24 h. The strain was subcultured for 3 generations to maximize its activity, and then used in subsequent experiments.

[0057] Example 2

[0058] I. Fermentation characteristics of the strain:

[0059] 1. Analysis of the coagulation characteristics of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289:

[0060] The time taken from inoculation to the appearance of coagulation was recorded as the coagulation time of the lactic acid bacteria, and the maximum time was 24 h. The activated Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 (strain IMAU13289) was inoculated into sterilized skim milk medium at a 2% volume fraction, and incubated at 37 °C for 12 h. The coagulation state of the strain was observed and recorded every 3 h. The activated strain IMAU12319 was inoculated into sterilized skim milk medium at a 2% volume fraction, and incubated at 37 °C for 12 h. The coagulation state of the strain was observed and recorded every 3 h.

[0061] Table 1 Coagulation of strain IMAU13289 at different time periods

[0062] ;

[0063] As shown in Table 1, the coagulation time of strain IMAU13289 was within 12 h, indicating that strain IMAU13289 has coagulation characteristics.

[0064] 2. Analysis of the acid production performance of strain IMAU13289:

[0065] Strain IMAU13289 was inoculated into skim milk at a 2% volume fraction, and incubated at 37 °C for 24 h. The pH value, titratable acidity, and coagulation time were measured. Strain IMAU12319 was inoculated into skim milk at a 2% volume fraction, and incubated at 37 °C for 24 h. The pH value, titratable acidity, and coagulation time were measured.

[0066] Table 2 Effect of fermentation of strain IMAU13289 on the quality of fermented milk

[0067] ;

[0068] Note: "+++" means that the curd is more solid, no whey precipitation, and has a unique flavor; "++" means that the curd is more solid, no whey precipitation, and has a fermented taste; "+" means that the curd is loose, less whey precipitation, and weak fermentation taste; " group is loose, whey precipitation is more serious, and has obvious fermented sour taste.

[0069] As shown in Table 2, strain IMAU13289 reached the fermentation end point after 11.3 h, and the pH value of the fermented milk of strain IMAU13289 at the fermentation end point was 4.23, and the titratable acidity was 98.7 °T, indicating that although the strain IMAU13289 had a slightly longer curd time, the acid production rate was fast.

[0070] Example 3

[0071] I. Results of simulated artificial gastrointestinal fluid tolerance test:

[0072] 1. pH 3.0 tolerance test:

[0073] The test bacteria (strain IMAU13289 or strain IMAU12319) were inoculated into MRS liquid medium with pH 3.0 at a volume fraction of 2%, and incubated at 37°C under anaerobic conditions for 24 h. The blank control was the corresponding pH MRS liquid medium without inoculation. The OD value was measured at 600 nm at 0 h and 24 h. The above fermented liquid was dipped with a disposable inoculation ring and streaked on MRS solid medium, and the growth of the plate was observed.

[0074] Table 3. Analysis of strain acid tolerance

[0075] ;

[0076] Note: The same column and different lowercase letters indicate significant differences (P < 0.05).

[0077] As shown in Table 3, strain IMAU13289 can survive under the condition of pH 3.0.

[0078] 2. 0.3% (w / v) bile salt tolerance test:

[0079] Strain IMAU13289 was inoculated into MRS liquid medium containing 0.3% bile salts at a volume fraction of 2%, and a disposable inoculation loop was used to take the liquid culture after the above culture and streaked on MRS solid medium with a 0.3% bile salt concentration, and the entire plate was cultured at 37°C under anaerobic conditions for 24h. The absorbance was measured at 600nm by UV spectrophotometer, and in order to ensure the accuracy and repeatability of the experiment, 3 parallel tests were repeated for each group, and whether the colony growth was observed. Strain IMAU12319 was inoculated into MRS liquid medium containing 0.3% bile salts at a volume fraction of 2%, and a disposable inoculation loop was used to take the liquid culture after the above culture and streaked on MRS solid medium with a 0.3% bile salt concentration, and the entire plate was cultured at 37°C under anaerobic conditions for 24h. The absorbance was measured at 600nm by UV spectrophotometer, and in order to ensure the accuracy and repeatability of the experiment, 3 parallel tests were repeated for each group, and whether the colony growth was observed.

[0080] Table 4 0.3% (w / v) bile salt tolerance test results

[0081] ;

[0082] Note: The same column indicates that there is a significant difference (P<0.05).

[0083] As shown in Table 4, strain IMAU13289 has certain bile salt tolerance under the condition of 0.3% (w / v) bile salt concentration.

[0084] 3. Analysis of the tolerance of the strain to artificial gastric and intestinal juice

[0085] The strains (strain IMAU13289 or strain IMAU12319) under acid stress and bile salt stress environment were continuously subcultured for three times to obtain the fermentation broth. Then, the supernatant was separated by centrifugation at a speed of 4000r / min for 10min. The precipitate was washed by centrifugation with phosphate buffered saline (PBS) treated by ultra-high pressure sterilization pot, and 5mL of sterilized PBS solution was added for centrifugal washing of the bacterial cells. The supernatant discarded twice was added to sterile PBS until washed for 3 times, and then 40mL of PBS solution was added to the bacterial slurry and mixed uniformly by blowing, and the concentration of the bacterial suspension was adjusted to 1.0×10 8 CFU / mL, which became the test bacterial solution.

[0086] 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 viable bacterial count was then determined. Next, 1000 μL of the bacterial culture after 3 hours of culture in simulated gastric fluid was mixed with 9 mL of simulated intestinal fluid. Samples were taken at 4 hours and 8 hours under anaerobic conditions at 37°C, and the viable bacterial count was counted at each time point. The calculation formula is as follows:

[0087] Artificial gastric fluid survival rate (%) = N1 / N0 × 100% (1);

[0088] Artificial intestinal fluid survival rate (%) = N2 / N1 × 100% (2);

[0089] Wherein, N0 represents the number of viable bacteria in the original bacterial solution at 0h (CFU / mL); N1 represents the number of viable bacteria in the artificial gastric fluid after 3h treatment (CFU / mL); and N2 represents the number of viable bacteria in the artificial intestinal fluid after 8h treatment (CFU / mL).

[0090] Table 5. Results of strain tolerance analysis to artificial gastrointestinal fluid.

[0091] ;

[0092] Note: The data in the table are mean (X) ± standard deviation (SD). Different lowercase letters in the same column indicate significant differences (P<0.05).

[0093] As shown in Table 5, the survival rates of different strains in gastric juice varied. Strain IMAU13289 showed a high survival rate of 86% after 3 hours of digestion, and still maintained a 52% survival rate after 11 hours, which was superior to strain IMAU12319. As probiotics, the presence of a certain number of live bacteria is a prerequisite for maintaining their function.

[0094] Example 4

[0095] I. Growth characteristics of strain IMAU13289:

[0096] 1. Morphological characteristics of strain IMAU13289:

[0097] like Figure 2 China A and Figure 2 As shown in Figure B, the single colony of strain IMAU13289 is round, small in diameter, slightly convex in the center, with neat edges, smooth surface, and transparent white color; it was confirmed to be a Gram-positive bacterium by observation under a Gram staining microscope.

[0098] 2. Growth curve and acid production performance analysis of strain IMAU13289:

[0099] The prepared test bacteria liquid was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and placed in a 37°C constant temperature incubator for culture. During the growth period, 200 μL of fermentation liquid was collected every 2 h (i.e. 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 h) to determine the optical density (OD) value of the bacterial suspension, and every 4 h (i.e. 0, 4, 8, 12, 16, 20, 24, 28, 32, 36 h) to determine the viable cell count and pH value.

[0100] Viable cell count determination: according to the range of total number of viable lactic acid bacteria, appropriate dilution tubes were selected, 1.0 mL of bacterial suspension was taken and placed in sterile petri dishes (two replicates for each dilution), and was poured into 15 mL of MRS solid medium cooled to 50°C, the petri dish was gently rotated for more than 20 times for mixing, after the agar was solidified, the plate was inverted and cultured anaerobically at 37°C for 48 h, if no colonies grew, the culture time could be extended to 72 h, after the culture ended, the number of colonies on the plate was counted, and the bacterial suspension was diluted to the point that the plate pouring operation needed to be completed within 15 min.

[0101] OD value determination: the bacterial suspension was thoroughly mixed, 200 μL was accurately taken with a pipette and injected into a 96-well enzyme-labeled plate, and the absorbance at 600 nm was determined.

[0102] pH value determination: the pH meter was used to determine the pH value at each time point.

[0103] The bacterial growth curve reflects the growth pattern of a single microbial population in a specific environment under liquid culture. The growth and acid-producing characteristics of strain IMAU13289 are shown in Figure 3 and Figure 4 . As shown in Figure 3 , strain IMAU13289 was in a lag phase from 0 to 2 h, and the OD 600nm value did not change significantly; after a 2 h delay, it entered the logarithmic growth phase, and the OD 600nm value and viable cell count rapidly increased from 4 h; then the OD 600nm growth rate gradually slowed down and tended to be stable, entering the stationary phase; the viable cell count peaked at 16 h, and the OD 600nm value tended to be stable after 18 h. As shown in Figure 4 , the pH of strain IMAU13289 did not change significantly from 0 to 2 h; from 2 to 14 h, it entered the logarithmic growth phase, and as the number of bacteria increased, the ability to produce lactic acid increased, and the pH decreased significantly; at 20 h, the pH of the strain was 3.93, and then it gradually stabilized; after 24 h, it entered the death phase, and the viable cell count decreased significantly, and the pH increased slightly. In summary, the overall growth status and acid-producing rate of strain IMAU13289 were good.

[0104] 3. Growth of strain IMAU13289 at different temperatures:

[0105] The activated bacterial solution was inoculated into 5 mL of MRS liquid medium at a volume fraction of 2%. Five temperature gradients were set, and the culture was carried out 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 strain IMAU13289.

[0106] Depend on Figure 5 It can be seen that the OD of the fermentation broth of strain IMAU13289 is... 600nm The OD value initially increased slowly and then decreased with increasing temperature. (OD value of fermentation broth for strain IMAU13289) 600nm The growth activity of the strain reached its maximum 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.

[0107] 4. Growth of strain IMAU13289 at different pH values:

[0108] The bacterial culture was inoculated into MRS liquid medium with initial pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 at a volume fraction of 2%, and then incubated at 37℃ for 24 h. The absorbance of the fermentation broth at 600 nm was then measured to investigate the effect of pH on the growth of the bacterial strain.

[0109] Depend on Figure 6 The results show that strain IMAU13289 can grow well in MRS liquid medium with pH values ​​ranging from 2 to 8. Specifically, growth is slow below pH 4.0; growth is good between pH 4.0 and 7.0; and bacterial density gradually decreases above pH 7.0. Overall, the optimal pH for the growth of strain IMAU13289 is 5.0, indicating that it thrives in a slightly acidic environment.

[0110] Example 5

[0111] I. Physiological and biochemical characteristics of strain IMAU13289:

[0112] 1. Identification of strain IMAU13289 API 50 CHL:

[0113] This experiment used 50 biochemical detection methods from the API 50CHL kit to identify and test the utilization of 49 sugars and their derivatives of strain IMAU13289.

[0114] Table 6. Determination of carbohydrate types available for strain IMAU13289

[0115] ;

[0116] Note: "+" means the reaction is positive, "-" means the reaction is negative.

[0117] As shown in the API 50CHL reaction results in Table 6, the strain IMAU13289 showed positive reaction to 6 kinds of carbohydrates, in which the strain IMAU13289 mainly utilized the following carbohydrates: D-galactose, D-glucose (GLU), N-acetyl-glucosamine (NAG), D-maltose (MAL), D-lactose (LAC), D-trehalose (TRE).

[0118] 2. β-galactosidase activity determination:

[0119] After 37°C, 24h culture of the strain IMAU13289 or the strain IMAU12319 in MRS liquid medium, the bacterial cells were collected by centrifugation (4°C, 8000r / min, 15min), and then the supernatant was obtained as crude enzyme solution by ultrasonic treatment (power 125W, working 3s, pause 8s, total time 15min). The total sugar content in the crude enzyme solution was determined by ultraviolet spectrophotometry.

[0120] When determining the β-galactosidase activity, first, 2.5mg / mL ONPG solution prepared with pH 6.5 PBS was placed in a 37°C water bath for 5min, and then 1mL ONPG solution and 1mL crude enzyme solution were sequentially added in a test tube, mixed well, and reacted at 37°C for 10min. After the reaction was completed, 4mL 500mmol sodium carbonate solution was quickly added, mixed well, and stood for 5min, and then the absorbance value of the solution at 420nm was determined. The blank determination was basically the same as the above process, except that the sodium carbonate solution was added before the crude enzyme solution to inactivate the reaction enzyme solution. At the same time, the standard curve of ortho-nitrophenol (ONP) was drawn (Figure 2), and the β-galactosidase activity in the sample was calculated according to the following formula: Figure 7

[0121] Enzyme activity (U / mL) = (c x v x d) / (t x v1) (3);

[0122] Wherein, c is the concentration of ONP; v is the total volume of the reaction solution; d is the dilution multiple of the sample; t is the reaction time; v1 is the volume of the reaction enzyme solution.

[0123] The amount of 1μg / mL ortho-nitrophenol (ONP) released per milligram of bacterial cell dry weight per minute was taken as the enzyme activity unit (U / 10 4 cells), which was used to measure the β-galactosidase activity. After ultrasonic crushing treatment of the bacterial cells of the test strain at different concentrations, the enzyme activity was detected by ONPG method.

[0124] Table 7 β-galactosidase activity

[0125] ​ ;

[0126] As shown in Table 7, the lactase activity of strain IMAU13289 was 2.93 U / 10 4 cell.

[0127] 3. Determination of proteolytic activity of strain IMAU13289:

[0128] A standard curve for casein hydrolysis activity reaction was constructed using o- phthaldehyde as a substrate (Fig. 3), which was used for subsequent evaluation of proteolytic activity. Figure 8

[0129] The activated strain IMAU13289 was inoculated into skim milk at a volume fraction of 2% inoculation amount and incubated at 37°C for 12 h, and skim milk without inoculum was set as a blank control. 2.5 mL of sample was accurately pipetted into a test tube, 0.5 mL of heavy water was added and mixed, followed by the addition of 0.5 mL of 0.75 mol / L trichloroacetic acid (TCA), vortex mixing, and room temperature standing for 10 min. The supernatant was obtained by filtration for standby. 150 μL of supernatant was taken into a test tube, 3 mL of OPA reagent was added, mixed thoroughly, and reacted at a specified temperature for 2 min, and then the absorbance at 340 nm was measured. Since the proteolytic activity in this system is equivalent to the tyrosine content, OD 340nm was taken as the OPA index, and the standard curve was used for comparative analysis.

[0130] As shown in Table 8, during the 0-6 h of culture, the OD 340nm absorbance changed gently; from 6 to 12 h, the OD 340nm absorbance showed a rapid upward trend; from 12 to 18 h, the upward rate was further intensified; after 18 h, the absorbance decreased slowly. Overall, the strain showed the strongest proteolytic activity in the logarithmic growth phase. After 24 h of culture, the final absorbance value of strain IMAU13289 was 0.3812, corresponding to a free amino acid content of 25.39 mg / L. Figure 10 4. Determination of exopolysaccharide of strain IMAU13289:

[0131] Based on the glucose standard curve drawn (Fig. 4), the subsequent exopolysaccharide determination work was carried out with reference to it.

[0132] Figure 9

[0133] ​​​The activated strain IMAU13289 or strain IMAU12319 was inoculated into 10 mL MRS medium at a volume fraction of 2% and incubated in a 37°C incubator for 24 h. The culture solution was centrifuged in a refrigerated centrifuge (8000 r / min, 4°C) for 20 min, and the supernatant was collected and cooled to room temperature after being boiled in a water bath for 5 min. Then, 80% trichloroacetic acid (TCA) was added to a final concentration of 10%, and the mixture was stirred thoroughly and left to stand overnight. Then, the mixture was centrifuged again at 8000 r / min for 20 min to remove the protein, and the supernatant was obtained. The supernatant was transferred to a conical flask, 3 times the volume of 95% ethanol was added, and the mixture was left to stand overnight at 4°C to precipitate the polysaccharide in the form of a flocculent precipitate. Then, the mixture was centrifuged at 8000 r / min for 20 min at 4°C, the supernatant was discarded, and the precipitate was dissolved in 5 mL distilled water. The solution was loaded into a dialysis bag and continuously stirred with a magnetic stirrer for 48 h, and the external aqueous solution was replaced every 8 h to ensure sufficient dialysis.

[0134] The precipitate obtained by dialysis was subjected to total sugar determination by the phenol-sulfuric acid method, with glucose as the standard. The extracellular polysaccharide content was quantified. The MRS liquid medium without inoculation was set as the blank control group. The final extracellular polysaccharide concentration of the strain was determined by subtracting the blank control value from the experimental value (unit: mg / L).

[0135] Table 8 Yield determination of extracellular polysaccharide of strain IMAU13289

[0136] ;

[0137] As shown in Table 8, the extracellular polysaccharide content of strain IMAU13289 was 151.69 mg / L, indicating that the strain had a high level of extracellular polysaccharide production.

[0138] Example 6

[0139] I. Probiotic properties of strain IMAU13289:

[0140] 1. Determination of antioxidant capacity of the strain:

[0141] (1) Determination of free radical scavenging rate of 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH):

[0142] 2 mL of cell suspension, cell-free extract, or fermentation broth was mixed with 1 mL of DPPH solution prepared with anhydrous ethanol, and the mixture was left to stand in the dark for 30 min. Then, the mixture was centrifuged at 10000 r / min for 10 min, and the supernatant was subjected to instrument zero adjustment. The absorbance value at 600 nm was measured using a UV spectrophotometer, and the value was recorded as B. At the same time, a control group was set up by replacing the DPPH-containing anhydrous ethanol with anhydrous ethanol, and the corresponding absorbance value was recorded as C. A blank group was also set up, and the absorbance value was recorded as A.

[0143] Cell suspension: Add 40 mL of PBS solution to the bacterial sludge and mix thoroughly by pipetting. Adjust the bacterial suspension concentration to 1.0 × 10⁻⁶. 8 CFU / mL.

[0144] Cell-free extract: Based on the cell suspension, the cells were sonicated in an ice bath (200W power, pulse for 30s / intermittent for 30s, for a total of 5min), then centrifuged at 9000r / min and 4℃ for 15min to collect the supernatant, and filtered through a 0.22μm filter membrane to remove bacterial cells.

[0145] Fermentation broth: The strain was inoculated into MRS broth and cultured for 36 hours. After centrifugation at 8000 r / min and 4℃ for 15 min, the supernatant was collected and filtered to remove bacteria.

[0146] Given that vitamin C (VC) possesses antioxidant activity and that this activity is positively correlated with its concentration, an aqueous VC 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 concentration in the aqueous VC solution increased, its DPPH scavenging rate increased accordingly. Subsequent calculations of relevant indicators were based on the following formulas:

[0147] DPPH free radical scavenging rate (%) = [1 - (AB) / C] × 100% (4);

[0148] The absorbance value of the blank group is A, the absorbance value of the cell suspension, cell-free extract or fermentation broth experimental group at 600 nm is recorded as B, and the absorbance value corresponding to anhydrous ethanol containing DPPH replaced by anhydrous ethanol is C.

[0149] Depend on Figure 11 The test results showed significant differences in the DPPH free radical scavenging rate of different solutions of strain IMAU13289. The DPPH scavenging efficiency of the positive control group (VC aqueous solution) reached 87%. For strain IMAU13289, the fermentation broth group showed a high DPPH free radical scavenging rate of 78%; the cell suspension group had a scavenging rate of 51%; and the cell-free extract group had a scavenging rate of 37%. The fermentation broth group showed the highest DPPH scavenging rate among the three, and it was closest to the DPPH scavenging rate of the positive control VC solution. In conclusion, strain IMAU13289 exhibited a strong overall DPPH free radical scavenging ability.

[0150] (2) Superoxide anion (O2) - Determination of scavenging ability:

[0151] Take 0.5 mL of cell suspension or 0.5 mL of cell-free extract or 0.5 mL of fermentation broth, add 1 mL of 3 mmol / L diethylenetriamine pentaacetic acid, 1 mL of 150 mmol / L pH 8.2 tris-hydroxymethyl methane hydrochloride and 1 mL of 1.2 mmol / L o-phenanthroline in turn, mix thoroughly, and then treat in a 25°C water bath for 10 min. After the water bath is over, measure the absorbance of the system at 325 nm. The calculation formula is:

[0152] Superoxide anion clearance rate = {1-[(G-F) / (E-D)]}x100% (5);

[0153] Where D represents the blank control absorbance, E represents the maximum absorbance, F represents the sample group absorbance, and G represents the sample blank absorbance.

[0154] By adding cell suspension or its cell-free extract, fermentation broth, the color change of colored intermediates reflects the generation and reduction of superoxide anion free radicals (O2 - ).

[0155] As shown in Figure 11 , the superoxide anion (O2 - ) clearance rate of the control group (VC) solution is 100%, among which the superoxide anion (O2 - ) clearance rate of the fermentation broth group of strain IMAU13289 is 82%, the superoxide anion (O2 - ) clearance rate of the cell suspension group of strain IMAU13289 is 51%, and the superoxide anion (O2 - ) clearance rate of the cell-free extract group of strain IMAU13289 is 21%.

[0156] (3) Determination of hydroxyl radical (-OH) clearance ability:

[0157] Measure 2 mL of cell suspension (or an equal amount of cell-free extract, fermentation broth), add 1 mL of 5 mol / mL salicylic acid ethanol solution, 1 mL of 5 mmol / L ferric sulfate solution, 1 mL of 3 mol / mL hydrogen peroxide solution and 5 mL of double distilled water in turn, mix well, and then treat in a 37°C water bath for 15 min. Then centrifuge at 4°C and 9000 r / min for 10 min to obtain the supernatant, measure the absorbance at 510 nm and record it as H, and use ultrapure water instead of the sample as a blank control, and the corresponding absorbance is recorded as I0. The calculation formula is:

[0158] Hydroxyl radical clearance rate (%) = [1-(H / I0)]x100% (6).

[0159] As shown in Figure 11As shown in the table 9, the surface hydrophobicity of bacteria is positively correlated with its adhesion and colonization in the organism, the stronger the hydrophobicity, the easier to achieve the process. The surface hydrophobicity of the strain IMAU13289 was determined, and the strain hydrophobicity was 30%-32%.

[0160] 2. Hydrophobicity test of the strain IMAU13289:

[0161] The surface hydrophobicity of the strain IMAU13289 was evaluated by carbon hydrophobic adsorption method. The bacterial slurry was collected by centrifugation at 4000 r / min for 5 min, washed twice with PBS buffer, resuspended in PBS, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL, and the initial absorbance J0 at 600 nm was measured.

[0162] 2 mL of xylene was mixed with an equal amount of bacterial suspension, vortexed for 10 min, and then left at room temperature for 30 min. The lower water phase was aspirated, and the absorbance J at 600 nm was measured. The formula is as follows:

[0163] Hydrophobicity (%) = (J-J0) / J0×100% (7).

[0164] Table 9. Results of hydrophobicity test of the strain IMAU13289

[0165] ;

[0166] As shown in the table 9, the surface hydrophobicity of bacteria is positively correlated with its adhesion and colonization in the organism, the stronger the hydrophobicity, the easier to achieve the process. The surface hydrophobicity of the strain IMAU13289 was determined, and the strain hydrophobicity was 30%-32%.

[0167] 3. Determination of antibacterial activity of the strain IMAU13289:

[0168] The antibacterial activity of the strain was determined by Oxford cup method. The strain was inoculated in LB solid medium and cultured for 12 h, then transferred to LB liquid medium and incubated at 37°C, 180 r / min for 12 h. The obtained fermentation broth was diluted 10 times and plated, and then incubated at 37°C for 24 h. Then, the supernatant with antibacterial activity was collected by centrifugation at 4000 r / min for 10 min for subsequent test.

[0169] In the antibacterial assay, the experimental group strain IMAU13289 was activated by LB solid and fermented by liquid shaking flask, and the supernatant was taken after treatment of the fermentation broth; the Oxford cup was placed in the medium containing the indicator bacteria Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922). Staphylococcus aureus ), Escherichia coli (Escherichia coli Listeria monocytogenes (Lm) Listeria monocytogenes Salmonella (S) Salmonella ) culture medium plate, add the supernatant, culture under the same conditions, and measure the diameter of the inhibition zone. In the blank group, the Oxford cup is added to the culture medium plate containing the indicated bacteria, MRS broth is added to the Oxford cup, and the culture is observed for the presence or absence of an inhibition ring.

[0170] Table 10 Determination results of the bacteriostatic performance of the strain

[0171] ;

[0172] As shown in Table 10, no inhibition ring was formed when only MRS broth was added in the blank control group, indicating that the composition of the culture medium does not interfere with the bacteriostatic determination. Strain IMAU13289 showed good bacteriostatic effect on the above four common pathogenic bacteria, especially the inhibition effect on Staphylococcus aureus was significant.

[0173] Example 7

[0174] Safety evaluation of strain IMAU13289:

[0175] A paper disc diffusion test was used to perform a drug sensitivity test on 26 kinds of antibiotics. First, the strain obtained by screening was activated, centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. The bacterial cells were resuspended in sterile saline solution, and the turbidity was accurately adjusted to 0.5 McFarland units. 100 μL of the bacterial suspension was uniformly coated on the surface of the MRS solid culture medium. After drying, the drug sensitivity paper was adhered to the plate coated with lactic acid bacteria by the paper disc method, and then placed in an anaerobic environment for culture. According to the size of the inhibition zone around the drug sensitivity disc, the sensitivity of the bacteria to each antibiotic was determined.

[0176] Table 11 Drug sensitivity test results

[0177] ;

[0178] Note: The diameter of the inhibition ring is judged as sensitive (S), intermediate (I), and resistant (R).

[0179] The test results of K-B drug sensitivity test on 20 commonly used antibacterial drugs are shown in Table 11. Strain IMAU13289 showed sensitive performance to 5 commonly used antibacterial drugs, and a large number of colonies were observed during culture. It showed moderate sensitivity to one of the antibacterial drugs, and an obvious inhibition ring was observed after 4 weeks of culture. It was highly sensitive to 14 antibiotics, no colonies were grown after 4 weeks of culture, and a large-diameter inhibition ring was formed, indicating that the strain had strong sensitivity to antibiotics.

[0180] Example 8

[0181] I. Application of strain IMAU13289 in fermented milk

[0182] The commercially available pure milk was preheated to 65℃ and stirred for 15 min, 6.5% white sugar was added slowly during the stirring period, and then homogenized (low pressure 50 bar, high pressure 190 bar), sterilized at 95℃ for 40 min, cooled at 42℃, to obtain a milk and white sugar mixture. Strain IMAU13289 was mixed with the base starter according to a volume ratio of 1:1 to obtain a fermentation starter, and the fermentation starter was inoculated into the milk and white sugar mixture according to a volume fraction of 4% of the milk and white sugar mixture to calculate the inoculum. The fermentation starter was inoculated into the milk and white sugar mixture for fermentation, and then placed in a constant temperature incubator at 42℃ until the pH was in the range of 4.5-4.6, and then the fermentation was stopped. The fermented milk was stored at 4℃, and the relevant indicators were measured after 4 weeks of storage.

[0183] 1. Determination of viable cell count of strain in fermented milk

[0184] The fermentation time and viable cell count of IMAU13289 fermented milk are shown in Table 1. Figure 12 During the experimental observation period, the viable cell count showed a trend of first decreasing and then increasing and tending to be stable. On the first day, the viable cell count was at a high level, reaching about 1.3×10 9 CFU / mL. By the 7th day, the viable cell count decreased significantly to about 1.2×10 9 CFU / mL. From the 7th day to the 14th day, the viable cell count increased to about 1.25×10 9 CFU / mL, and the viable cell count on the 21st day remained at a similar level to that on the 14th day, about 1.25×10 9 CFU / mL. The International Dairy Federation (IDF) stipulates that the viable cell count of lactic acid bacteria in fermented milk should reach more than 10 7 CFU / mL during storage, transportation and sales. The total viable cell count of the fermented milk sample during storage meets this standard.

[0185] After entering the storage period, it is possible that the growth and reproduction of IMAU13289 consumed the nutrients in the fermented milk, resulting in a decrease in the viable cell count. For Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, in addition to the insufficient nutrients in the fermented milk, they will produce a large amount of lactic acid when decomposing lactose, and excessive lactic acid will cause the acidity of the fermented milk to rise, thereby affecting the activity of microorganisms. Therefore, when the acidity is too high, the viable cell count will show a decreasing trend.

[0186] 2. Determination of micro-rheology of strain during fermentation

[0187] (1) Determination of elastic factor (EI) of strain

[0188] Elasticity factor is a parameter used to evaluate and analyze the elasticity characteristics of fermented milk. By reflecting the degree of change in the elasticity of fermented milk during fermentation, it reveals the changes in its structure and texture. The size of the EI value is closely related to the elastic state of the gel structure. When the EI value rises, it indicates that the gel network of fermented milk is more tightly combined, resulting in increased elasticity and a more stable state of the sample.

[0189] The changes in the EI value of IMAU13289 complex fermented milk during fermentation are shown in Figure A. Figure 13 As shown in Figure A, from the start of inoculation to the fermentation stage, the EI value of the fermented milk sample was relatively stable within the initial 0-8 h, with no significant difference (P>0.05). During this stage, the protein inside the fermented milk had not yet formed a network structure, resulting in high fluidity and low viscosity of the liquid properties. As the fermentation process continued, the 8h complex fermented milk reached the turning point first, i.e., the gel point, which corresponds to the sharp rise in the viscosity factor in the figure. This marks the first transition of the fermented milk system from liquid to solid, with increased intermolecular interaction and the beginning of the construction of the gel network structure. In the subsequent process, the sharp rise in the viscosity factor is similar to the near-vertical rise in the EI curve. After the EI value reaches the peak, it quickly enters the high elasticity stage, followed by a slight decline, and then approaches a relatively stable level from 14-16 h. This change reflects that during the acidification process of fermented milk, the original electrostatic repulsion balance is broken, prompting the rearrangement of casein particles to form a three-dimensional network structure, and then a stable gel structure is formed.

[0190] (2) Determination of strain viscosity factor (MVI):

[0191] The viscosity factor (MVI) is measured in microns, representing the static viscosity of the sample. The MVI value is positively correlated with the time of particle movement and the viscosity of the system under a given area. As shown in Figure B, Figure 13 During the fermentation of IMAU13289 fermented milk, the viscosity factor fluctuated frequently between 10 -6 and 10 -4 from 0-9 h, with low and unstable values. From 9-10 h, the viscosity factor sharply rose. From 10-16 h, it tended to be relatively stable and maintained at a high level, indicating that the casein micelles at this time aggregated into a stable three-dimensional network structure, and the gel system was stable, with the viscosity reaching the peak.

[0192] (3) Determination of strain solid-liquid balance value (SLB):

[0193] The solid-liquid balance value is used to represent the solid-liquid state of the fermented milk sample under static conditions at different fermentation times. When SLB=0, the sample is solid; when 0<SLB<0.5, the sample is mainly solid with initial liquid characteristics; when 0.5<SLB<1, the liquid characteristics gradually increase and the solid characteristics gradually decrease; and when SLB=1, the sample is liquid. As shown in Figure C, Figure 13As shown in Figure C, the SLB value of IMAU13289 fermented milk was high and fluctuating in the early stage of fermentation, indicating that the sample had strong fluidity and was in a liquid state. It reached the gel point at 9 hours, and the texture transformed into a gel structure. Subsequently, the SLB value increased slightly and then stabilized, meaning that the gel structure tended to stabilize, eventually becoming an elastic, viscous solid. During this process, acid production by the strain promoted the dissociation of casein aggregates, making the sample slightly viscous. At the end of fermentation, the SLB value was 0.50. Adding IMAU13289 can significantly improve the viscosity and elasticity of fermented milk, which is beneficial to product stability.

[0194] (4) Determination of bacterial flow factor (FI):

[0195] Flow factor (FI) is a key indicator for measuring the flowability of fermented milk samples, directly revealing the rate of particle movement within the sample. A higher FI value indicates faster particle movement, primarily exhibiting liquid characteristics, while a lower FI value indicates slower particle movement, more closely resembling solid characteristics. This corresponds to the SLB value. The FI value changes during the fermentation process of IMAU13289 fermented milk are shown below. Figure 13 As shown in Figure D, in the initial stage of fermentation, the FI value of the fermented milk is relatively high, accompanied by some fluctuations, indicating that the particles in the sample are moving at a relatively fast speed, resulting in increased overall fluidity of the sample. The gel point is reached at 9 hours, after which the FI value drops sharply, indicating that the sample is transitioning from a liquid to a solid state. As the fermentation process continues, dehydration and condensation gradually intensify, and the FI value of the samples remains stable at around 0.01, at which point the fermented milk samples exist in a stable solid state.

[0196] 3. Continuous pH measurement of the strain and pH measurement during storage:

[0197] The acid-producing capacity of lactic acid bacteria is a key indicator for measuring the growth and activity of bacterial strains. It reflects both their efficiency in utilizing nutrients and indirectly their adaptability to specific environments, making it 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, and can indicate microbial activity and affect product quality and taste. The continuous pH and storage pH values ​​for IMAU13289 fermented milk are shown in [reference needed]. Figure 14 China A and Figure 14 In product B, during storage (4℃), because the activity of lactic acid bacteria is not completely inhibited, they still ferment and produce acid using nutrients in the milk as a substrate, leading to a decrease in pH value. Figure 14 The pH changes of the fermented milk during storage are shown in Figure B. The pH value was 4.5 after 1 day of storage and 4.1 after 21 days. Studies have shown that the optimal drinking pH value for fermented milk during storage is above 4.0, and the pH value of this fermented milk during storage meets the requirements.

[0198] 4. Determination of water-holding capacity and viscosity of the strain during storage:

[0199] Water holding capacity reflects the ability of fermented milk to retain water, the higher the water holding capacity, the stronger the gel structure to free water binding force, the less whey separation, the higher the system stability, the better the product sensory and flavor retention. The change of water holding capacity of IMAU13289 fermented milk during storage is shown in Figure 15 A, the water holding capacity of fermented milk increased during 1-7d storage, and then decreased during 7-21d storage, which was due to the continuous decrease of pH value and the large amount of whey separation, which destroyed the tight gel structure. The water holding capacity of IMAU13289 fermented milk reached a peak of 66% after 7d storage, indicating that IMAU13289 can enhance the water holding capacity of fermented milk, improve the quality of fermented milk, and is beneficial to the storage of fermented milk.

[0200] Viscosity, as a core physical parameter representing the degree of fluid viscosity, not only reflects the continuous stability and organization state of fermented milk system, but also is a key indicator affecting its storage and transportation performance. High-quality fermented milk usually presents uniform organization structure, good stringiness, and low fluidity of semi-solid state. The change of viscosity of IMAU13289 fermented milk during storage is shown in Figure 15 B, the change trend is similar to the result of water holding capacity experiment, both showing an increase followed by a decrease. At the early stage of storage, the exopolysaccharide produced by lactic acid bacteria metabolism, or the pH value decrease induced casein precipitation and formed gel structure, and the coagulation particles gradually aggregated to promote the increase of viscosity; at the later stage of storage, with the continuous decrease of pH value, the dissolution of colloidal calcium phosphate destroyed the protein cross-linking structure, accompanied by whey separation, leading to the decrease of system viscosity. The viscosity of the fermented milk reached a peak of 1950mPa·s after 14d storage, indicating that IMAU13289 can significantly improve the viscosity of fermented milk, optimize the organization state, and improve the product texture.

[0201] 5. Changes in texture of the strain during storage:

[0202] Texture properties are important indicators for evaluating the quality of fermented milk, which are not only closely related to the taste, but also are the key basis for measuring the stability of product quality. The texture characteristics of fermented milk, such as hardness, consistency, cohesiveness and viscosity index, are independent of each other and are related to each other. Among them, the content of milk protein and fat dominates the hardness of the sample, and the type and amount of starter culture significantly affect the consistency and cohesiveness. Hardness, as the core representation parameter of gel structure strength, the higher the value, the more stable the gel network structure and the stronger the resistance to deformation; consistency is quantified by the force-time positive peak area during the simulation of stirring process; cohesiveness reveals the internal structure bonding strength of the sample. The texture detection data of IMAU13289 fermented milk during storage are shown in Table 12, the hardness of which reaches 33.176±3.11g, which reflects a relatively solid gel structure; the consistency is 246.653±16.27g·sec, which reflects that a larger force needs to be applied during the stirring process; the cohesiveness is-21.211±4.24g, the viscosity index is-20.907±5.00g·sec, and the particle index is 0. These quantitative indicators reveal the texture properties of the fermented milk from multiple dimensions, providing a scientific basis for its quality evaluation.

[0203] Table 12 Texture of fermented milk during storage

[0204] ;

[0205] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. *Lactobacillus malutulatus* subsp. *malutulatus* ( Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289, characterized in that, The Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 is deposited with the China General Microbiological Culture Collection Center on December 04, 2024, and the deposit address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32897.

2. Use of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in the preparation of a probiotic agent with high exopolysaccharide yield according to claim 1.

3. A high exopolysaccharide-producing probiotic agent, characterized in that, The Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 is deposited with the China General Microbiological Culture Collection Center on December 04, 2024, and the deposit address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32897.

4. Use of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in the preparation of a probiotic agent with rapid lactic acid production according to claim 1.

5. A fast lactic acid producing probiotic agent, characterized by, The Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 is deposited with the China General Microbiological Culture Collection Center on December 04, 2024, and the deposit address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32897.

6. Use of the Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 in the preparation of a probiotic agent resistant to gastrointestinal juice digestion according to claim 1.

7. A probiotic agent resistant to digestion by gastrointestinal fluids, characterized in that, The Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 is deposited with the China General Microbiological Culture Collection Center on December 04, 2024, and the deposit address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32897.

8. Use of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 as claimed in claim 1 for the preparation of a probiotic agent resistant to antibiotics, characterized in that, The drug-resistant probiotic agent is resistant to neomycin.

9. A resistant probiotic agent, characterized in that, The Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 is deposited with the China General Microbiological Culture Collection Center on December 04, 2024, and the deposit address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32897.

10. Use of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 as claimed in claim 1 for the preparation of a fermented milk having excellent renneting properties and excellent storage properties, characterized in that, The fermented milk with excellent renneting properties and excellent storage properties is prepared by: 1) Preheat the milk to 65℃, maintain 65℃ for 15 min, continuously stir during the period and add 6.5% white sugar by mass fraction based on the mass of the milk, mix and homogenize, sterilize at 95℃ for 40 min, and cool to 42℃ to obtain a mixture of milk and white sugar; 2) Mix Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU13289 and the base starter according to the volume ratio of 1:1 to obtain a fermentation inoculum; 3) inoculate the fermentation inoculum obtained in step 2) into the mixture of milk and white granulated sugar obtained in step 1), and stop the fermentation when the pH reaches 4.5-4.6 at 42°C to obtain fermented milk. The inoculation amount of the fermentation inoculum in step 3) is calculated according to 4% of the volume fraction of the mixture of milk and white granulated sugar.

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