Lactobacillus amyloliquefaciens FMNS13 and application thereof

Abstract

The invention discloses lactobacillus amyloliquefaciens FMNS13 and application thereof, and belongs to the technical field of probiotics. The invention provides a strain of lactobacillus amyloliquefaciens FMNS13, the strain has good endurance capacity to gastrointestinal fluid and inhibition capacity to pathogenic bacteria, and the strain and metabolites thereof also have good oxidation resistance, can be used for preparing antioxidant drugs and health care products, or can be used for preparing freeze-dried fruit and vegetable products, and has wide application. The invention also optimizes the freeze-drying protective agent of the lactobacillus amyloliquefaciens FMNS13, so that the survival rate of viable bacteria in the lactobacillus amyloliquefaciens FMNS13 freeze-dried powder prepared by using the freeze-drying protective agent can reach 89.72%.

Description

Technical Field

[0001] The invention relates to Lactobacillus amylovora FMNS13 and application thereof, belonging to the technical field of probiotics. Background Art

[0002] "Probiotics" are live microorganisms that promote or support a beneficial balance of the indigenous microbial population of the gastrointestinal fluid. These microorganisms are not necessarily permanent residents of the gastrointestinal fluid, but they should have a beneficial effect on the general and health status of humans and animals. Research on probiotics is not only about maintaining the balance of intestinal microorganisms, but also about treating various diseases related to the immune system, such as obesity, type II diabetes and cancer. Probiotics are used in treatments for more than just health reasons and disease prevention. Therefore, there is a growing economic interest in probiotics in medicines, fermented foods, cosmetics and non-chemotherapeutic drugs.

[0003] Numerous in vitro and in vivo studies have clearly pointed out that probiotics adjust the structure of microbial communities by producing metabolites that are beneficial to health and selectively proliferating in the intestine, thereby showing multiple health-promoting effects. However, it is worth noting that some research reports have raised objections, pointing out that the application of probiotics, especially in the human environment, has not significantly observed the improvement of health benefits. This phenomenon may also be related to the changes in the physiological state of probiotic products, especially the freeze-drying technology widely used in the production process. Freeze-drying, as a key step in the industrial production of probiotic products, has effectively extended the shelf life of the product, but in this process, microbial cells need to withstand great environmental pressure, including the dual extreme conditions of freezing and drying, which may cause multiple damages to the cells. Specifically, freeze-drying not only inhibits the metabolic activity of microorganisms, which is manifested as a significant decrease in intracellular enzyme activity and metabolic rate, which may interfere with their normal metabolic pathways, but also directly affects their growth potential and function in the ecosystem. Further analysis shows that the physical stability of the cell membrane is impaired, the effects caused by changes in solute concentration, the destruction of cell structure caused by dehydration, the damage caused by thermal stress, the damage at the DNA level, and the overall decline of metabolic function are all severe challenges that microbial cells may encounter during the freeze-drying process. In order to alleviate these adverse factors and improve the survival rate and functional integrity of probiotics after freeze-drying, a series of sophisticated protection strategies must be implemented, including but not limited to optimizing the process parameters of freeze-drying, using protective agents to enhance cell resistance, and exploring more gentle drying technologies. These measures aim to maximize the activity and efficacy of probiotics and ensure that they can still effectively exert their potential to promote health when they reach consumers.

[0004] Among the strains currently disclosed for probiotic products, there are relatively few Lactobacillus amylovorus strains, and their probiotic abilities are generally weak, and there is no technical description of the protective measures for the processing of Lactobacillus amylovorus strains. Therefore, the main technical problem to be solved by the present invention is to develop Lactobacillus amylovorus strains with stronger probiotic properties and provide protective measures for the subsequent application and processing of the strains. Summary of the invention

[0005] The present invention provides a strain of Lactobacillus amylovorus FMNS13, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on August 20, 2024, with a deposit number of GCMCC No: 65020.

[0006] The present invention also provides a probiotic preparation containing the Lactobacillus amylovora FMNS13.

[0007] In one embodiment, the number of Lactobacillus amylovora FMNS13 in the probiotic preparation is ≥ 1×10 12 CFU / g.

[0008] The invention also provides freeze-dried powder containing the Lactobacillus amylovora FMNS13.

[0009] In one embodiment, the freeze-dried powder is prepared by freeze-drying after mixing the bacterial suspension of Lactobacillus amylovora FMNS13 with a freeze-drying protectant containing skim milk, sodium L-glutamate and D-trehalose.

[0010] In one embodiment, the lyoprotectant contains 40-200 g / L skim milk, 30-70 g / L sodium L-glutamate, and 20-100 g / L trehalose.

[0011] In one embodiment, the freeze-drying protective agent is specifically: 114 g / L skim milk, 32 g / L sodium L-glutamate, and 65 g / L D-trehalose.

[0012] The present invention also provides a method for preparing probiotic freeze-dried powder, which comprises mixing the bacterial suspension of Lactobacillus amylovora FMNS13 with a freeze-drying protective agent containing skim milk, sodium L-glutamate and D-trehalose, and vacuum freezing at -80°C for at least 24 hours, or 24 to 48 hours.

[0013] The present invention also provides the use of the Lactobacillus amylovorus FMNS13 or its postbiotics in preparing health products or medicines with antioxidant function.

[0014] In one embodiment, the postbiotics include but are not limited to the supernatant collected after centrifugation of the cell culture fluid after fermentation of the Lactobacillus amylophagous FMNS13.

[0015] The present invention also provides application of Lactobacillus amylovora FMNS13 in preparing freeze-dried vegetables.

[0016] In one embodiment, the vegetable includes but is not limited to okra.

[0017] In one embodiment, the application is to control the bacterial load of Lactobacillus amylovora FMNS13 on freeze-dried okra to reach 1×10 8 CFU / g.

[0018] Beneficial effects:

[0019] 1. The present invention screened out the Lactobacillus amylovorus FMNS13 strain, which has a good tolerance to gastrointestinal fluid and is conducive to colonization in the gastrointestinal environment; the strain has an inhibitory ability against pathogenic bacteria and can effectively inhibit Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella typhimurium; the strain and its metabolites also have good antioxidant properties and can be used as an effective ingredient for probiotic preparations.

[0020] 2. The present invention optimizes the freeze-drying protective agent of Lactobacillus amylovora FMNS13, and can make the survival rate of live bacteria in the freeze-dried powder of Lactobacillus amylovora FMNS13 prepared by using the freeze-drying protective agent reach 89.72%.

[0021] 3. The amylovorax lactobacillus prepared by the present invention can also be used to prepare antioxidant drugs, health products, or freeze-dried fruit and vegetable products, and has a wide range of uses.

[0022] Biomaterial Deposit

[0023] Lactobacillus amylovorus FMNS13, classified and named Lactobacillus amylovorus FMNS13, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 20, 2024, with the deposit number GDMCC No: 65020, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the phylogenetic tree of lactic acid bacteria screened from kumiss.

[0025] Figure 2These are the results of survival rate determination of different LAB strains screened out in simulated gastrointestinal fluid in vitro; a is the survival rate of LAB in simulated gastric fluid; b is the survival rate of LAB in simulated intestinal fluid.

[0026] Figure 3 The results of the colonization ability test of different LAB strains screened out; a is the hydrophobic property; b is the self-aggregation ability; c is the co-aggregation ability.

[0027] Figure 4 The results of the determination of the ability of different LABs screened out to inhibit common pathogenic bacteria; a is the inhibitory ability of LAB against Escherichia coli; b is the inhibitory ability of LAB against Staphylococcus aureus; c is the inhibitory ability of LAB against Listeria monocytogenes; d is the inhibitory ability of LAB against Salmonella.

[0028] Figure 5 are the results of LAB antioxidant capacity determination; a1~a3 are the scavenging abilities of microbial fermentation supernatant, bacterial suspension and cell-free extract on DPPH; b1~b3 are the scavenging abilities of microbial fermentation supernatant, bacteria and cell-free extract on superoxide anions; c1~c3 are the abilities of microbial fermentation supernatant, bacteria and cell-free extract on hydroxyl radicals; d1~d3 are the reducing abilities of microbial fermentation supernatant, bacteria and cell-free extract.

[0029] Figure 6 Figure a is the hemolytic evaluation of strains; Figure b is the hemolytic evaluation of Staphylococcus aureus, and Figure 5 is the hemolytic evaluation of Lactobacillus amylovorus FMNS13.

[0030] Figure 7 a is the effect of a single protective agent on the survival rate of Lactobacillus amylovorus FMNS13 strain; a is the effect of skim milk on the freeze-dried survival rate of Lactobacillus amylovorus FMNS13 strain; b is the effect of sodium L-glutamate on the survival rate of Lactobacillus amylovorus FMNS13 strain; c is the effect of D-trehalose on the survival rate of Lactobacillus amylovorus FMNS13 strain.

[0031] Figure 8The three-dimensional surface map and contour map of the interaction between skim milk, sodium L-glutamate and D-trehalose on the freeze-dried survival rate of Lactobacillus amylovorus FMNS13; a is a three-dimensional surface map of the effect of skim milk and sodium L-glutamate on the survival rate of the strain; b is a contour map of the effect of skim milk and sodium L-glutamate on the survival rate of the strain; c is a three-dimensional surface map of the effect of skim milk and D-trehalose on the survival rate of the strain; d is a contour map of the effect of skim milk and D-trehalose on the survival rate of the strain; e is a three-dimensional surface map of the effect of D-trehalose and sodium L-glutamate on the survival rate of the strain; f is a contour map of the effect of D-trehalose and sodium L-glutamate on the survival rate of the strain.

[0032] Fig. 9 This is the morphological characterization of the strain Lactobacillus amylovorus FMNS13 after freeze-drying; a is the strain morphology before freeze-drying, b is the direct morphology after freeze-drying with the addition of a protective agent, c is the strain morphology after freeze-drying without the addition of a protective agent, d is the direct morphology after freeze-drying without the addition of a protective agent, e is the strain morphology after freeze-drying with the addition of a protective agent, and f is the direct morphology after freeze-drying without the addition of a protective agent.

[0033] Fig.10 The cell membrane integrity and fluidity of FMNS13 after freeze-drying; a is the fluorescence absorption spectrum of ANS probe; b is the fluorescence intensity of ANS probe at λ=480nm; c is the staining result of the strain before freeze-drying; d is the staining result of the strain without protective agent; e is the staining result of the strain with cryoprotectant.

[0034] Fig.11 are the results of key enzyme activity detection of lactic acid bacteria metabolism; a is the result of Na+ / K+-ATPase detection; b is the result of β-galactosidase detection; c is the result of phosphoketolase detection; d is the result of L-lactate dehydrogenase detection.

[0035] Fig.12 Application of Lactobacillus amylovorus FMNS13 strain in freeze-drying of okra; a and c are okra samples before freeze-drying; b is the freeze-dried okra sample without adding strain and compound protective agent; d is the freeze-dried okra sample with adding strain and compound protective agent.

[0036] Error bars represent mean ± standard deviation (n = 3), and different letters indicate significant differences (P < 0.05). DETAILED DESCRIPTION

[0037] The strain Lactobacillus amylovorus FMNS13 is a strain obtained by screening in this application. In order to facilitate the common description and horizontal comparison with other strains during the screening process, its original number FMNS13 is retained and the number is used to describe the relevant application of the strain in each embodiment. Unless otherwise specified, the strain Lactobacillus amylovorus FMNS13 mentioned in the embodiment is the strain with the deposit number GDMCC No: 65020 for which protection is requested in this application.

[0038] Activation of strains:

[0039] All materials involved in this application (including pipette tips, inoculation loops, culture media, conical flasks, saline and other strain culture materials and culture media) have been thoroughly sterilized for 15 minutes at 121°C. Subsequently, under aseptic conditions, MRS (De Man, Rogosa, and Sharpe) broth was placed in a 37°C environment and introduced into the target strain at a 1% inoculation ratio. After 18 hours of preliminary culture, two generations of continuous activation culture processes were carried out to ensure the growth vitality of lactic acid bacteria. Finally, the lactic acid bacteria culture was recovered from the MRS culture media, and the total culture time at this time reached 36 hours.

[0040] Example 1: Screening of Lactobacillus amylovorus FMNS13

[0041] The samples used for LAB separation and purification were traditional kumis products. Fresh Ulan Kumis, a specialty drink in Inner Mongolia (Kongjiaying East District, no lactic acid fermentation agent was added during traditional winemaking), was purchased. After sampling, all samples were stored in plastic containers and transported to the laboratory at 4°C. LAB was separated by standard dilution method. Take 100 μl aliquots of dilution (dilution degree is 10 -5 , 10 -6 , 10 -7 and 10 -8 ) and coated on the surface of MRS agar containing calcium carbonate. Finally, the plate was incubated at 37°C for 72 hours. According to the morphological and culture characteristics, typical colonies were selected and then subcultured in MRS liquid medium and incubated at 37°C for 18 hours.

[0042] Bacterial genomic DNA was extracted according to the manufacturer's instructions (reagent company Solebol). The extracted DNA was used as a template for PCR amplification. Each PCR reaction (50 μL) consisted of 1.5 μL (10 μmol / L) upstream and downstream primers, 2.0 μL genomic DNA, 5.0 μL 10× buffer, 4.0 μL dNTP (2.5 mmol / L), 0.5 μL Taq DNA polymerase and 35.5 μL distilled water. The 16S primer (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′′) was used to amplify the DNA. The PCR amplification was performed as follows: pre-denaturation at 95°C for 5 minutes, followed by 30 cycles (denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 15 seconds), and finally end extension at 72°C for 5 minutes.

[0043] The amplified products were obtained and stored at 4°C to prevent degradation. The amplified products were sent to Biotechnology Co., Ltd. (Beijing Qingke) for sequencing. BLAST analysis was performed using MEGA 11 software, and the obtained gene sequences were used to construct a phylogenetic tree with a demarcation threshold of homology by the neighbor-joining method. The selected microorganisms were stored at -80°C.

[0044] The identification results are as follows Figure 1 As shown. Blast comparison results showed that the 14 lactic acid bacteria screened from the traditional fermented kumis in Inner Mongolia belonged to seven different species. According to the similar physiological characteristics of strains of the same genus, seven non-homologous lactic acid bacteria, including Lactobacilluskitasatonis strain FMNS5, Ligilactobacillus acidipiscis FMNS10, Nialliacirculans strain FMNS11, Pediococcus acidilactici FMNS12, Lactobacillusamylovorus FMNS13, Pediococcus acidilactici FMNS15 and Laciicaseibacillusrhamnosus FMNS18, were selected to verify the probiotic properties of the strains in vitro.

[0045] Example 2: Preparation of bacterial suspension

[0046] The strains screened in Example 1 were cultured in MRS medium at 37°C for 36 hours, during which they underwent two generations of activation to enhance their growth activity. Subsequently, the microbial culture solution was taken out from the cultured lactic acid bacteria solution, centrifuged at 8000×g, 4°C for 5 minutes, and the supernatant was discarded. For the precipitate obtained after centrifugation (i.e., lactic acid bacteria cells), it was first washed twice with phosphate buffered saline (PBS) with a pH value of 7.2 to remove residual culture medium and other impurities. Afterwards, the washed lactic acid bacteria cells were resuspended in an equal volume of PBS (pH 7.2), and the concentration of the microorganisms was adjusted to 1.0×10 12 CFU / mL, and prepare the complete lactic acid bacteria cell suspension for subsequent experiments.

[0047] Example 3: Preparation of fermentation supernatant

[0048] The strains screened in Example 1 were cultured in MRS (De Man, Rogosa, and Sharpe) medium at 37°C for 18 hours, during which they underwent two generations of activation to enhance their growth activity. Subsequently, 10 mL of microbial culture solution was taken from the cultured lactic acid bacteria solution and centrifuged at 8000×g and 4°C for 5 minutes to separate the fermentation supernatant, which was then collected and prepared for subsequent detection.

[0049] Example 4: Preparation of cell-free extracts

[0050] The cell suspension prepared in Example 2 was ultrasonically disrupted in EP tubes. The ultrasonic disruption conditions were set as follows: power 120 W, working for 3 seconds, stopping for 8 seconds, and repeating the operation 30 times. The ultrasonic disruption liquid was collected and centrifuged at 12000×g, 4°C for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter membrane to successfully prepare the microbial cell-free extract.

[0051] The lactic acid bacteria cell suspension prepared above was transferred to an EP tube and then subjected to ultrasonic disruption. The parameters of ultrasonic disruption were set as follows: 120W power, working for 3s, stopping for 8s, and repeating 30 times. After ultrasonic disruption, the resulting liquid was collected and centrifuged at 12000×g at 4°C for 10 minutes. After centrifugation, the supernatant was carefully collected and filtered through a 0.22μμm pore size filter membrane to remove any residual cell debris or particulate matter. After the above steps, the cell-free extract of the microorganism was successfully prepared.

[0052] Example 5: Verification of LAB survival rate in simulated gastrointestinal fluid in vitro

[0053] To prepare artificial gastric fluid, 3.0 g / L pepsin (1:10000, Solebao) was dissolved in sterile saline (0.5% w / v), and the pH was adjusted to 2.0 with hydrochloric acid. 1.0 g / L trypsin (1:250, Solebao) was dissolved in sterile saline (0.5% w / v), and the pH was adjusted to 7.0 with NaOH, and bile salts were added to make the final concentration of bile salts reach 0.3%, which is the simulated intestinal fluid. The simulated gastrointestinal fluids were filtered using a 0.22 μm filter membrane. The suspension was mixed with artificial gastric fluid, incubated at 37°C for 2 hours, transferred to artificial intestinal fluid, and incubated anaerobically at 37°C for 5 hours. Finally, the survival rate (%) was calculated by plate counting. The average of 3 independent experiments was calculated:

[0054] Survival rate (%) = logCFUN0 / logCFUNt×100%,

[0055] Where N0 is the number of viable bacteria (CFU / mL) at 0h, and Nt is the number of viable bacteria in the artificial gastrointestinal fluid at 2h and 7h. All tests were repeated three times, and the data are expressed as mean ± SD (standard deviation)

[0056] After preliminary screening, the 7 lactic acid strains screened in Example 1 were subjected to tolerance to simulated gastric fluid (pH 2) and intestinal transport (pH 7) tests. The effects of simulated gastrointestinal fluid on the viability of the test strains were shown in Figure 2. Figure 2 As shown. Although most of the test strains can grow in an acidic environment, the growth of different strains in artificial gastric fluid at pH 2 for 3 hours varies greatly. The survival rate of the FMNS13 strain in simulated gastric fluid (88.83±1.30%) was significantly greater than that of the positive control LGG strain (78.07±1.30%). There was no significant difference in the survival rate between the FMNS10 strain (82.10±1.68%) and the LGG strain (78.07±1.30%). The survival rates of the other test strains in simulated gastric fluid were significantly lower than those of the LGG strain ( Figure 2 a, p<0.05).

[0057] After incubation in simulated intestinal fluid for 5 hours, the survival rates of FMNS18 (69.27±0.93%) and FMNS13 (61.02±1.87%) strains were significantly higher than that of the positive LGG strain (52.64±2.14%). There was no significant difference between the survival rates of FMNS05 strain (57.45±1.23%) and LGG strain (52.64±2.14%). The survival rates of other test strains in simulated intestinal fluid were significantly lower than those of LGG strain ( Figure 2 b, p<0.05).

[0058] Example 6: Verification of LAB colonization ability in the intestine

[0059] In this example, the hydrophobicity, self-cohesion and co-cohesion of the strain were evaluated to determine the colonization ability of the strain.

[0060] 1. Determination of self-cohesion ability

[0061] Take 10 mL of the activated lactic acid bacteria culture solution and centrifuge it at 4°C, 8000 g for 5 min. Discard the supernatant and collect the precipitate. Wash the cells twice with phosphate buffered saline (PBS, pH 7.2), resuspend them in the same volume of buffer, and adjust the microbial concentration to 10 7 Up to 10 8 CFU / mL, the initial absorbance of the bacterial solution was measured at 600 nm. The bacterial suspension was then incubated at room temperature and monitored for 6 h. Each group was repeated three times.

[0062]

[0063] Where t0 represents the OD at t = 0h 600 Value, Δ t =OD at 6h 600 .

[0064] 2. Determination of hydrophobicity

[0065] In order to carry out the LAB microbial surface hydrophobicity determination, a bacterial suspension was prepared according to the method of Example 2. After obtaining the cell suspension, the liquid OD was adjusted. 600 The value is about 0.5. Add an equal volume of xylene to the microbial solution, shake vigorously and mix well, incubate at room temperature for 1 hour, remove the organic phase, and measure the absorbance of the aqueous phase at 600nm. PBS is used as a control. The experiment is repeated three times. The surface hydrophobicity of lactic acid bacteria is calculated according to the following formula:

[0066]

[0067] Where OD0 represents the OD before mixing with xylene 600 Value, OD1 represents the OD after mixing with xylene 600 Value co-condensation.

[0068] 3. Determination of co-coagulation ability

[0069] Escherichia coli O157:H7 (accession number CICC21530) was used as an indicator strain to evaluate the coaggregation ability of lactic acid bacteria screened from traditional fermented kumiss. The bacterial suspension was prepared according to the method of Example 2 and the OD 600The absorbance of the single bacterial suspension was measured as a control. The OD600nm value of the mixture and the single bacterial suspension was monitored and the measurement was repeated 3 times.

[0070]

[0071] OD2 and OD3 represent the OD of the test bacteria and indicator bacteria suspensions treated separately for 2 hours. 600 OD0 represents the OD value of the mixed suspension of the test bacteria and the indicator bacteria for 2 hours. 600 value.

[0072] Colonization ability of strains Figure 3 As shown. There is a certain correlation between the surface hydrophobicity of lactic acid bacteria and their adhesion. The hydrophobicity of FMNS18 (79.06±1.79%), FMNS15 (72.60±2.12%) and FMNS05 (65.59±1.19%) strains has no significant difference with the positive control LGG strain (70.12±4.28%). The hydrophobicity of other test strains is significantly lower than that of LGG strain ( Figure 3 a, p>0.05).

[0073] After lactic acid bacteria self-aggregate, a biofilm will be formed on the surface of the intestine, which is equivalent to a biological barrier. This barrier not only allows lactic acid bacteria to adhere more firmly in the intestine, but also effectively blocks the adhesion and colonization of pathogenic bacteria to the intestine, thereby protecting intestinal health. The self-aggregation of the strains FMNS18 (77.76±2.47%) and FMNS13 (61.30±1.43) screened in Example 1 was not significantly different from that of the LGG strain (88.02±1.75%). The self-aggregation of the other test strains was significantly lower than that of the LGG strain ( Figure 3 b, p>0.05).

[0074] The coaggregation ability of Lactobacillus species may enable them to form a barrier, which prevents the colonization of pathogens by coaggregation, and may help the host resist infection and protect the host from the invasion of pathogens. Escherichia coli was used as an indicator bacteria to determine the coaggregation rate of the test strains. The strains FMNS13 (64.57±2.13%) and FMNS12 (66.02±1.98%) had strong coaggregation ability with pathogens, and there was no significant difference between the two and the positive control LGG strain (63.40±1.60%) ( Figure 3 c, p<0.05). The coaggregation rates of the other test strains were significantly lower than that of the LGG strain ( Figure 3c, p>0.05). Probiotic colonization is the key and basis for its function. Highly adherent lactic acid bacteria can firmly adhere to the intestinal mucosa, forming a stable biological barrier, which helps to maintain the balance of intestinal microecology.

[0075] Example 7: LAB is used to inhibit pathogenic bacteria

[0076] The in vitro inhibitory effects of the seven LAB strains selected in Example 1 on four common enteric pathogens were quantitatively evaluated using the well diffusion assay. The four pathogenic bacteria were obtained from the China Industrial Microbiological Culture Collection: Listeria monocytogenes (collection number CICC 21635), Staphylococcus aureus (collection number CICC 21600), Escherichia coli O157:H7 (collection number CICC 21530) and Salmonella typhimurium (CICC 21483). Sterile LB medium was used for the activation of the strains, and two generations of Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus were selected and activated, and they were ensured to be in the logarithmic growth phase before the experiment.

[0077] MRS agar was used as the basal culture medium. After sterilization, it was cooled to about 45°C and vigorously mixed with 200μl of overnight culture of pathogenic bacteria. The mixed agar was quickly poured into a sterile microbial culture dish, and a sterile puncher was used to punch a circular hole (Wells hole) with a diameter of 8mm on the agar layer to be solidified. To avoid cross contamination, the straight-line distance between the Wells holes was not less than 30mm, and it was allowed to cool and solidify naturally. After cooling and solidification, 100μL of the LAB strain culture to be tested was placed in each well, and the plate was incubated aerobically at 37°C for 24 hours. At the end of the incubation, check whether there is a clear antibacterial zone around the Wells hole, and use a vernier caliper to measure and record the diameter of each antibacterial zone as a quantitative indicator of inhibition ability. The test was repeated three times.

[0078] The results showed that in the inhibition of Escherichia coli ( Figure 4 a, p<0.05), the inhibitory abilities of FMNS18 (18.6±0.92mm), FMNS12 (20.36±0.74mm), FMNS10 (19.59±0.67mm) and FMNS05 (19.13±1.14mm) strains were significantly higher than the positive control LGG strain (14.39±0.74mm), while the inhibitory abilities of FMNS13 (15.50±1.25mm) and FMNS11 (15.19±0.59mm) strains against Escherichia coli were not significantly different from those of LGG strain ( Figure 4a, p>0.05); in terms of the inhibition of Staphylococcus aureus, the strains FMNS13 (17.43±0.59mm) and FMNS10 (17.98±1.21mm) were significantly better than the positive control LGG strain (14.59±0.72mm) ( Figure 4 b, p<0.05), there was no significant difference in the ability of other test strains to inhibit Staphylococcus aureus compared with LGG strain ( Figure 4 b, p>0.05). The inhibitory ability of FMNS18 (21.44±0.58mm), FMNS13 (15.18±0.60mm) and FMNS12 (23.09±0.96mm) against Listeria monocytogenes was significantly better than that of the positive control LGG strain (11.85±0.70mm) ( Figure 4 c, p<0.05), there was no significant difference in the ability of other test strains to inhibit Listeria monocytogenes compared with LGG strain ( Figure 4 c, p>0.05). The inhibitory ability of FMNS15 (12.31±1.20mm) and FMNS11 (10.47±0.73mm) against Salmonella was significantly lower than that of the positive control LGG strain (14.56±0.94mm) ( Figure 4 d, p<0.05), there was no significant difference in the ability of other test strains to inhibit Salmonella with LGG strain ( Figure 4 d, p>0.05). The antibacterial ability of probiotics is of great significance in maintaining intestinal health, enhancing immune function, promoting nutrient absorption, preventing and treating diseases, and promoting intestinal recovery.

[0079] Example 8: Determination of antioxidant capacity of LAB

[0080] Cell-free extracts, microbial fermentation supernatants and cell suspensions were prepared according to the methods of Examples 2 to 4. The antioxidant capacity of LAB was evaluated by DPPH scavenging capacity, hydroxyl radical scavenging capacity, superoxide anion scavenging capacity and reducing capacity.

[0081] 1. DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) scavenging ability

[0082] Take 1mL of cell-free extract in an amber EP tube, mix 0.2mM DPPH-anhydrous ethanol solution (Sigma) and the sample in a 1:1 (v / v) ratio in the amber tube, mix thoroughly, and react in the dark at 37°C for 30 minutes, take the supernatant solution and measure the absorbance at 517nm. When measuring complete cells and cell-free extract samples, mix DPPH with PBS (pH7.2) and culture under the same conditions as a control sample. When measuring lactic acid bacteria microbial fermentation supernatant samples, mix DPPH with blank MRS and culture under the same conditions as a control sample. Calculate according to the following formula:

[0083] DPPH free radical scavenging rate (%) = [1-A1 / A2] × 100%;

[0084] Where: A1 and A2 are the OD values ​​of the sample group and the control group measured at 517 nm, respectively. 517 value.

[0085] 2. Hydroxyl free radical scavenging ability

[0086] 0.75mM 1,10-phenanthroline (Sigma), PBS (pH 7.2) and sample were placed in an amber tube (referred to as "Mixture 1") at a ratio of 1:1 (v / v / v), mixed thoroughly, and then 1mL H2O2 (0.01%, v / v) was added. The mixture was incubated at 37°C for 1.5h and the absorbance was measured at 536nm, which was indicated as A1. A3 was the complete cell suspension in "Mixture 1" replaced with the same volume of distilled water, cultured under the same conditions and the absorbance was measured at 536nm. A2 was the H2O2 added to "Mixture 1" replaced with the same volume of distilled water, cultured under the same conditions and the absorbance was measured at 536nm. The ability of lactic acid bacteria to scavenge hydroxyl radicals was calculated according to the following formula:

[0087] Hydroxyl radical scavenging rate (%) = [A1-A3] / [A2-A3] × 100%;

[0088] Where: A1, A2 and A3 are the OD values ​​measured at 536 nm for the sample group, control group and blank group respectively. 536 value

[0089] 3. Determination of superoxide anion free radical scavenging ability

[0090] Take 1mL of the extract to be tested in an amber tube, mix the sample and 0.05mol / L phosphate buffer solution (pH 8) in a ratio of 1:9 (v / v) in the amber tube, mix thoroughly in a 25℃ constant temperature water bath for 15min, take 3mL of the mixed solution in a cuvette to measure the absorbance A2 at 420nm, add 0.1mL45 mmol / L pyrogallol (Sigma) solution, shake well and react for 3min, and measure the absorbance A1 at 420nm again; the control group is 1mL of distilled water, and the degree of self-oxidation of pyrogallol is measured, and the absorbance value is A3.

[0091] The clearance rate of superoxide anions P (%) = [1-(A1-A2) / A3] × 100%;

[0092] Where: A1, A2 and A3 are the OD values ​​measured at 420nm for the sample group, control group and blank group respectively. 420 value

[0093] 4. Determination of reducing ability of lactic acid bacteria

[0094] Mix 0.5 mL of the extract to be tested with the same volume of potassium ferricyanide (1%) and PBS buffer (0.2 mol / L, pH 6.6), and shake to make the system uniform. Mix distilled water with potassium ferricyanide and PBS as a blank control. The mixed system is incubated at 50°C for 20 minutes, cooled rapidly, and 0.5 mL of trichloroacetic acid (10%) is added. After centrifugation at 2000×g for 5 minutes, 1 mL of the supernatant is mixed with 1 mL of ferric chloride (1%) and reacted for 10 minutes. The absorbance is then measured at a wavelength of 700 nm, and cysteine ​​is used as a standard to characterize reducing power.

[0095] Reducing ability (%) = [(A1-A3) / A2] × 100%;

[0096] Where: A1, A2 and A3 are the OD values ​​measured at 700 nm for the sample group, standard and blank group respectively. 700 value.

[0097] The results are as follows Figure 5 As shown, the scavenging ability of LAB strains on DPPH free radicals is 9%-50% ( Figure 5 a). The DPPH free radical scavenging ability of FMNS18 (52.46±1.86%, 25.89±0.488% and 12.41±1.35%) was not significantly different from that of the positive control LGG strain (54.26±1.36%, 27.66±1.60% and 13.98±1.46%). Figure 5a1~a3, p<0.05). The DPPH free radical scavenging ability of other strains was significantly lower than that of LGG strain ( Figure 5 a1~a3, p>0.05).

[0098] like Figure 5 As shown in b, the superoxide anion scavenging activity of the LAB strain was between 8% and 32%, among which the active metabolites of the FMNS10 strain (32.93±1.58%), the FMNS11 strain suspension (24.21±1.12%) and the metabolites and suspensions of the LGG strain had no significant difference (35.70±0.93%, 25.95±1.30%) ( Figure 5 b1~b2, p<0.05). The superoxide anion scavenging activity of the cell-free extracts of the seven test strains had no significant difference from that of the positive control LGG strain ( Figure 5 b3, p<0.05).

[0099] like Figure 5 As shown in c, FMNS13 had the best hydroxyl radical scavenging ability of LAB strain fermentation metabolites, and its microbial fermentation activity products (24.28±1.48%) and microbial cell suspension (16.36±1.02%) were not significantly different from those of LGG strains (26.39±1.02%, 12.56±1.36%) ( Figure 5 c1~c2, p>0.05). However, the hydroxyl radical scavenging ability of the cell-free extract of FMNS13 strain (30.14±0.14%) was significantly higher than that of the control strain LGG (24.98±0.30%) (p<0.05).

[0100] like Figure 5 As shown in Figure d, the reducing ability of LAB strains was between 6% and 30%, among which FMNS10 and FMNS13 had the highest scavenging activity. The reducing abilities of their microbial fermentation active products (31.11±1.30%, 30.18±1.61%), microbial cell suspensions (10.83±1.50%, 9.70±1.95%) and microbial cell-free extracts (10.89±1.09%, 10.04±2.20%) were not significantly different from those of the positive control strain LGG (28.17±1.23%, 10.11±1.38% and 15.09±1.00%) (p<0.05).

[0101] Example 9: Analysis of in vitro probiotic properties of LAB

[0102] SPSS Statistics 25 was used to analyze the in vitro probiotic properties of 7 lactic acid bacteria isolated from kumis: principal component analysis (PCA) was performed on 21 indicators, including colonization ability (hydrophobicity, self-aggregation and co-aggregation), antibacterial activity (including inhibition of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella), antioxidant capacity (DPPH scavenging ability, hydroxyl radical scavenging ability, superoxide anion scavenging ability and total reducing power) and tolerance to gastrointestinal fluid survival rate. PCA can reduce the dimensionality of the data set while retaining most of the information in the data.

[0103] Table 1 Eigenvalues ​​and contribution rates of principal components

[0104]

[0105] Table 2 Score load matrix and ranking of each component of experimental lactic acid bacteria

[0106]

[0107] As shown in Table 1, five principal components (PCs) are proposed after dimensionality reduction of the 21 indicators, and the cumulative contribution rate of variance of these five principal components is 95.03%, that is, these five principal components can represent most of the information of the aforementioned 21 indicators.

[0108] The PCA analysis showed that the loading matrix of the scores of each component of the experimental lactic acid bacteria (Table 2) and the scores of each principal component Y x , calculation formula:

[0109] Y A =-0.333Y1+0.16Y2+0.084Y3+0.415Y4+0.051Y5+0.624Y6+0.301Y7+0.84Y8+0.814Y9+0.141Y 10 +0.801Y 11 +0.778Y 12 +0.823Y 13 +0.572Y 14 +0.748Y 15 +0.335Y 16 -0.022Y 17 +0.868Y 18 +0.083Y 19 +0.122Y 20 +0.369Y 21 ;

[0110] Y B<h2 style=";text-align:left;direction:ltr">=-0.037Y1+0.907Y2+0.1Y3+0.602Y4+0.811Y5+0.675Y6+0.913Y7-0.074Y8+0.452Y9+0.243Y<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> +0.418Y<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> +0.249Y<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> -0.136Y<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> +0.393Y<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> +0.435Y1+0.1155Y<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> +0.732Y<h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> +0.35Y<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> +0.192Y<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> -0.01Y<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> -0.009Y<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> ;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0111] <h2 style=";text-align:left;direction:ltr"> Y<h2 style=";text-align:left;direction:ltr"> C <h2 style=";text-align:left;direction:ltr"> =0.101Y1+0.031Y2+0.912Y3-0.176Y4+0.01Y5+0.293Y6+0.211Y7-0.121Y8-0.246Y9-0.118Y<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> +0.296Y<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> +0.232Y<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> +0.447Y<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> +0.036Y<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> +0.102Y<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> -0.648Y<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> -0.209Y<h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> -0.053Y<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> +0.656Y<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> +0.9Y<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> +0.526Y<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> ;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0112] <h2 style=";text-align:left;direction:ltr"> Y<h2 style=";text-align:left;direction:ltr"> D <h2 style=";text-align:left;direction:ltr"> = 0.176Y1 + 0.002Y2 + 0.289Y3 + 0.631Y4 + 0.496Y5 - 0.238Y6 - 0.1Y7 - 0.007Y8 - 0.259Y9 - 0.943Y<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> +0.01Y<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> +0.351Y<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> +0.039Y<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> +0.679Y<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> +0.443Y<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> +0.021Y<h2 style=";text-align:left;direction:ltr"> 16 <h2 style=";text-align:left;direction:ltr"> -0.047Y<h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> +0.334Y<h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> +0.379Y<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> -0.268Y<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> +0.708Y<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> ;

[0113] Y E =0.912Y1-0.306Y2+0.011Y3+0.048Y4+0.302Y5-0.054Y6+0.144Y7-0.089Y8+0.045Y9-0.126Y 10 -0.241Y 11 +0.23Y 12 +0.033Y 13 -0.131Y 14 -0.207Y15+0.669Y 16 -0.642Y 17 -0.09Y 18 -0.334Y 19 +0.311Y 20 +0.269Y 21 ;

[0114] The variance contribution rate of each principal component is used as the weight to construct a comprehensive evaluation model:

[0115] Y=0.43Y1+0.18Y2+0.13Y3+0.12Y4+0.09Y5The comprehensive total score of each group was calculated according to the model (Table 2). The 16S rRNA gene sequencing results of FMNS13 and 1.1 strains, which ranked first in the comprehensive total score of lactic acid bacteria screened out from traditional kumiss, showed that the sequence identity of FMNS13 strain and Lactobacillus amylovorus strain LAB16 strain was high (96.78%). Lactobacillus amylovorus FMNS13 strain was selected for the next safety experiment and freeze-drying protection.

[0116] Example 10: LAB Microbial Safety Evaluation

[0117] The safety of lactic acid bacteria curd isolates depends on their hemolytic activity and antibiotic susceptibility. For hemolytic activity, overnight grown MRS broth cultures of lactobacillus strains were streaked on blood agar plates (Haibo Biotechnology, Qingdao, China) and incubated at 37°C for 72 hours; thereafter, the plates were observed for the formation of any clean (β-hemolysis) or green (α-hemolysis) hemolytic zones around the lactic acid bacteria colonies, or the absence of such hemolytic zones (γ-hemolysis). Antibiotic susceptibility analysis Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method (KB method). The microorganisms to be tested were inoculated into MRS broth and cultured at 37°C for 18 hours and activated for two generations. The bacterial solution was collected with a sterile cotton swab and evenly spread on an agar (MHA) plate. Then, a paper disc containing antibiotics was attached to the MHA. The plates were placed in an incubator and cultured at 37°C for 24 hours, and the diameter of the inhibition zone was measured by a vernier caliper. The drug sensitivity test was conducted using 13 antibiotic sensitivity papers (Aisi Microbiology Co., Ltd., Hangzhou, China). The drug names and doses are shown in Table 3.

[0118] Table 3 Antibiotic sensitivity evaluation

[0119]

[0120]

[0121] Hemolysis test is one of the important means to evaluate the safety of lactic acid bacteria. By testing whether lactic acid bacteria are hemolytic, it can be determined whether they may cause red blood cells to rupture, thereby evaluating their safety in food, health products or medical fields. Hemolysis is divided into three categories: α-hemolysis is characterized by a grass-green hemolysis circle around the colony; β-hemolysis is characterized by a transparent hemolysis circle around the colony; γ-hemolysis is no hemolysis, and the culture medium around the colony does not change

[0122] like Figure 6 As shown in the figure, after the positive control Staphylococcus aureus was streaked and cultured in Columbia blood agar medium for 48 hours, a grass-green hemolytic ring (α-hemolysis) appeared around it. In addition to its hemolytic effect, α-hemolysin can also damage platelets, macrophages and leukocytes, cause vascular smooth muscle contraction, and lead to local tissue ischemia and necrosis; while FMNS13 did not have a hemolytic ring (γ-hemolysis), that is, the FMNS13 strain would not destroy sensitive components such as red blood cells, thereby reducing its risk as a potential pathogen.

[0123] The sensitivity of FMNS13 strain to antibiotics is shown in Table 3. The results of drug sensitivity tests showed that FMNS13 strain was sensitive to five antibiotics, tetracycline, penicillin, azithromycin, kanamycin and gentamicin, and had strong tolerance to four antibiotics, cefotaxime, ceftazidime, erythromycin and amikacin. When taking antibiotics, both beneficial and harmful bacteria in the intestines are often killed, which may lead to an imbalance in the intestinal flora and cause adverse reactions such as diarrhea. If probiotics themselves have a certain resistance to antibiotics, they can survive during antibiotic treatment and help maintain the balance of intestinal flora, thereby alleviating these adverse reactions. Drug sensitivity tests can evaluate the inhibitory or killing effects of different antibiotics on lactic acid bacteria, thereby understanding the potential impact of these antibiotics on intestinal microecology.

[0124] Example 11: Preparation of lyophilized powder using Lactobacillus amylovorus FMNS13

[0125] Among proteins, sugars and amino acid salts, skim milk powder (Biotopped, China, Bioreagent), D-trehalose (Aladdin, China, Bioreagent) and sodium L-glutamate (McLean, China, Bioreagent) were selected as ultra-low temperature freeze-drying protective agents for strains, respectively. Ultrapure water was used as negative control. Skim milk was sterilized at 108℃ for 15 minutes, and other experimental supplies were sterilized by high pressure sterilization at 115℃ for 15 minutes. The bacterial suspension was prepared according to the above description, and the suspension: cryoprotectant (W:V) ratio was 1:1 and mixed evenly. The mixture was pre-frozen at -80℃ (Panasonic, Japan) for 24 hours and frozen at -80℃ using a vacuum freeze dryer SCIENTZ-10N (Ningbo Xinzhi Biotechnology Co., Ltd., China) for 48 hours.

[0126] Skim milk powder was mixed with the bacterial suspension prepared in Example 2 at a ratio of 1:1 (V / V) according to a final concentration of 40 g / L, 80 g / L, 100 g / L, 160 g / L, 200 g / L, trehalose was mixed with a final concentration of 20 g / L, 40 g / L, 60 g / L, 80 g / L, 100 g / L, and sodium L-glutamate was mixed with a final concentration of 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L. The freeze-drying test was performed on the Lactobacillus amylovorus FMNS13 strain using the above-mentioned freeze-drying technical operation and procedure method. The test results were evaluated by viable cell counting.

[0127] The survival rate of Lactobacillus amylovorus FMNS13 strain was determined by the standard MRS agar plate counting method (GB 4789.35-2016, China). The recovered microbial cells or microbial freeze-dried powder were diluted 10 times in sterile saline (0.85%). Select the appropriate dilution gradient to spread on the MRS agar plate, culture at 37°C for 48h, and select the plate with 30 to 300 colonies and no scattered colony growth for counting. The number of microbial cells in the original sample was calculated together with the corresponding dilution multiple before spreading. This test was repeated three times.

[0128] Survival rate (%) = number of viable cells after lyophilization / number of viable cells before lyophilization × 100%.

[0129] The results are as follows Figure 7 As shown, when the mass fraction of skim milk powder was 12% (survival rate was 72.67±8.5%), or when the mass fraction of D-trehalose was 6% (survival rate was 83±3.61%), or when the mass fraction of L-glutamate was 4% (survival rate was 80.67±3.05%), the survival rate of strain FMNS13 after freeze-drying was relatively the highest. Based on the optimal concentration range obtained by single factor experiment, response surface experiment was designed.

[0130] The contents of skimmed milk powder (A), sodium glutamate (B), and trehalose (C) were used as independent variables, and three levels of high, medium, and low were set for each independent variable (A, B, C), represented by coded values ​​1, 0, and -1, respectively, and entered into the Desi-Expert13.0 program software.

[0131] Table 4 Results of single factor affecting strain survival rate guiding response interview design

[0132]

[0133] Table 5 Response surface experiment designed by Design-Expert 13.0 program and experimental results

[0134]

[0135] Table 6 Statistical parameters of response surface analysis

[0136]

[0137] Table 7 Analysis of variance for response surface experiment regression simulation

[0138]

[0139] Significance analysis: ** represents extremely significant (P < 0.01); * represents significant (0.01 < P < 0.05); no * represents not significant (P > 0.05).

[0140] Table 8 Verification of Response Surface Prediction Results

[0141]

[0142] A total of 15 independent experiments were arranged to observe and quantify the effects of different combinations of cryoprotectant concentrations on the survival rate (Y response) of freeze-dried strains by changing the concentration combinations of different cryoprotectants. In the experiment, the number of live cells was selected as the key quality index to measure the effect of cryoprotectants because it directly reflects the ability of cryoprotectants to maintain the activity of strains.

[0143] Through in-depth analysis of these data, the best quadratic polynomial regression model was fitted using Desi-Expert 13.0 software, and then the optimal combination of cryoprotectant concentrations to improve the freeze-drying survival rate of strains was identified.

[0144] The results of variance analysis and significance test of the quadratic model of the response surface of the freeze-drying survival rate (Y) of strain FMNS13 are shown in Table 8. This analysis reveals the complex relationship among the survival rate (Y), skim milk powder (A), sodium L-glutamate (B), and D-trehalose (C), and the regression equation of the quadratic interaction is obtained: Survival rate (Y) = 85.1 - 5.34A - 5.45B - 2.01C + 1.33AB - 1.4AC + 2.77BC - 16.82A 2 - 3.85B 2 - 1.58C 2 .

[0145] This regression model verified its extremely significant statistical significance through a highly significant P value (P < 0.0001), indicating that the constructed model can accurately describe the effects of various factors and their interactions on the freeze-drying survival rate of strain FMNS13. In addition, the test results of the response surface lack-of-fit term show no significant difference (P > 0.05), which proves that the quadratic polynomial model has an excellent fit with the experimental data, the model error is small, and the interference of unconsidered factors on the model prediction results can be ignored. Therefore, this model can effectively use the regression equation to replace the complex actual test process and provide a powerful tool for process optimization and condition screening.

[0146] Further analysis of the influence of each factor revealed that skim milk powder (A) and D-trehalose (C) had a significant effect on the freeze-dried survival rate (Y) of strain FMNS13 (p<0.05), indicating that the survival rate of the strain can be significantly changed when adjusting the concentration of these factors. In contrast, sodium L-glutamate (B) had a smaller effect on the survival rate and did not reach a statistically significant level (p>0.05), which may provide some flexibility in formulating the production process, that is, adjusting the dosage of sodium L-glutamate within a certain range may not have a significant effect on the final survival rate.

[0147] Taking the survival rate (Y) of freeze-dried strain FMNS13 as the response value, the response surface diagram and contour diagram between skimmed milk powder (A), sodium L-glutamate (B) and D-trehalose (C) can be obtained according to the ternary quadratic regression equation to reflect the strength of the interaction between the two factors, such as Fig.10 As shown. From the response surface diagram, it can be seen that the three-dimensional surface diagrams of skim milk (A) and D-trehalose (C) and skim milk (A) and sodium L-glutamate (B) are steeper than the three-dimensional surfaces of sodium L-glutamate (B) and D-trehalose (C), and the corresponding contour diagrams are closer to ellipses. This feature shows that the interaction between skim milk (A) and sodium L-glutamate (B) and D-trehalose (C) has a more significant effect on the freeze-dried survival rate (Y) of strain FMNS13. In contrast, the interaction between sodium L-glutamate (B) and D-trehalose (C) is relatively mild. And the openings of the three-dimensional surface of the response surface are all downward, which means that the effect of the selected protective agent on the freeze-dried survival rate of strain FMNS13 is a trend of increasing first and then decreasing, with the maximum response value.

[0148] For the response surface prediction group, the mass fraction ratio of the optimal compounding scheme was obtained by fitting the regression equation, so that the survival rate could reach up to 89.72%. It has been verified that under the action of the compound protective agent of skim milk (final concentration of 114g / L), sodium L-glutamate (final concentration of 32g / L) and D-trehalose (final concentration of 65g / L), the survival rate of strain FMNS13 was 90.77±1.88%, which was not significantly different from the response surface prediction value. It can be proved that the protective agent formula optimized in this experiment has a significant protective effect on the freeze-dried survival rate of Lactobacillus amylovorus FMNS13 strain screened out from Inner Mongolia fermented kumis.

[0149] Example 12: Scanning electron microscopy examination of Lactobacillus amylovorus FMNS13 after freeze drying

[0150] The surface structure of the freeze-dried lactic acid bacteria powder was observed by scanning electron microscopy (SEM TM3000, Hitachi, Japan). Fig. 9 As shown. To evaluate the effect of dehydration on bacterial morphology, the lyophilized powder was first washed twice with 0.1M PBS buffer and then fixed overnight with 2.5% glutaraldehyde in PBS at 4°C. After the samples treated overnight were washed with PBS, the samples were dehydrated in a series of ethanol solutions, including 30%, 50%, 70%, 80% and 90% ethanol, soaked for 10 minutes at each concentration, and finally treated with 100% ethanol twice, each for 10 minutes. Next, the bacterial samples were dried in a dryer for about 4 hours. All samples were then adhered to an aluminum plate with double-sided conductive tape and sprayed with a gold-palladium alloy layer using an ion sputtering coater to enhance their conductivity. Finally, the samples were morphologically observed by SEM.

[0151] Since freeze-drying includes freezing, primary drying (sublimation) and secondary drying (desorption), the water in the solution sublimates directly from ice to water vapor in a vacuum environment, so the surface of the powder becomes microporous due to water loss. Fig.10 a shows the normal morphology of the FMNS13 strain, which is long and stick-shaped with a full appearance and no deformation or damage. Fig.10 c is the freeze-dried powder without lyoprotectant. After fixation and gradient elution, the freeze-dried powder was observed by SEM. The cells were severely deformed and the cell membrane was broken. Fig.10 e is freeze-dried powder with added composite freeze-dried protective agent. After fixation and gradient elution, the freeze-dried powder was observed by SEM. The lactic acid bacteria had a smooth morphology and the cell membrane had no obvious damage.

[0152] Fig.10 c and d show the freeze-dried powder without adding antifreeze. The powder was directly observed by SEM. The cells were severely deformed and the cells were piled together, which may be caused by cell deformation or rupture and leakage of intracellular substances. Fig.10 b is the freeze-dried powder with the addition of composite cryoprotectant, with a smooth surface and the powder completely covered by the coating, which reduces the contact area between the bacteria and the outside world to a certain extent, while also isolating oxygen, avoiding oxidative damage, and maintaining the integrity of the cell structure. SEM shows that the cryoprotectant in this study can effectively maintain the integrity of the cell membrane structure and the permeability barrier, preventing leakage of cell contents.

[0153] Example 13: Detection of cell membrane fluidity and integrity of Lactobacillus amylovorus FMNS13

[0154] PI dye (Propidium Iodide, PI), is an analog of ethidium bromide, which cannot pass through intact cell membranes, but can enter the cell through damaged cell membranes. Once inside the cell, PI will be embedded between the bases of double-stranded DNA, and after binding to DNA, it will release strong red fluorescence under excitation light.

[0155] SYTO 9 dye has excellent cell membrane permeability, high affinity and fluorescence properties. Unlike PI, SYTO 9 dye can only enter living cells with intact cell membranes. After binding to DNA (and RNA), it will emit bright green fluorescence at an excitation wavelength of 498nm, thus clearly marking the presence of living cells. When SYTO 9 and PI dyes are used together, it is possible to distinguish whether microbial cells contain intact cell membranes.

[0156] To prepare the sample suspension, first take out 1 mL of the pre-freeze-dried bacterial solution. At the same time, prepare two freeze-dried bacterial powders (both of which need to be rehydrated in 0.1M PBS), one is 0.08g treated with a compound protective agent, and the other is 0.01g without a compound protective agent, to restore the dissolved state of both. Wash the precipitates obtained after centrifugation of the three samples (0.1M PBS) to remove residual impurities or protective agents. After washing, resuspend the precipitate in an equal volume of 0.1M PBS to ensure the formation of a uniform and stable suspension. The prepared sample suspension can be used for staining observation, cell membrane fluidity analysis, and determination of key enzymes in lactic acid bacteria metabolism.

[0157] To mark the integrity of the microbial cell membrane, take the sample suspension prepared above, add 5 μL of PI (propidium iodide, used to mark cells with damaged cell membranes) and 5 μL of SYTO9 (a green fluorescent dye that can penetrate and bind to living cells) to the suspension. After gently mixing, incubate at room temperature for 10 minutes to allow the dye to fully bind to the cells. After incubation, wash the stained suspension twice with 0.1M PBS to remove unbound dye. After that, take an appropriate amount of the stained suspension and drop it onto a clean glass slide, and gently cover it with a coverslip (be careful to avoid bubbles). Finally, use a laser confocal microscope (LSM 9000, ZEISS, Germany). Observe the staining of microbial cells under the dual fluorescence staining technique. PI will mark and display red fluorescence, indicating cells with damaged cell membranes; while SYTO9 makes living cells show green fluorescence. By contrasting these two colors, the integrity of the microbial cell membrane can be intuitively evaluated.

[0158] The cell membrane fluidity was determined using the 8-anilino-1-naphthalenesulfonate (ANS) fluorescence probe method. In the cell membrane, the fluorescence intensity (IF) of ANS reflects the changes in the membrane lipid binding site environment. The increase in the fluidity of microbial cell membrane lipids will make the ANS molecules move more freely in the membrane, thereby reducing the quantum yield of its fluorescence emission, that is, the IF value of ANS is negatively correlated with the cell membrane fluidity. The smaller the fluorescence intensity value, the higher the fluidity of the membrane lipid; conversely, the larger the fluorescence intensity value, the lower the fluidity of the membrane lipid. As mentioned above, the sample was washed to remove the protective agent and resuspended in PBS (PH7.2, 50mM, containing 0.5mM dithiothreitol DTT and 0.1% Tween 20). The cell suspension was ultrasonically disrupted in an ice bath (Ningbo Xinyi Ultrasonic Equipment Co., Ltd., China), with an ultrasonic power of 200W, turned on for 3 seconds, turned off for 6 seconds, and a total of 6 minutes. The disrupted solution was centrifuged at 10,000 g for 20 min at 4 °C, and the supernatant was added to the ANS solution (10 μmol / L). The mixed solution was incubated at room temperature for 20 min, and then the fluorescence emission spectrum of ANS was recorded at an excitation wavelength of 385 nm and an emission wavelength of 480 nm using a fluorescence spectrophotometer (Hitachi F-7000, China).

[0159] Evaluation of the cell membrane integrity and fluidity of strain FMNS13 in lyophilized powder protected by skim milk (final concentration of 114 g / L), sodium L-glutamate (final concentration of 32 g / L) and D-trehalose (final concentration of 65 g / L) lyophilization protectants, such as Fig.10 As shown in the figure, these two characteristics are key indicators of cell survival status and are crucial to understanding how microorganisms respond to external environmental pressures. By implementing dual fluorescent staining technology, we intuitively observed the significant impact of freeze-drying, an extreme treatment condition, on the integrity of the cell membrane of strain FMNS13. In this experiment, living cells exhibited green fluorescence due to intact cell membranes, while dead cells emitted red fluorescence due to damaged cell membranes ( Fig.10 d). It is worth noting that when the compound lyoprotectant was introduced during the freeze-drying process, the cell membrane integrity of strain FMNS13 was effectively protected, as manifested by an increase in the proportion of green fluorescent cells. Furthermore, using the characteristics of the ANS probe binding to the cell membrane, we indirectly evaluated the fluidity of the cell membrane. The fluorescence intensity of the ANS probe is negatively correlated with the fluidity of the cell membrane, that is, the higher the fluorescence intensity, the lower the fluidity of the cell membrane. The experimental results showed that the cell membrane fluidity of the unprotected strain FMNS13 decreased significantly after freeze-drying, as manifested by a significant increase in fluorescence intensity at a wavelength of 480nm, and this change was statistically different from that before freeze-drying ( Fig.10b, p<0.05). However, when the compound protective agent was used, the cell membrane fluidity of strain FMNS13 was well maintained, and its fluorescence intensity at a wavelength of 480 nm was not significantly different from that before freeze-drying ( Fig.10 b, p>0.05). In summary, the dual evidence of dual fluorescence staining and ANS probe detection strongly supports the positive protective effect of cryoprotectants on the cell membrane integrity and fluidity of strain FMNS13 during freeze-drying. This discovery not only deepens the understanding of the survival mechanism of microbial cells under adverse conditions by cryoprotectants, but also provides a valuable reference for optimizing freeze-drying processes and improving the stability of biological products.

[0160] Example 14: Determination of key enzyme activities in lactic acid bacteria metabolism

[0161] In order to evaluate the effect of lyoprotectant on enzyme activity during freeze drying of FMNS13 strain, this example measured the activity of key metabolic enzymes of lactic acid bacteria in three strains after strain activation, freeze drying without protective agent, and freeze drying with protective agent. Including the enzyme activities of four enzymes: phosphate lyase (PKase), β-galactosidase (β-galactosidase), L-lactate dehydrogenase (L-LDH) and Na+-K+-ATPase. Among them, the lyoprotectant formula is: skim milk 114g / L, L-sodium glutamate 32g / L, D-trehalose 65g / L.

[0162] The activities of key enzymes in lactic acid bacteria metabolism during the freeze-drying process were determined using a PKase activity assay kit, a β-galactosidase activity assay kit, an L-LDH activity assay kit, and a Na+ / K+-ATPase activity assay kit (four activity assay kits, Beijing Soleb Technology Co., Ltd.).

[0163] Effects of freeze drying on the activities of key metabolic enzymes in strain FMNS13 Fig.11 As shown. In the preparation process of microbial powder without compounding drying protective agent, the key enzyme activities of the powder obtained after freeze-drying decreased significantly, specifically, Na+ / K+-ATPase activity decreased by 39.63%, β-galactosidase activity decreased by 47.36%, PKase activity decreased by 44.62%, and L-LDH activity decreased by 44.85%. These changes were statistically significant (p<0.05).

[0164] Example 15: Application of Lactobacillus amylovorus FMNS13 in the preparation of freeze-dried okra

[0165] Wash the fresh okra, cut off the inedible parts at both ends, and cut the middle section of the okra into small sections with a thickness of about 7 mm for later use. After the FMNS13 strain is activated, it is resuspended in the protective agent to prepare a bacterial-protective agent suspension (so that the bacterial concentration reaches 10 12 CFU / mL), soak the okra segments cut into small pieces in the bacteria-protectant suspension at 4°C for 8 hours. After soaking, take out the okra and pre-freeze it in a -80°C refrigerator for 12 hours, and freeze it at -80°C using a vacuum freeze dryer SCIENTZ-10N (Ningbo Xinzhi Biotechnology Co., Ltd., China) for 48 hours. After freeze drying, take out the sample for gradient dilution and spread on the plate to verify the bacterial load of probiotic freeze-dried okra.

[0166] The experimental results show that ( Fig.12 ), the bacterial load of freeze-dried okra can reach 1×10 8 CFU / g. This means that the FMNS13 strain can be well attached to freeze-dried okra. From the morphology of okra after freeze-drying, the addition of the protective agent will not have a negative impact on the freeze-dried morphology of okra, but can also resist the cold damage index of okra caused by the harsh environment during freeze-drying to a certain extent, and prevent the oxidative browning caused by direct contact between the okra body and the air. This provides a theoretical basis for the application of strains in freeze-dried fruits and vegetables.

[0167] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactobacillus amylovorus FMNS13 was deposited in Guangdong Microbiological Culture Collection Center on August 20, 2024, with the deposit number GCMCC No: 65020.

2. A probiotic preparation containing the Lactobacillus amylovora FMNS13.

3. The probiotic preparation according to claim 2, characterized in that The number of Lactobacillus amylovora FMNS13 in the probiotic preparation is ≥1×10 12 CFU / g.

4. A freeze-dried powder containing the Lactobacillus amylovora FMNS13 according to claim 1.

5. A method for preparing the freeze-dried powder according to claim 4, characterized in that: The bacterial suspension of Lactobacillus amylovora FMNS13 according to claim 1 is mixed with a freeze-drying protective agent containing skim milk, sodium L-glutamate and D-trehalose, and then vacuum-freeze-dried at -80°C.

6. The method according to claim 5, characterized in that The freeze-drying protective agent contains 40-200 g / L of skim milk, 30-70 g / L of sodium L-glutamate, and 20-100 g / L of trehalose.

7. The method according to claim 5, characterized in that The concentration of skim milk in the freeze-drying protective agent is 110-115 g / L, the concentration of sodium L-glutamate is 30-35 g / L, and the concentration of D-trehalose is 64-68 g / L.

8. Use of the Lactobacillus amylovora FMNS13 or its postbiotics according to claim 1 in the preparation of health products or medicines with antioxidant function.

9. The use according to claim 8, characterized in that: The postbiotics include but are not limited to the fermentation supernatant of the Lactobacillus amylovora FMNS13.

10. Use of the Lactobacillus amylovora FMNS13 according to claim 1 in preparing freeze-dried vegetables.

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