Lactobacillus kefiranofaciens IMAU12319 and application thereof

Abstract

The invention discloses lactobacillus kefiranofaciens IMAU12319 and application thereof, belongs to the technical field of microorganisms, and particularly relates to lactobacillus kefiranofaciens IMAU12319 which is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 8, 2024, the preservation address is No. 3, No.1 yard, West Beichen Road, Chaoyang District, Beijing, and the number is CGMCC No.31224. The lactobacillus kefiranofaciens IMAU12319 has the advantages that the lactobacillus kefiranofaciens IMAU12319 can be used for preparing the lactobacillus kefiranofaciens IMAU12319, and the lactobacillus kefiranofaciens IMAU12319 can be used for preparing the lactobacillus kefiranofaciens IMAU12319; the invention also relates to an application of the lactobacillus kefiranofaciens IMAU12319 in preparation of a probiotic agent capable of resisting gastrointestinal digestion. The strain disclosed by the invention not only shows good gastrointestinal tract endurance capacity, but also shows relatively strong potential in the aspects of intestinal adhesion, oxidation resistance and bacteriostasis, and has a wide application prospect in leavening agent strains.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to kuvialobacillus IMAU12319 and applications thereof. Background Art

[0002] Probiotic functional products have received widespread attention due to their increasing popularity. Probiotics refer to a class of living microorganisms that are planted in the host body and have probiotic functions such as being able to regulate the host's intestinal mucosal system and maintain the balance of intestinal flora. Ideal probiotics basically meet the following conditions: they are acid-resistant and bile-resistant, have strong reproductive ability in the intestine, have strong adhesion to the host's digestive system, have no pathogenicity and harmful side effects on host cells, and can reduce the number of pathogenic microorganisms. In recent years, the role of probiotics has been studied more and more deeply. There have been a large number of comprehensive research results on the role of probiotics in the human body, how they affect the biological functions of the intestine, and the immune relationship between probiotics and intestinal mucosa.

[0003] Lactobacillus kefiranofaciens As a new type of edible probiotic strain, it has been proven to have the effects of adjusting metabolism, reducing allergies, enhancing immunity, anti-oxidation, improving diabetes and promoting brain health. Its beneficial properties are closely related to the specificity of the strain.

[0004] Based on the above-mentioned problem of lack of high-quality strains in the current probiotics industry, it is crucial to screen and discover probiotics with excellent characteristics. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes Kruskal Lactobacillus IMAU12319 and its application. This strain not only exhibits good gastrointestinal tolerance, but also shows strong potential in intestinal adhesion, anti-oxidation and antibacterial properties. The present invention broadens its application prospects in fermentation agent strains.

[0006] To achieve the above object, the present invention provides a kumis-like Lactobacillus ( Lactobacillus Kefiranofaciens )IMAU12319, the kumiss-like Lactobacillus IMAU12319 was deposited in the General Microbiology Center of China Culture Collection Administration on July 8, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO.31224.

[0007] The present invention also provides the use of the kumiss-like Lactobacillus IMAU12319 in preparing a probiotic agent resistant to gastrointestinal digestion.

[0008] The present invention also provides a probiotic agent resistant to gastrointestinal digestion, wherein the probiotic agent comprises the kumiss-like Lactobacillus IMAU12319.

[0009] The present invention also provides the use of the kumiss-like Lactobacillus IMAU12319 in preparing a probiotic with antioxidant activity.

[0010] The present invention also provides a probiotic with antioxidant activity, wherein the probiotic comprises the kuvialobacillus IMAU12319.

[0011] The present invention also provides the use of the kumiss-like Lactobacillus IMAU12319 in preparing a probiotic with antibacterial properties.

[0012] The present invention also provides a probiotic with antibacterial properties, wherein the probiotic comprises the kefir-like Lactobacillus IMAU12319.

[0013] The present invention also provides the use of the kumiss-like Lactobacillus IMAU12319 in preparing a probiotic with high beta-galactosidase activity.

[0014] The present invention also provides the use of the kumiss-like Lactobacillus IMAU12319 in preparing a probiotic agent with a carbohydrate metabolism function.

[0015] The present invention also provides the use of the kumiss-like Lactobacillus IMAU12319 in preparing a probiotic agent with high proteolytic activity.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention provides a strain of Kühm-like Lactobacillus IMAU12319 with excellent probiotic properties, the survival rate of which is 51.66% after 3h digestion in artificial gastric juice and 41.66% after 8h digestion in artificial intestinal juice; it has a positive reaction to 13 kinds of carbohydrates; its β-galactosidase activity is 3.12 (U / 10 4 cell); has good proteolytic activity; is resistant to 4 antibiotics and highly sensitive to 16 antibiotics; has good inhibitory effect on 4 common pathogens; has strong antioxidant capacity, with a DPPH scavenging capacity of 83%, a hydroxyl free radical scavenging capacity of 56%, and a superoxide anion (O2 - ) The clearance rate can reach 132%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0018] Figure 1 The colony morphology of Kühm-like Lactobacillus IMAU12319, wherein A is a Gram microscope observation image and B is the colony morphology; Figure 2 is a growth curve of Kühm-like Lactobacillus IMAU12319; Figure 3 is the acid production curve of Kumiss-like Lactobacillus IMAU12319; Figure 4 The effect of different temperatures on the growth of Kühm-like Lactobacillus IMAU12319; Figure 5 The effect of different pH values ​​on the growth of Kühm-like Lactobacillus IMAU12319; Figure 6 It is the standard curve of OPN concentration; Figure 7 is the standard curve of tyrosine; Figure 8 This is a graph showing the changes in the proteolytic activity of Kühm-like Lactobacillus IMAU12319; Fig. 9 is the standard curve of glucose concentration; Fig.10 This is a graph showing the results of the antioxidant capacity test of Kühm-like Lactobacillus IMAU12319; Fig.11 This is the phylogenetic tree of Kühm-like Lactobacillus IMAU12319. DETAILED DESCRIPTION

[0019] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] The V9 strain used in the present invention is derived from the lactic acid bacteria germplasm resource bank of Inner Mongolia Agricultural University.

[0025] Example 1 In 2020, the Key Laboratory of Dairy Biotechnology and Engineering of Inner Mongolia Agricultural University collected traditional fermented yogurt samples in Xilinhot, Inner Mongolia, and isolated and identified the lactic acid bacteria in them based on traditional pure culture technology. A total of 20 mL of samples were collected, and a protective agent (starch and calcium carbonate mass ratio of 50:1) was added to the enzyme-free sterile centrifuge tube sampling tube to protect the microorganisms in the sample. The collected samples were kept at a constant low temperature and stored in an ultra-low temperature refrigerator at -80°C.

[0026] The collected samples were thoroughly mixed, and 0.5 mL of the sample was taken and added to 0.85% saline to prepare a bacterial suspension for gradient dilution, and the mixing and shaking time of the bacterial suspension should be controlled to about 20 seconds. Then, a suitable dilution gradient bacterial suspension was selected for spreading culture, and 0.2 mL of the bacterial suspension was added to MRS and M17 culture media for spreading culture, and different culture temperatures (37°C / 30°C) and oxygen conditions (aerobic / anaerobic) were set for culture. After the colony was formed, the colony morphology (edge ​​outline, color, middle bulge and size, etc.) was recorded, and a single colony was picked for 2-3 times of streaking separation until a pure strain culture was obtained, and the pure culture was inoculated into a sterile liquid culture medium and cultured using the corresponding culture conditions.

[0027] In order to maintain sufficient nutrients in the culture environment of the strain and thus ensure the vitality of the strain, 0.5 mL of bacterial solution was aspirated every 24 hours and placed in the corresponding sterile liquid culture medium for subculture of the strain. The pure culture obtained after three subcultures of lactic acid bacteria was in the period of strongest vitality, and the bacteria were then collected for later use.

[0028] Collect 1 mL of the third generation bacterial solution after thorough washing and extract DNA. 260 / 280 The DNA of qualified strains was amplified by PCR, and the amplification primers were 16S rRNA bacterial universal primers: FA-27F: 5′-GAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO.2), RA-1495R: 5′-CTACGGCTACCTTGTTACGA-3′ (SEQID NO.3).

[0029]

[0030] The amplification conditions were: FA-27F 1.5μL, RA-1495R 1.5μL, DNA polymerase 0.5μL, dNTP 4μL, ddH2O35.5μL, template DNA 2μL, 10× buffer 5μL; 30 cycles of amplification at 94℃, 5min (pre-denaturation); 94℃, 1min (denaturation); 58℃, 1min (annealing); 72℃, 2min (extension); 72℃, 10min (terminal extension). After amplification, the PCR product was detected by agarose gel electrophoresis, and the electrophoresis bands were observed. If the bands were clear and without tailing, it indicated that the PCR product met the requirements for subsequent sequencing.

[0031] The PCR amplification products that meet the sequencing requirements were kept in a low temperature state and sent to Shanghai Paisonno Biotechnology Co., Ltd. for sequencing. The sequences obtained by sequencing were spliced ​​and quality tested using Seqman. The qualified strain sequences were compared with the known strain sequences uploaded in NCBI (https: / / www.ncbi.nlm.nih.gov) to preliminarily determine the taxonomic status of the strains. Then the strain type strain sequence was downloaded from the online website LPSN, and the phylogenetic tree of the isolated strains and the type strains was constructed using MEGA software, and the strains were identified by combining the NCBI database comparison and phylogenetic relationship.

[0032] The preserved strains were screened to obtain the strain Lactobacillus kumiss-like ( Lactobacillus kefiranofaciens )IMAU12319, phylogenetic tree between kumiss-like Lactobacillus IMAU12319 strain and closely related species Fig.11 shown.

[0033] Example 2 Tolerance test of simulated gastrointestinal fluid Preparation of artificial gastric juice: Take 20mL of 1mol / L HCl, adjust the pH to 2.0 with distilled water and 1mol / L NaOH solution, then add 0.3% pepsin to dissolve it, filter with a 0.22μm sterile filter membrane to obtain artificial gastric juice, which can be used immediately after preparation.

[0034] Preparation of artificial intestinal fluid: Weigh 0.27 g of KH2PO4, dissolve it in 20 mL of distilled water, adjust the pH to 6.8 with 1 mol / L NaOH solution, then add 0.1% trypsin, dissolve and mix thoroughly, filter with a 0.22 μm sterile filter membrane to obtain artificial intestinal fluid, which can be used immediately after preparation.

[0035] The method is as follows: 8 strains were subcultured three times, and the fermentation broth and supernatant were separated under centrifugal conditions of 4000r / min for 10min. The culture was sterilized by centrifugation and washed three times with PBS sterilized by ultra-high pressure sterilization pot, and the culture was prepared into a suspension with 5mL of saline. After diluting the suspension 10 times, the number of live bacteria in the starting fermentation broth at 0h was counted by selecting an appropriate concentration. 1mL of the suspension was mixed with 9mL of simulated gastric fluid with a pH of 2.5, and anaerobically fermented at 37℃ for 3h, and the number of live bacteria was counted. 1mL of simulated gastric fluid cultured for 3h was combined with 9mL of simulated intestinal fluid, and samples were collected at 4h and 8h under anaerobic conditions at 37℃, and the number of live bacteria was counted. The calculation formula is as follows: Survival rate in artificial gastric juice (%) = N1 / N0 × 100%; Survival rate in artificial intestinal fluid (%) = N2 / N1 × 100%; In the formula, N0 represents the number of viable bacteria in the original bacterial solution (CFU / mL); N1 represents the number of viable bacteria in gastric juice cultured for 3 hours (CFU / mL); N2 represents the number of viable bacteria in intestinal juice cultured for 4 hours and 8 hours (CFU / mL).

[0036] Table 1 Test results of resistance to simulated artificial gastrointestinal fluid ; Note: The data in the table are mean values. ) ± standard deviation (SD), different lowercase letters in the same column indicate significant differences (P<0.05).

[0037] As shown in Table 1, the survival rate of IMAU12319 (Kumiss-like Lactobacillus IMAU12319) in artificial gastrointestinal fluid at pH 2.0 for 3 hours was 51.66%. After 8 hours of digestion in artificial intestinal fluid, the survival rate of IMAU12319 was 41.66%, which was closer to the control bacteria V9, indicating that IMAU12319 had good gastrointestinal fluid tolerance.

[0038] Example 3 Study on the growth characteristics of Kumiss-like Lactobacillus IMAU12319 1. Morphological characteristics of strains The method is as follows: streak IMAU12319 (kumiss-like Lactobacillus IMAU12319) on MRS solid culture medium, let it stand at 37°C for 24 hours, take a single colony, and observe its staining changes and morphological characteristics using Gram staining.

[0039] like Figure 1 As shown in B, the single colony of strain IMAU12319 showed a typical round shape, relatively small diameter, slight central bulge, neat edges, smooth surface, and milky white color. Figure 1As shown in A, Gram-positive lactobacilli were confirmed by Gram microscopy.

[0040] 2. Growth curve and acid production curve of strains The method is as follows: strain IMAU12319 was cultured continuously in MRS medium at 37°C for 36 h, and viable bacteria were counted and pH was measured every 2 h.

[0041] like Figure 2 As shown, the strain IMAU12319 was in the growth retardation period from 0 to 4 hours. 600nm There was no significant change in activity under the condition of OD 2. 600nm The value and viable count began to increase rapidly at 14 h; subsequently, OD 600nm The growth rate of IMAU12319 gradually slowed down, gradually stabilized, and entered the stable period. At 16h, the number of viable bacteria of IMAU12319 reached the peak value, and OD 600nm There was a clear downward trend after 18 hours.

[0042] like Figure 3 The pH curve shown shows that the pH of the strain hardly changes between 0 and 2 hours; within 2 to 14 hours, the strain reaches the logarithmic growth stage, and as the number of bacteria increases, its ability to produce lactic acid significantly increases, and the pH decreases significantly; at 20 hours, the pH value of the strain is 4.34, and then the pH value gradually stabilizes; after 24 hours, the strain enters the death stage, during which the number of live bacteria significantly decreases, and there is a slight increase in pH. Therefore, the overall growth and acid production rate of strain IMAU12319 are good.

[0043] 3. Optimal growth temperature of strains The method is as follows: the activated bacterial solution is inoculated into 5 mL of MRS liquid culture medium at a 2% inoculation volume, and cultured at a constant temperature for 24 h at 5 temperature gradients, and then the absorbance of the fermentation broth at 600 nm is measured.

[0044] The results are as follows Figure 4 As shown in Figure 2, the biomass of strain IMAU12319 increased with the increase of temperature and then showed a downward trend. 600nm The value slowly decreases with the increase of temperature. The strain IMAU12319 grows fastest and best at 30℃, followed by 37℃. When the temperature exceeds 55℃, the growth of the strain is inhibited and it does not grow. This shows that high temperature has a certain inhibitory effect on its growth vitality.

[0045] 4. Optimal pH of the strain The method is as follows: inoculate the bacterial liquid at an inoculum size of 2% into MRS liquid culture medium with an initial pH value of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, culture at 37°C for 24 hours, and measure the absorbance of the fermentation liquid at 600nm.

[0046] The results are as follows Figure 5 As shown, strain IMAU12319 has a good proliferation ability at around pH 6.0 and is suitable for growth under weakly acidic conditions.

[0047] Example 4 Study on biochemical characteristics of kumiss-like Lactobacillus IMAU12319 1.API 50CHL test The method is as follows: According to the API 50CHL lactic acid bacteria identification standard, the strain was subjected to a sugar fermentation test using the API 50CHL kit (purchased from BioMérieux, France) to identify the test strain's metabolism of 49 carbohydrates. The operation was performed according to the instructions.

[0048] Table 2 Carbohydrate fermentation determination results ; ;

[0049] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0050] The results are shown in Table 2. The strain IMAU12319 showed positive reactions to 13 carbohydrates. The strain IMAU12319 was able to utilize the following carbohydrates: D-galactose (GAL), D-glucose (GLU), D-fructose (FRU), D-mannose (SAC), N-acetyl-glucosamine (NAG), arbutin (ARB), salicyclic alcohol (SAL), D-maltose (MAL), D-lactose (LAC), D-melibiose (MEL), D-sucrose (SAC), D-trehalose (TRE), and D-raffinose (RAF).

[0051] 2. β-galactosidase activity of strains Preparation of crude enzyme solution: The activated strain was cultured in MRS liquid medium at 37°C for 24 h, and the bacteria were collected under centrifugal conditions (4°C, 8000 r / min, 15 min) and subjected to ultrasonic treatment to obtain the supernatant.

[0052] The method is as follows: Place ONPG solution (2.5 mg / mL, prepared with pH 6.5 phosphate buffer) in a water bath at 37°C for 5 minutes. Add 1 mL of ONPG solution and 1 mL of crude enzyme solution to the test tube, mix well, and react at 37°C for 10 minutes. After the reaction, quickly add 4 mL of 500 mmol sodium carbonate solution, mix well and let stand for 5 minutes, and measure the absorbance of the solution at 420 nm. The blank determination method is basically the same as the above operation. In order to inactivate the reaction enzyme solution, sodium carbonate solution is added before adding the crude enzyme solution. At the same time, draw a standard curve for o-nitrophenol (ONP), see Figure 6 The β-galactosidase activity in the sample was calculated according to the following formula: Enzyme activity (U / mL) = (c×v×D) / (t×v1); 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.

[0053] Table 3 β-galactosidase activity test results ; 3. Determination of proteolytic activity of strains The method is as follows: the activated IMAU12319 strain is inoculated into skim milk at a 2% inoculation rate and cultured at 37°C for 12 hours. The blank control group is the corresponding milk sample without inoculum. Accurately draw 2.5 mL of the sample into the tube, then add 0.5 mL of deionized water and mix, then add 0.5 mL of 0.75 mol / L TCA, mix in a vortex, keep at room temperature for 10 minutes, filter, and obtain the supernatant for use. Take 150 μL of the supernatant and add it to a test tube, then add 3 mL of OPA reagent, mix well, react at standard temperature for 2 minutes, and measure the absorbance at 340 nm. Compare with the standard curve Figure 7 Correspondingly, the proteolytic activity is considered to be equal to the tyrosine content, expressed as OD 340nm For the OPA index, direct comparison can also be made.

[0054] The results are as follows Figure 8 As shown, the strain had the strongest proteolytic activity in the logarithmic growth phase. The final photometric value of strain IMAU12319 was 0.4936 at 24h, and the corresponding free amino acid was 33.09mg / L.

[0055] 4. Determination of Extracellular Polysaccharides Table 4 Determination of extracellular polysaccharide content ; As shown in Table 4, the extracellular polysaccharide content of strain IMAU12319 was 52.79 mg / L. Studies have found that the extracellular polysaccharide content of lactic acid bacteria in synthetic and semi-synthetic culture media is usually in the range of 40-600 mg / L, and IMAU12319 is consistent with this conclusion.

[0056] Example 5 Safety evaluation of kefir-like Lactobacillus IMAU12319 The method is as follows: Use the paper diffusion test method to conduct drug sensitivity tests on 26 antibiotics. Activate the strains obtained by screening, centrifuge at 4000r / min for 10min, discard the supernatant, suspend the bacteria in sterile salt solution, and adjust its turbidity to 0.5 McFarland units. Spread 100μL of the bacterial suspension evenly on the MRS solid culture medium, let it dry, and then use the paper method to adhere it to the plate coated with lactic acid bacteria for anaerobic culture. Determine the sensitivity of the bacteria to the antibiotics based on the size of the inhibition zone around the drug sensitivity sheet.

[0057] Table 5 Results of drug sensitivity test ; ; Note: The criteria for judging the diameter of the inhibition zone are sensitive (S), intermediate (I), and resistant (R).

[0058] The results are shown in Table 5. The results of the drug sensitivity test of IMAU12319 showed that the strain IMAU12319 was resistant to 4 antibiotics and was extremely sensitive to 16 antibiotics.

[0059] Example 6 Antibacterial ability determination The method is as follows: The Oxford cup method was used to conduct an antibacterial test on the strain, and the antibacterial ability of IMAU12319 against four common foodborne pathogens (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium) was determined.

[0060] Table 6 Determination results of antibacterial properties ; As can be seen from Table 6, IMAU12319 has good inhibitory effects on these four common pathogens, and has the best antibacterial ability against Staphylococcus aureus. The diameter of the inhibition zone of IMAU12319 is 23.84 mm, and the antibacterial effect is good.

[0061] Example 7 Evaluation of probiotic properties 1. Antioxidant activity study DPPH free radical scavenging ability assay The method is as follows: 2 mL of cell suspension or cell-free extract, fermentation broth and other solutions are mixed with 1 mL of DPPH anhydrous ethanol, placed in a place without light for 30 minutes, and then centrifuged at 10000r / min for 10 minutes, the supernatant solution is extracted, adjusted to zero, and the OD is detected using a UV spectrophotometer. 600nm The value is expressed as B; the control (using anhydrous ethanol to replace DPPH) is expressed as C; the absorbance value of the blank group is A. Vitamin C (Ascorbic acid, VC) has a certain antioxidant effect, and the content is proportional to the antioxidant effect. VC aqueous solution is used as a positive control for DPPH scavenging rate. The results show that the scavenging rate of DPPH in VC aqueous solution increases with the increase of vitamin C content. The calculation is based on the following equation: DPPH free radical scavenging rate (%) = [1-(AB) / C] × 100%.

[0062] The results are as follows Fig.10 As shown, the DPPH free radical scavenging ability of strain IMAU12319 in the fermentation broth group can reach 83%, the DPPH free radical scavenging ability of strain IMAU12319 in the cell suspension group can reach 64%, and the DPPH free radical scavenging ability of strain IMAU12319 in the cell-free extract group can reach 58%. Among the three, the DPPH scavenging rate of the fermentation broth was the highest, and the DPPH scavenging rate of the fermentation broth was closest to the DPPH scavenging rate of the VC solution in the control group. IMAU12319 showed a high DPPH scavenging ability.

[0063] Determination of hydroxyl radical scavenging ability The method is as follows: add 1mL of 5mol / mL salicylic acid ethanol solution, 1mL of 5mmol / L ferric sulfate solution, 1mL of 3mol / mL hydrogen peroxide solution and 5mL of secondary evaporated water to 2mL of cell suspension (or 2mL of cell-free extract or fermentation broth), and mix them thoroughly. After 15 minutes in a 37℃ water bath, centrifuge at 4℃, 9000r / min, 10min to obtain the supernatant. Measure its 510nm absorbance and indicate it as B; the blank control (using ultrapure water instead of the sample to be tested) is indicated by A. The calculation formula is: Hydroxyl free radical scavenging rate (%) = [1-(B / A0)] × 100%.

[0064] The results are as follows Fig.10 As shown, the hydroxyl radical scavenging rate of strain IMAU12319 in the fermentation broth group can reach 56%, the hydroxyl radical scavenging rate of strain IMAU12319 in the cell suspension group can reach 42%, and the hydroxyl radical scavenging rate of strain IMAU12319 in the cell-free extract group can reach 22%. IMAU12319 showed good hydroxyl radical scavenging activity.

[0065] Superoxide anion (O2 - )Determination of scavenging ability The method is as follows: add 1 mL of 3 mmol / L diethylenetriaminepentaacetic acid, 1 mL of 150 mmol / L trimethylolmethane hydrochloride (pH 8.2), and 1 mL of 1.2 mmol / L pyrogallol to 0.5 mL of cell suspension (or 0.5 mL of cell-free suspension and fermentation broth), respectively, incubate in a water bath at 25°C for 10 min, and measure the absorbance at 325 nm after bathing.

[0066] The results are as follows Fig.10 As shown, different strains reacted with superoxide anion (O2 - ) showed significant differences in superoxide anion scavenging ability. The superoxide anion scavenging rate of the control group (VC) solution was 100%, among which the superoxide anion scavenging rate of the strain IMAU12319 in the fermentation broth group could reach 132%, the superoxide anion scavenging rate of the strain IMAU12319 in the cell suspension group could reach 78%, and the superoxide anion scavenging rate of the strain IMAU12319 in the cell-free extract group could reach 35%. IMAU12319 showed an excellent superoxide anion scavenging rate.

[0067] 2. Results of the cell surface hydrophobicity test The method is as follows: the surface hydrophobicity of the bacteria is determined by hydrocarbon adsorption method, the bacteria activated to the third generation are centrifuged at 4000r / min for 5min, the bacterial sludge is collected, washed twice with PBS buffer, and then suspended in PBS, and the liquid is adjusted to 1×10 8 CFU / mL, measure the initial absorbance A0 at 600nm. Mix 2mL of xylene with 2mL of bacterial suspension, vortex for 10min, and let it stand in the room for 30min. Take the lower aqueous phase and measure the absorbance A at 600nm. The formula is as follows: Hydrophobicity (%) = (A-A0) / A0×100%.

[0068] Table 7 Hydrophobicity test results ; The results are shown in Table 7. Good hydrophobicity indicates high intestinal adhesion. The hydrophobicity of strain IMAU12319 can be as high as 43.26%. It has good hydrophobicity and self-aggregation ability and can be used as a potential probiotic.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens )IMAU12319, characterized in that The kumiss-like Lactobacillus IMAU12319 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 8, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 31224.

2. Use of the kumiss-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic agent resistant to gastrointestinal digestion.

3. A probiotic agent resistant to gastrointestinal digestion, characterized in that: The probiotic comprises the kefir-like Lactobacillus IMAU12319 according to claim 1.

4. Use of the kefir-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic with antioxidant activity.

5. A probiotic with antioxidant activity, characterized in that: The probiotic comprises the kefir-like Lactobacillus IMAU12319 according to claim 1.

6. Use of the kumiss-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic with antibacterial properties.

7. A probiotic with antibacterial properties, characterized in that: The probiotic comprises the kefir-like Lactobacillus IMAU12319 according to claim 1.

8. Use of the kumquat-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic with high β-galactosidase activity.

9. Use of the kumquat-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic having the function of metabolizing carbohydrates.

10. Use of the kumquat-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic with high proteolytic activity.

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