Lactobacillus kefiranofaciens IMAU12319 and its applications

Abstract

The present invention discloses Lactobacillus kefiranofaciens IMAU12319 and its application, belonging to the technical field of microorganisms, and particularly relates to Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) IMAU12319, which was deposited on July 8, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the accession number being: CGMCC No. 31224. The application of Lactobacillus kefiranofaciens IMAU12319 in the preparation of probiotic agents resistant to gastrointestinal digestion. The bacterium of the present invention not only exhibits good gastrointestinal tolerance ability, but also shows strong potential in intestinal adhesion, antioxidant and antibacterial aspects, and has broad application prospects in starter cultures.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to Lactobacillus kefiranofaciens IMAU12319 and its application. Background Art

[0002] Probiotic functional products have received extensive attention due to their increasing popularity. Probiotics refer to a class of live microorganisms implanted in the host body, and also have probiotic functions such as being able to regulate the host intestinal mucosal system and maintain the balance of the intestinal flora. Ideal probiotics basically meet the following conditions: having the characteristics of acid resistance and bile salt resistance, having strong reproductive ability in the intestine, having a strong adhesion effect on the host digestive system, having no pathogenicity and harmful side effects on host cells, and being able to reduce the number of pathogenic microorganisms. In recent years, the role of probiotics has been studied more and more deeply, and there have been a large number of comprehensive research results on the role of probiotics in the human body, how to affect the biological functions of the intestine, and the immune relationship between probiotics and the 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, antioxidant, improving diabetes, and promoting brain health, and its beneficial characteristics are closely related to the specificity of the strain.

[0004] Based on the current problem of lack of excellent strains in the probiotic industry as described above, it is crucial to screen and excavate probiotics with excellent characteristics. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes Lactobacillus kefiranofaciens IMAU12319 and its application. This strain not only exhibits good gastrointestinal tolerance ability, but also shows strong potential in intestinal adhesion, antioxidant and antibacterial aspects. The present invention broadens its application prospect in starter cultures.

[0006] To achieve the above object, the present invention provides a Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) IMAU12319, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 8, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO. 31224.

[0007] The present invention also provides the application of the Lactobacillus kefiranofaciens IMAU12319 in the preparation of probiotic agents resistant to gastrointestinal digestion.

[0008] The present invention also provides a probiotic agent resistant to gastrointestinal digestion, and the probiotic agent includes the Lactobacillus kefiranofaciens IMAU12319.

[0009] The present invention also provides the use of Lactobacillus kefiranofaciens IMAU12319 in the preparation of a probiotic agent having antioxidant activity.

[0010] The present invention also provides a probiotic agent having antioxidant activity, wherein the probiotic agent comprises Lactobacillus kefiranofaciens IMAU12319.

[0011] The present invention also provides the use of Lactobacillus kefiranofaciens IMAU12319 in the preparation of a probiotic agent having antibacterial property.

[0012] The present invention also provides a probiotic agent having antibacterial property, wherein the probiotic agent comprises Lactobacillus kefiranofaciens IMAU12319.

[0013] The present invention also provides the use of Lactobacillus kefiranofaciens IMAU12319 in the preparation of a probiotic agent having high β-galactosidase activity.

[0014] The present invention also provides the use of Lactobacillus kefiranofaciens IMAU12319 in the preparation of a probiotic agent having the function of metabolizing carbohydrates.

[0015] The present invention also provides the use of Lactobacillus kefiranofaciens IMAU12319 in the preparation of a probiotic agent having high protein hydrolysis activity.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] The present invention provides a strain of Lactobacillus kefiranofaciens IMAU12319 with excellent probiotic characteristics. Its survival rate is 51.66% after 3 hours of digestion in artificial gastric juice and 41.66% after 8 hours of digestion in artificial intestinal juice; it shows positive reactions to 13 kinds of carbohydrates; its β-galactosidase activity is 3.12 (U / 10 4 cell); it has good protein hydrolysis activity; it is resistant to 4 kinds of antibiotics and highly sensitive to 16 kinds of antibiotics; it has good inhibitory effects on 4 common pathogenic bacteria; it has strong antioxidant ability, and its DPPH scavenging ability can reach 83%, its hydroxyl radical scavenging ability can reach 56%, and its superoxide anion (O 2 - ) scavenging rate can reach 132%. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 Colony morphology of Lactobacillus kefiranofaciens IMAU12319, where A is the observation diagram under a Gram microscope and B is the colony morphology;

[0020] Figure 2 Growth curve of Lactobacillus kefiranofaciens IMAU12319;

[0021] Figure 3 Acid production curve of Lactobacillus kefiranofaciens IMAU12319;

[0022] Figure 4 Effect of different temperatures on the growth of Lactobacillus kefiranofaciens IMAU12319;

[0023] Figure 5 Effect of different pH values on the growth of Lactobacillus kefiranofaciens IMAU12319;

[0024] Figure 6 Standard curve of OPN concentration;

[0025] Figure 7 Standard curve of tyrosine;

[0026] Figure 8 Change diagram of proteolytic activity of Lactobacillus kefiranofaciens IMAU12319;

[0027] Figure 9 Standard curve of glucose concentration;

[0028] Figure 10 Determination result diagram of antioxidant capacity of Lactobacillus kefiranofaciens IMAU12319;

[0029] Figure 11 Phylogenetic tree diagram of Lactobacillus kefiranofaciens IMAU12319. Detailed implementation manners

[0030] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise specified, 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 invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0035] The V9 strain used in the present invention is derived from the Germplasm Resource Bank of Lactic Acid Bacteria, Inner Mongolia Agricultural University.

[0036] Example 1

[0037] In 2020, the Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University collected traditional fermented koumiss samples in Xilingol City, Inner Mongolia, and isolated and identified the lactic acid bacteria therein based on traditional pure culture techniques. A total of 20 mL of the sample was collected, and a protective agent (the mass ratio of starch to calcium carbonate was 50:1) was added to the enzyme-free and sterile centrifuge sampling tube to protect the microorganisms in the sample. The collected sample was kept at a constant low temperature and stored in an ultra-low temperature refrigerator at -80 °C.

[0038] Fully mix the collected samples, pipette 0.5 mL of the sample into 0.85% physiological saline to prepare a bacterial suspension for gradient dilution, and the mixing and shaking time of the bacterial suspension should be controlled for about 20 s. Then select the bacterial suspension with an appropriate dilution gradient for spread plating culture. Pipette 0.2 mL of the bacterial suspension onto MRS and M17 media for spread plating culture, and set different culture temperatures (37 °C / 30 °C) and oxygen conditions (aerobic / anaerobic) for culture. After colony formation, record the colony morphology (edge contour, color, central elevation, size, etc.), pick single colonies for streak isolation 2 - 3 times until a pure strain culture is obtained, and inoculate the pure culture into a sterile liquid medium for culture under the corresponding culture conditions.

[0039] To maintain sufficient nutrients in the strain culture environment and thus ensure the vitality of the strain, pipette 0.5 mL of the bacterial liquid into the corresponding sterile liquid medium every 24 h for subculture of the strain. The pure culture obtained after 3 subcultures of lactic acid bacteria is in the strongest vitality period, and then collect the bacterial cells for standby.

[0040] Collect 1 mL of the third-generation bacterial liquid after thorough washing of the bacteria, and extract DNA. The OD of the DNA 260 / 280 is considered qualified when it is between 1.8 and 2.0. Amplify the qualified strain DNA by PCR, and the amplification primers are the universal primers for 16S rRNA bacteria. The primers are: FA-27F: 5′-GAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO.2), RA-1495R: 5′-CTACGGCTACCTTGTTACGA-3′ (SEQ ID NO.3).

[0041]

[0042] The amplification conditions were as follows: 1.5 μL of FA-27F, 1.5 μL of RA-1495R, 0.5 μL of DNA polymerase, 4 μL of dNTP, 35.5 μL of ddH 2 O, 2 μL of template DNA, and 5 μL of 10× buffer; pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min; annealing at 58°C for 1 min; extension at 72°C for 2 min; final extension at 72°C for 10 min, and amplification was carried out for 30 cycles under these conditions. After the amplification was completed, the PCR products were detected by agarose gel electrophoresis, and the bands in the electrophoresis pattern were observed. If the bands were clear and there was no trailing phenomenon, it indicated that the PCR products met the requirements for subsequent sequencing.

[0043] The PCR amplification products that met the sequencing requirements were sent to Shanghai Personal Biotechnology Co., Ltd. at a low temperature 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 sequences of the type strains of the species were downloaded from the online website LPSN, and a phylogenetic tree of the isolates and the type strains was constructed using MEGA software. The species were identified comprehensively by combining the comparison in the NCBI database and the phylogenetic relationship.

[0044] The preserved strains were screened to obtain the strain Lactobacillus kefiranofaciens subsp. kefiranofaciens ( Lactobacillus kefiranofaciens ) IMAU12319 with potential probiotic characteristics. The phylogenetic tree between Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU12319 and related species is as Figure 11 shown.

[0045] Example 2

[0046] Determination of tolerance to simulated artificial gastrointestinal fluids

[0047] Preparation of artificial gastric juice: Take 20 mL of 1 mol / L HCl, adjust the pH to 2.0 with distilled water and 1 mol / L NaOH solution, then add pepsin at 0.3% and dissolve it, and filter it with a 0.22 μm sterile filter membrane to obtain artificial gastric juice, which is prepared and used immediately.

[0048] Preparation of artificial intestinal juice: Weigh 0.27 g of KH 2 PO 4 , 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 well, and filter it with a 0.22 μm sterile filter membrane to obtain artificial intestinal juice, which is prepared and used immediately.

[0049] The method is as follows: Subculture 8 strains of bacteria three times, separate the fermentation broth and supernatant under the centrifugation conditions of 4000 r / min for 10 min, wash 3 times by centrifugation with PBS sterilized using an autoclave, prepare the culture into a suspension with 5 mL of saline, after diluting the suspension 10-fold, select an appropriate concentration to count the viable bacteria count in the initial fermentation broth at 0 h. Mix 1 mL of the suspension with 9 mL of simulated gastric juice with a pH of 2.5, ferment anaerobically at 37 °C for 3 h, and count the viable bacteria count. Mix 1 mL of the simulated gastric juice cultured for 3 h with 9 mL of simulated intestinal fluid, sample at 4 h and 8 h under anaerobic conditions at 37 °C, and count the viable bacteria count. The calculation formula is as follows:

[0050] Survival rate in artificial gastric juice (%) = N 1 / N 0 × 100%;

[0051] Survival rate in artificial intestinal fluid (%) = N 2 / N 1 × 100%;

[0052] In the formula, N 0 represents the viable bacteria count of the original bacterial solution (CFU / mL); N 1 represents the viable bacteria count after culturing in gastric juice for 3 h (CFU / mL); N 2 represents the viable bacteria count after culturing in intestinal fluid for 4 h and 8 h (CFU / mL).

[0053] Table 1 Results of the test for tolerance to simulated artificial gastrointestinal fluids

[0054] ;

[0055] Note: The data in the table are the mean ( ) ± standard deviation (SD), and different lowercase letters in the same column indicate significant differences (P < 0.05).

[0056] As can be seen from Table 1, the survival rate of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU12319 after digestion in artificial gastrointestinal fluid with a pH of 2.0 for 3 h was 51.66%. After 8 h of digestion in artificial intestinal fluid, IMAU12319 was 41.66%, which was closer to the control strain V9. Therefore, it was shown that IMAU12319 had good tolerance to gastrointestinal fluids.

[0057] Example 3

[0058] Study on the growth characteristics of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU12319

[0059] 1. Morphological characteristics of the strain

[0060] The method is as follows: Streak IMAU12319 (Lactobacillus kefiranofaciens IMAU12319) on MRS solid medium, leave it static at 37°C for 24 h, pick a single colony, and observe its staining changes and morphological characteristics by Gram staining method.

[0061] As Figure 1 shown in B, it was observed that the single colony of strain IMAU12319 presented a typical round shape, relatively small diameter, slightly convex center, neat edge, smooth surface, and showed milky white color, as Figure 1 shown in A. It was confirmed by Gram microscope observation that it was a Gram-positive Lactobacillus.

[0062] 2. Growth curve and acid production curve of the strain

[0063] The method is as follows: Continuously culture strain IMAU12319 in MRS medium at 37°C for 36 h, and perform viable cell counting and pH measurement every 2 h.

[0064] As Figure 2 shown, the period from 0 to 4 h for strain IMAU12319 was the lag phase of strain growth, and there was no obvious change in its activity under OD 600nm . After a 2-h lag phase, logarithmic growth began, and the OD 600nm value and the number of viable cells began to increase rapidly at 14 h; subsequently, the growth rate of OD 600nm gradually slowed down, gradually tended to be stable, and entered the stationary phase. The number of viable cells of IMAU12319 reached the peak at 16 h, and the OD 600nm showed an obvious downward trend after 18 h.

[0065] As Figure 3 shown by the pH curve, between 0 and 2 h, the pH of the strain hardly changed; within 2 to 14 h, the strain reached the logarithmic growth stage, and with the increase in the number of bacteria, its lactic acid production ability increased significantly, and the pH decreased significantly; the pH value of the strain was 4.34 at 20 h, and then the pH value gradually stabilized; after 24 h, the strain entered the death stage, in which the number of live bacteria decreased significantly, and there was a slight increase in pH. Therefore, the overall growth and acid production rate of strain IMAU12319 were good.

[0066] 3. Optimum growth temperature of the strain

[0067] The method is as follows: Inoculate the activated cell solution into 5 mL of MRS liquid medium at an inoculation volume of 2%, and culture it under constant temperature conditions at 5 temperature gradients for 24 h, and then measure the absorbance value of the fermentation broth at 600 nm.

[0068] The results are as Figure 4As shown, the biomass of strain IMAU12319 increased with the increase of temperature and then showed a downward trend, and the OD 600nm value decreased slowly with the increase of temperature. Strain IMAU12319 grew fastest and had the best growth at 30 °C; followed by 37 °C. When the temperature exceeded 55 °C, the growth of the strain was inhibited and it did not grow. This shows that high temperature has a certain inhibitory effect on its growth vitality.

[0069] 4. Optimal pH of the strain

[0070] The method is as follows: Inoculate the bacterial liquid at an inoculation amount of 2% into MRS liquid medium with initial pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, and incubate statically at 37 °C for 24 h, and measure the absorbance value of the fermentation broth under the condition of 600 nm.

[0071] The results are as Figure 5 shown. Strain IMAU12319 has better proliferation ability at about pH 6.0 and is suitable for growth under weakly acidic conditions.

[0072] Example 4

[0073] Study on the biochemical characteristics of Lactobacillus kefiranofaciens subsp. kefiranofaciens IMAU12319

[0074] 1. API 50CHL test

[0075] The method is as follows: According to the API 50CHL lactic acid bacteria identification standard, use the API 50CHL kit (purchased from bioMérieux, France) to conduct a sugar fermentation test on the strain to identify the metabolism of 49 carbohydrates by the test strain. Operate according to the instructions.

[0076] Table 2 Results of carbohydrate fermentation determination

[0077] ; ;

[0078] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0079] The results are shown in Table 2. Strain IMAU12319 showed positive reactions to a total of 13 carbohydrates. The carbohydrates that strain IMAU12319 can utilize include: D-galactose (GAL), D-glucose (GLU), D-fructose (FRU), D-mannose (SAC), N-acetyl-glucosamine (NAG), arbutin (ARB), salicin (SAL), D-maltose (MAL), D-lactose (LAC), D-melibiose (MEL), D-sucrose (SAC), D-trehalose (TRE), D-raffinose (RAF).

[0080] 2. β - galactosidase activity of the strain

[0081] Preparation of crude enzyme solution: In MRS liquid medium, the activated strain was cultured at 37 °C for 24 h. The cells were collected under centrifugation conditions (4 °C, 8000 r / min, 15 min) and subjected to ultrasonic treatment to obtain the supernatant.

[0082] The method is as follows: Place the ONPG solution (2.5 mg / mL, prepared with phosphate buffer at pH 6.5) in a water bath at 37 °C for 5 min. Add 1 mL of ONPG solution and 1 mL of crude enzyme solution to the test tube respectively. After mixing evenly, react at 37 °C for 10 min. After the reaction, quickly add 4 mL of 500 mmol sodium carbonate solution, mix evenly and let stand for 5 min, and measure the absorbance value of the solution at 420 nm. The blank determination method is basically the same as the above operation. To inactivate the reaction enzyme solution, add sodium carbonate solution before adding the crude enzyme solution. At the same time, draw the standard curve of o - nitrophenol (ONP), see Figure 6 . The β - galactosidase activity in the sample is calculated according to the following formula:

[0083] Enzyme activity (U / mL) = (c × v × D) / (t × v 1 )

[0084] In the formula, c is the concentration of test ONP; v is the total volume of the reaction solution; D is the sample dilution factor; t is the reaction time; v 1 is the volume of the reaction enzyme solution.

[0085] Table 3 Determination results of β - galactosidase activity

[0086] ;

[0087] 3. Determination of proteolytic activity of the strain

[0088] The method is as follows: The activated IMAU12319 strain was inoculated into skim milk at an inoculation amount of 2% and cultured at 37 °C for 12 h. The blank control group was the corresponding milk sample without inoculum. Precisely aspirate 2.5 mL of the test sample into a tube, then add 0.5 mL of double - distilled water, mix, then add 0.5 mL of 0.75 mol / L TCA, mix in a vortex, keep at room temperature for 10 min, filter to 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 evenly, react at the standard temperature for 2 min, and measure the absorbance at 340 nm. Corresponding to the standard curve Figure 7 , it is considered that the proteolytic activity is equal to the tyrosine content, with OD 340nmIt is the OPA index and can also be directly compared.

[0089] The results are as Figure 8 shown. The proteolytic activity of the strain is the strongest during the logarithmic growth phase. The final photometric value of strain IMAU12319 at 24 h is 0.4936, and the corresponding free amino acids are 33.09 mg / L.

[0090] 4. Determination of exopolysaccharides

[0091] Table 4 Determination of exopolysaccharide content

[0092] ;

[0093] As shown in Table 4, the exopolysaccharide content of strain IMAU12319 is 52.79 mg / L. Some studies have found that the exopolysaccharide content of lactic acid bacteria in synthetic and semi-synthetic media is usually in the range of 40 - 600 mg / L, and IMAU12319 conforms to this conclusion.

[0094] Example 5

[0095] Safety evaluation of Lactobacillus kefiranofaciens IMAU12319

[0096] The method is as follows: Using the disk diffusion test method, susceptibility tests were performed on 26 antibiotics. The selected strain was activated, centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the cells were suspended in sterile saline solution, and its turbidity was adjusted to 0.5 McFarland units. 100 μL of the bacterial suspension was evenly spread on the MRS solid medium, allowed to dry, and then adhered to the plate coated with lactic acid bacteria by the disk method for anaerobic culture. The sensitivity of the bacteria to antibiotics was determined according to the size of the inhibition zone around the drug susceptibility disk.

[0097] Table 5 Results of drug sensitivity test

[0098] ;

[0099] ;

[0100] Note: The judgment criteria for the diameter of the inhibition zone are sensitive (S), intermediate (I), and resistant (R).

[0101] The results are shown in Table 5. The drug sensitivity test results of IMAU12319 indicate that the strain IMAU12319 is resistant to 4 antibiotics and highly sensitive to 16 antibiotics.

[0102] Example 6

[0103] Determination of antibacterial ability

[0104] The method is as follows: The Oxford cup method was used to conduct antibacterial tests on the strains, and the antibacterial ability of IMAU12319 against 4 common foodborne pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium) was determined.

[0105] Table 6 Determination results of antibacterial performance

[0106] ;

[0107] As can be seen from Table 6, IMAU12319 has good inhibitory effects on these 4 common pathogenic bacteria, and has the best antibacterial ability against Staphylococcus aureus. The diameter of the antibacterial circle of IMAU12319 is 23.84 mm, and the antibacterial effect is good.

[0108] Example 7

[0109] Evaluation of probiotic characteristics

[0110] 1. Research on antioxidant activity

[0111] Determination of DPPH free radical scavenging ability

[0112] The method is as follows: Mix 2 mL of cell suspension or cell-free extract, fermentation broth and other solutions with 1 mL of anhydrous ethanol of DPPH, place it in a place without light irradiation for reaction for 30 min, then centrifuge at 10000 r / min for 10 min, extract the supernatant solution, zero it, and use an ultraviolet spectrophotometer to detect the OD 600nm value and represent it as B; the control (using anhydrous ethanol to replace the anhydrous ethanol of DPPH) is represented as C; the absorbance value of the blank group is A. Vitamin C (Ascorbic acid, VC) has certain antioxidant effects, and the content is proportional to the antioxidant effect. Use the VC aqueous solution as the positive control for the DPPH scavenging rate. The results show that the scavenging rate of DPPH in the VC aqueous solution increases with the increase of vitamin C content. The calculation is based on the following equation:

[0113] DPPH free radical scavenging rate (%) = [1 - (A - B) / C] × 100%.

[0114] The results are as Figure 10 shown. The DPPH free radical scavenging of the fermentation broth group of the strain IMAU12319 can reach 83%, the DPPH free radical scavenging of the strain IMAU12319 in the cell suspension group can reach 64%, and the DPPH free radical scavenging of the strain IMAU12319 in the cell-free extract group can reach 58%. Among the three, the DPPH scavenging rate of the fermentation broth is the highest, and the DPPH scavenging rate of the fermentation broth is closest to the DPPH scavenging rate of the control VC solution. IMAU12319 shows a very high DPPH scavenging ability.

[0115] Determination of Hydroxyl Radical Scavenging Ability

[0116] The method is as follows: 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 to 2 mL of cell suspension (or 2 mL of cell-free extract or fermentation broth), and mix well. After 15 min in a 37 °C water bath, centrifuge at 4 °C, 9000 r / min for 10 min to obtain the supernatant. Measure the absorbance at 510 nm and denote it as B; use A to represent the blank control (ultrapure water is used to replace the sample to be tested). The calculation formula is as follows:

[0117] Hydroxyl radical scavenging rate (%) = [1 - (B / A 0 )] × 100%.

[0118] The results are as Figure 10 shown. The hydroxyl radical scavenging rate of the fermentation broth group of strain IMAU12319 can reach 56%, the hydroxyl radical scavenging rate of the cell suspension group of strain IMAU12319 can reach 42%, and the hydroxyl radical scavenging rate of the cell-free extract group of strain IMAU12319 can reach 22%. IMAU12319 shows good hydroxyl radical scavenging activity.

[0119] Determination of Superoxide Anion (O 2 - ) Scavenging Ability

[0120] The method is as follows: Add 1 mL of 3 mmol / L diethylenetriaminepentaacetic acid, 1 mL of 150 mmol / L tris(hydroxymethyl)aminomethane 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), and perform a 10 min water bath at 25 °C. Measure the absorbance at 325 nm after the bath.

[0121] The results are as Figure 10 shown. There are significant differences in the scavenging ability of different strains against superoxide anion (O 2 - ). The superoxide anion scavenging rate of the control group (VC) solution is 100%. Among them, the superoxide anion scavenging rate of the fermentation broth group of strain IMAU12319 can reach 132%, the superoxide anion scavenging rate of the cell suspension group of strain IMAU12319 can reach 78%, and the superoxide anion scavenging rate of the cell-free extract group of strain IMAU12319 can reach 35%. IMAU12319 shows excellent superoxide anion scavenging rate.

[0122] 2. Results of the Determination of Cell Surface Hydrophobicity of Strains

[0123] The method is as follows: The surface hydrophobicity of the bacteria is determined by the hydrocarbon adsorption method. The bacteria activated to the 3rd generation are centrifuged at 4000 r / min for 5 min, and the bacterial sludge is collected, washed twice with PBS buffer, and then resuspended in PBS. The liquid is adjusted to 1×10 8 CFU / mL, and the initial absorbance A at 600 nm is measured 0 . 2 mL of xylene is mixed with 2 mL of the bacterial suspension, vortexed for 10 min, and left standing indoors for 30 min. The absorbance A of the lower aqueous phase is measured at 600 nm. The formula is as follows:

[0124] Hydrophobicity rate (%) = (A - A 0 ) / A 0 × 100%.

[0125] Table 7 Results of the hydrophobicity determination test

[0126] ;

[0127] As shown in Table 7, good hydrophobicity indicates high intestinal adhesiveness. The hydrophobicity rate of strain IMAU12319 can be as high as 43.26%, and it has good hydrophobicity rate and self-aggregation ability, and can be used as a potential probiotic.

[0128] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) Use of IMAU12319 in the preparation of a probiotic with antioxidant activity, characterized in that: The kefir-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 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is: CGMCC No.31224. The probiotic agent is a cell suspension of kefir-like Lactobacillus IMAU12319.

2. A probiotic with antioxidant activity, characterized in that: The probiotic comprises the kefir-like Lactobacillus IMAU12319 described in claim 1, and the probiotic is a cell suspension of kefir-like Lactobacillus IMAU12319.

3. The use of the kumquat-like Lactobacillus IMAU12319 as claimed in claim 1 in the preparation of a probiotic having β-galactosidase activity, characterized in that: The β-galactosidase activity of the kumiss-like Lactobacillus IMAU12319 is 3.12±0.012 U / 10 4 cell.

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