A strain of Lactobacillus rhamnosus SAL2 and its preparation and application

Abstract

The present invention provides a new Lactobacillus rhamnosus SAL2 which is deposited in CGMCC with the deposit number of CGMCC No.32935 and its preparation and application. Lactobacillus rhamnosus SAL2 tolerates the human digestive tract environment, does not produce hemolysin, and has good biological safety; it has strong carbohydrate anabolism, anti-oxidation and exopolysaccharide secretion capabilities, the self-aggregation of the strain reaches more than 99%, and it is hydrophilic and has excellent probiotic properties.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a strain of Lactobacillus rhamnosus SAL2 and a preparation and application thereof. Background Art

[0002] Lactobacillus rhamnosus is a Gram-positive bacillus that plays an important role in balancing intestinal flora, regulating the immune system, anti-oxidation and resisting intestinal pathogens. Due to its probiotic properties and safety, Lactobacillus rhamnosus is one of the most intensively studied and widely used probiotics, with extremely high application value and development prospects. Summary of the invention

[0003] The present invention provides a new Lactobacillus rhamnosus SAL2 product and its preparation. Lactobacillus rhamnosus SAL2 tolerates the human digestive tract environment, does not produce hemolysin, has good biological safety, has strong carbohydrate anabolism, anti-oxidation and exopolysaccharide secretion capabilities, has a self-aggregation of more than 99%, is hydrophilic, and has excellent probiotic properties.

[0004] In order to achieve the first invention object of the present invention, the present invention provides Lactobacillus rhamnosus SAL2 which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee CGMCC, with a deposit number of CGMCC No.32935 and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 6, 2024, and the classification name is Lactobacillus rhamnosus; Latin name: Lactobacillus rhamnosus.

[0005] To achieve the second object of the present invention, the present invention provides the use of Lactobacillus rhamnosus SAL2 in inhibiting bacterial growth. Preferably, the bacteria include Staphylococcus aureus, Enterococcus faecium, Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella typhimurium, and Shigella flexneri.

[0006] In order to achieve the third object of the present invention, the present invention provides the use of Lactobacillus rhamnosus SAL2 in anti-oxidation.

[0007] In order to achieve the fourth object of the present invention, the present invention provides an antibacterial agent, wherein the antibacterial agent contains Lactobacillus rhamnosus SAL2 as its effective ingredient.

[0008] In order to achieve the fifth object of the present invention, the present invention provides an antioxidant, wherein the antioxidant contains Lactobacillus rhamnosus SAL2 as its effective ingredient.

[0009] In order to achieve the sixth object of the present invention, the present invention provides the use of Lactobacillus rhamnosus SAL2 in the preparation of chemical drugs, foods and health products.

[0010] Different from the prior art, the above technical solution includes at least the following beneficial effects: providing a new Lactobacillus rhamnosus SAL2 and its antibacterial agent which are preserved in CGMCC with the preservation number of CGMCC No.32935. Lactobacillus rhamnosus SAL2 tolerates the human digestive tract environment, does not produce hemolysin, and has good biological safety; it has antibiotic resistance and strong carbohydrate anabolism, antioxidant and exopolysaccharide secretion capabilities; the self-aggregation of the strain reaches more than 99%, and it is hydrophilic and has excellent probiotic properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the Gram staining result of Lactobacillus rhamnosus SAL2.

[0012] Figure 2 This is a graph showing the results of the hemolysis test.

[0013] Figure 3 This is the result of drug sensitivity test.

[0014] Figure 4 Comparison of high salt tolerance. A. Colony growth under different concentrations of NaCl. L. rhamnosus SAL2 and L. rhamnosus GG were diluted to 10 -5 B. Survival rate of strains under different concentrations of NaCl. Error bars represent Mean±SD of data from three repeated experiments, ns indicates p>0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0015] Figure 5 Comparison of tolerance to artificial intestinal fluid. A. Bacterial colony growth in artificial intestinal fluid. L. rhamnosus SAL2 and L. rhamnosus GG were diluted to 10 -4 B. Survival rate of strains in artificial intestinal fluid. Error bars represent Mean±SD of data from three repeated experiments, ns indicates p>0.05, and * indicates p<0.05.

[0016] Figure 6 Comparison of antibacterial activity. A. Antibacterial effect of Lactobacillus rhamnosus on each indicator bacteria. B. Antibacterial test results. Error bars represent Mean±SD of 3 repeated test data, ns indicates p>0.05, * indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001.

[0017] Figure 7Comparison of H2O2 tolerance. A. Colony growth under different H2O2 concentrations. L. rhamnosus SAL2 was diluted to 10 at 1-9 mM H2O2 concentration. -4 , diluted to 10 at 10mM H2O2 concentration -3 L. rhamnosus GG was diluted to 10 at 1-2 mM H2O2 concentration. -4 , diluted to 10 at 3 mM H2O2 concentration -3 , diluted to 10 at 4-7mM H2O2 concentration -2 , 8-10mM H2O2 concentration is the original solution. B. Survival rate of strains under different H2O2 concentrations. Error bars represent Mean±SD of 3 repeated experiments, * indicates p<0.05, **** indicates p<0.0001.

[0018] Figure 8 Comparison of DPPH radical scavenging ability of GG. A. DPPH radical scavenging rate of fermentation supernatant at different culture times. B. DPPH radical scavenging rate of bacterial suspension at different culture times. Error bars represent Mean±SD of data from 3 repeated experiments, ns means p>0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001. Fig. 9 The figure is a comparison of hydroxyl radical scavenging ability. A. Hydroxyl radical scavenging rate of fermentation supernatant at different culture times. B. Hydroxyl radical scavenging rate of bacterial suspension at different culture times. Error bars represent Mean±SD of 3 repeated experiments, ns indicates p>0.05, ** indicates p<0.01.

[0019] Fig.10 The figure is a comparison of superoxide anion radical scavenging ability. A. Superoxide anion radical scavenging rate of fermentation supernatant at different culture times. B. Superoxide anion radical scavenging rate of bacterial suspension at different culture times. Error bars represent Mean±SD of 3 repeated experiments, ns means p>0.05, * means p<0.05, **** means p<0.0001.

[0020] Fig.11 This is a circle map of the L. rhamnosus SAL2 genome. Note: From the inside to the outside, the first circle represents the gene size; the second circle represents the GC Skew; the third circle represents the GC content; the fourth and seventh circles represent the COG to which each CDS belongs; the fifth and sixth circles represent the positions of CDS, tRNA and rRNA on the genome. DETAILED DESCRIPTION

[0021] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0022] 1. Separation and purification:

[0023] 1. Isolation and purification of Lactobacillus:

[0024] The Lactobacillus rhamnosus SAL2 strain was isolated from fresh stool specimens of patients with colon cancer who were treated with antibiotics, hormones, and other drugs for a long time. The feces of the patients were selected and inoculated on MRS solid plates containing calcium carbonate for separation and streaking, and placed in a LOCK fresh-keeping box (with a built-in Mitsubishi anaerobic gas-producing bag). After anaerobic culture at 37°C for 48 hours, a single colony of medium size, milky white, and with a calcium-dissolving ring was selected for Gram staining and catalase test. The strains with negative catalase test, positive Gram staining, and long rod-shaped without spores were suspected lactobacilli, and they were subcultured on MRS medium for preservation.

[0025] The test results are as follows Figure 1 As shown, these are its morphological characteristics under the microscope after Gram staining. The bacteria are straight rod-shaped with blunt ends and exist individually or in short chains.

[0026] 2. Identification of Lactobacillus species

[0027] With reference to Bergey's Manual of Bacterial Identification (8th edition), the purified strains isolated were tested to determine whether each strain belonged to the genus Lactobacillus. The results are as follows: the isolated strain did not liquefy gelatin, and the nitrate reduction test, indole test, and hydrogen sulfide test were negative, and it was preliminarily identified as a Lactobacillus bacterium. The API identification result determined that it was Lactobacillus rhamnosus, and was named Lactobacillus rhamnosus SAL2.

[0028] Lactobacillus rhamnosus SAL2 originates from the feces of patients with colon cancer. Due to the long-term treatment of patients, their immune system function is impaired, making them more susceptible to infection. In addition, the frequent use of antibiotics or hormones in clinical treatment will further aggravate the imbalance of intestinal flora, making it difficult for probiotics to colonize in the body. Therefore, probiotics isolated in this living environment may have stronger tolerance and biological activity.

[0029] 2. Performance Testing

[0030] This study used in vitro tests to comprehensively analyze the probiotic properties and safety of L. rhamnosus SAL2 through high salt, artificial gastric juice, artificial intestinal juice tolerance tests, adhesion tests, antibacterial tests, drug sensitivity tests, hemolysis tests, and antioxidant tests.

[0031] 2.1. Strains

[0032] (1) The strain of the present application: L. rhamnosus SAL2. (SAL2)

[0033] (2) Classic strain: L. rhamnosus GGATCC53103 (GG) was purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0034] (3) Indicator strains: Staphylococcus aureus, Enterococcus faecium, Listeria monocytogenes, Klebsiella pneumoniae, Salmonella typhimurium, Shigella flexneri, Pseudomonas aeruginosa, and Acinetobacter baumannii were purchased from Wenzhou Kangtai Biotechnology Co., Ltd.

[0035] 2.2 Preparation of culture medium and main reagents

[0036] (1) MRS agar medium: peptone 10.0 g, yeast extract powder 4.0 g, glucose 20.0 g, beef extract powder 8.0 g, magnesium sulfate 0.2 g, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2 g, sodium acetate 2.5 g, diammonium hydrogen citrate 2.0 g, manganese sulfate 0.04 g, Tween 80 1.0 g, agar 14.0, adjust pH to 6.5±0.2, heat and dissolve in 1000 mL of deionized water, sterilize by high pressure at 121°C for 20 min, and pour into a sterile plate when cooled to about 60°C.

[0037] (2) MRS broth medium: peptone 10.0 g, yeast extract powder 4.0 g, glucose 20.0 g, beef extract powder 8.0 g, magnesium sulfate 0.2 g, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, diammonium hydrogen citrate 2.0 g, manganese sulfate 0.04 g, Tween 80 1.0 g, adjust pH to 5.7 ± 0.2, dissolve in 1000 mL of deionized water by heating, and sterilize by high pressure at 121 °C for 20 min.

[0038] (3) Solid agar medium: 5 g sodium chloride, 15 g agar, 10 g peptone, adjust pH to 7.3±0.1, heat and dissolve in 1000 mL deionized water, sterilize by high pressure at 121°C for 20 min, and pour into a sterile plate when cooled to about 60°C.

[0039] 2.3 Strain activation: Under sterile conditions, Lactobacillus rhamnosus was inoculated into MRS broth medium, and indicator bacteria were inoculated into nutrient broth medium for culture. After three consecutive passages, a single colony with typical colony characteristics was selected for subsequent testing.

[0040] 2.4 Activation of seed solution: Pick single colonies of L. rhamnosus SAL2 and L. rhamnosus GG and inoculate them into MRS broth medium respectively, and culture them in a shaking incubator at 37°C and 150 r / min for 16 h to obtain the seed solution.

[0041] 2.5 Hemolysis test

[0042] Use Staphylococcus aureus as the quality control bacteria, divide and streak with a sterile inoculation loop, inoculate Lactobacillus rhamnosus and the quality control bacteria on Columbia blood agar plates, culture at 37°C for 24 hours, and observe whether there is a hemolytic zone around the colonies on the plates.

[0043] The results are as follows Figure 2 As shown, Staphylococcus aureus showed a transparent hemolytic ring, which was β-hemolysis; L. rhamnosus SAL2 and L. rhamnosus GG did not show hemolysis.

[0044] 2.6 Drug sensitivity test

[0045] The KB drug sensitivity paper diffusion method was used for drug sensitivity test. Figure 3 As shown in Table 1, L. rhamnosus SAL2 was sensitive to tetracycline, erythromycin, penicillin, gentamicin, clindamycin, cefotaxime, levofloxacin, and linezolid; intermediate to cefepime; and resistant to vancomycin.

[0046] Table 1 Drug sensitivity of Lactobacillus rhamnosus SAL2

[0047]

[0048] Note: S: sensitive; I: intermediate; R: resistant

[0049] Antibiotic resistance helps strains survive in an antibiotic environment and maintain the balance of intestinal flora. Different Lactobacillus rhamnosus has different sensitivities to antibiotics, and this difference is closely related to its genetic structure and metabolic pathway. Because L. rhamnosus SAL2 has no plasmid, its inherent resistance genes do not have the ability to transfer horizontally to other bacteria, so this strain can be used in parallel with certain drugs. L. rhamnosus SAL2 is resistant to vancomycin and can be used in parallel with vancomycin to continue to play its probiotic role during treatment.

[0050] 2.7 High salt tolerance test

[0051] MRS broth medium containing NaCl concentrations of 0, 1, 2, 4, 6, and 8 g / 100 mL was prepared, dispensed into test tubes, sterilized by high pressure steam at 121°C for 20 min, and cooled to room temperature. L. rhamnosus SAL2 and L. rhamnosus GG seed liquid were inoculated into MRS broth medium containing different NaCl concentrations at a 10% inoculum, cultured at 37°C and 150 r / min on a shaking incubator for 24 h, and after gradient dilution, colony counts were performed using the spotting method, and the survival rate was calculated according to formula (1).

[0052] Survival rate (%) = N t / N0×100% (1)

[0053] Where: N t is the number of viable bacteria of the test strain after cultivation (lg CFU / mL); N0 is the number of viable bacteria in the seed solution of the test strain (lg CFU / mL).

[0054] The results are as follows Figure 4 As shown in the figure, the survival rates of the two strains of Lactobacillus rhamnosus gradually decreased with the increase of NaCl concentration. When the NaCl concentration was higher than 4 g / 100 mL, the survival rate of L. rhamnosus SAL2 increased. The survival rate of L. rhamnosus GG was higher than that of L. rhamnosus SAL2 when the NaCl concentration was 1, 2, and 4 g / 100 mL, but at high NaCl concentrations (6 and 8 g / 100 mL), the survival rate of L. rhamnosus SAL2 was significantly higher than that of L. rhamnosus GG.

[0055] In the human body, natural ecology and food processing, high salt conditions and the accompanying high osmotic pressure environment are common phenomena. When the salt concentration increases, the external osmotic pressure increases, and the concentration of the solution inside the bacteria is relatively low, which causes a large amount of water loss in the bacteria, leading to significant changes in the internal biochemical reaction environment, and then causing various metabolic activities in the cell to become disordered, and even causing cell dehydration and death. L. rhamnosus SAL2 has a strong tolerance to high salt and has a higher survival rate under high concentration NaCl conditions.

[0056] 2.8 Artificial gastric juice tolerance test

[0057] Use 37% concentrated hydrochloric acid to prepare acid solution with pH of 2.0 and 2.5, filter and sterilize with a syringe filter with a pore size of 0.22 μm, and add pepsin at a concentration of 1 g / 100 mL to obtain artificial gastric juice. Inoculate L. rhamnosus SAL2 seed solution into artificial gastric juice at a 10% inoculum, and culture at 37°C, 150 r / min, shaker for 1 h, 2 h, and 4 h. Take out the bacterial solution, and dilute it stepwise (10 -4 ), colony counts were performed using the clock method, and the survival rate was calculated according to formula (1).

[0058] The results showed that the survival rates of L. rhamnosus SAL2 in artificial gastric juices of pH 2.0 and pH 2.5 fluctuated slightly and were both higher than 87%.

[0059] 2.9 Artificial intestinal fluid tolerance test

[0060] Weigh 3.4 g of KH2PO4, dissolve it in distilled water and make up to 250 mL, adjust the pH to 6.8 with 0.4% NaOH solution, dilute it with distilled water to make up to 500 mL, and sterilize it with high pressure steam at 121°C for 20 min. Add trypsin at a concentration of 1 g / 100 mL, filter and sterilize it with a syringe filter with a pore size of 0.22 μm, and then divide it into packages to obtain artificial intestinal fluid. Inoculate L. rhamnosus SAL2 and L. rhamnosus GG seed liquids in artificial intestinal fluid at a 10% inoculation rate, and culture them in a shaking incubator at 37°C and 150 r / min for 2 h, 4 h, and 6 h. Take out the bacterial liquid, and after gradient dilution, count the colonies using the clock method, and calculate the survival rate according to formula (1).

[0061] The results are as follows Figure 5 As shown in the figure, the survival rate of L.rhamnosus SAL2 after culture in artificial intestinal fluid was lower than that of L.rhamnosusGG, but the number of live bacteria of L.rhamnosus SAL2 showed an increasing trend with the extension of culture time; the number of live bacteria of L.rhamnosus GG increased after 2 hours of culture in artificial intestinal fluid, while the number of live bacteria decreased after 4 hours and 6 hours of culture, and the survival rate was lower than that of L.rhamnosusSAL2 at 6 hours. The survival rates of both strains were higher than 87%, indicating that both strains grew stably in artificial intestinal fluid. The number of live bacteria of L.rhamnosusSAL2 increased with the extension of culture time, indicating that L.rhamnosus SAL2 has strong adaptability and reproduction ability. In the intestinal environment, this characteristic helps L.rhamnosus SAL2 to gain an advantage in competition and better colonize in the host intestine.

[0062] 2.10 Antibacterial test

[0063] Take 2 μL of L. rhamnosus SAL2 and L. rhamnosus GG seed solution respectively, set 3 parallel points on each plate, and inoculate them on MRS agar plates, and incubate them at 37°C overnight. Take 8 indicator bacteria, inoculate them in nutrient broth, and incubate them at 37°C and 150 r / min overnight. Take 200 μL of indicator bacteria solution and mix it with 7 mL of soft nutrient agar medium, pour it into the MRS plate inoculated with Lactobacillus rhamnosus, incubate it aerobically at 37°C for 24 hours, and observe and measure the size of the inhibition zone.

[0064] The results are as follows Figure 6 As shown in Table 2, L. rhamnosus SAL2 and L. rhamnosus GG had different degrees of antibacterial activity against the eight indicator bacteria. The antibacterial effect of L. rhamnosus SAL2 was slightly better than that of L. rhamnosus GG.

[0065] Table 2 Diameters of inhibition zones of two strains of Lactobacillus rhamnosus against eight indicator bacteria (mm)

[0066]

[0067] 2.11 Adhesion test

[0068] Preparation of bacterial suspension: Pick single colonies of L. rhamnosus SAL2 and L. rhamnosus GG, inoculate them into MRS broth medium, culture them in a shaking incubator at 37°C and 150 r / min for 16 hours, remove the bacterial solution, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the bacterial cells. Wash the bacterial cells twice with sterile saline and resuspend them, and adjust the absorbance A0 at 600 nm to 0.6±0.05, which is the bacterial suspension.

[0069] 2.11.1 Self-cohesion

[0070] Vortex the suspension to mix evenly, let it stand at 37°C for 24 hours, and measure the absorbance of the sample supernatant at 600nm. Calculate the self-cohesion force according to formula (2):

[0071]

[0072] Where: A0 is the initial absorbance of bacterial suspension at 0h; A 24 It is the absorbance value of the upper layer of the bacterial suspension after standing for 24 hours.

[0073] 2.11.2 Hydrophobicity

[0074] Take 3 mL of bacterial suspension in a test tube, add 3 mL of xylene, vortex for 2 min, let stand at room temperature for 30 min, and measure the absorbance of the water phase at 600 nm. Calculate the hydrophobicity according to formula (3):

[0075]

[0076] Where: A0 is the initial absorbance of bacterial suspension at 0h; A 30 It is the absorbance of the aqueous phase after standing for 30 min.

[0077] The self-cohesion of L. rhamnosus SAL2 and L. rhamnosus GG were 99.27±1.26% and 74.65±1.02% respectively. The hydrophobicity of L. rhamnosus SAL2 and L. rhamnosus GG were 17.09±3.53% and 53.88±4.31% respectively.

[0078] The adhesion of Lactobacillus rhamnosus was indirectly evaluated by in vitro testing of self-aggregation ability and hydrophobicity. The self-aggregation of the strain is conducive to its adhesion to the host epithelial and mucosal surfaces, thereby forming a protective barrier on the host cell surface, and self-aggregation is an important characteristic for the formation of biofilms. Lactobacillus biofilms are conducive to the intestinal colonization of strains. The self-aggregation of L.rhamnosusSAL2 is close to 100%. The hydrophobicity of the bacterial surface is one of the key factors affecting the nonspecific adhesion process, and it also affects the bacterial absorption and degradation of hydrophobic organic substances. L.rhamnosus SAL2 is hydrophilic, which helps it to better interact with water-soluble nutrients in the intestinal environment and promote its survival and function in the host intestine.

[0079] 2.12 Antioxidant test

[0080] Preparation of fermentation supernatant and bacterial suspension: Pick single colonies of L. rhamnosus SAL2 and L. rhamnosus GG, inoculate them into MRS broth medium, culture them in a shaking incubator at 37°C and 150 r / min for 16 h, 24 h, 48 h, and 72 h, then take out the bacterial suspension, centrifuge at 4000 r / min for 15 min, and the obtained supernatant is the fermentation supernatant. Wash the bacteria twice with sterile saline and resuspend them, and adjust the bacterial suspension concentration to 10 9 CFU / mL, that is, bacterial suspension.

[0081] 2.12.1 H2O2 tolerance test

[0082] The seed solution of L.rhamnosus SAL2 and L.rhamnosus GG was centrifuged at 8000r / min for 5min. The supernatant was discarded, and the bacteria were resuspended in physiological saline. After centrifugation at 8000r / min for 5min, the supernatant was discarded. The above operation was repeated twice. The bacteria were collected and resuspended in 10mL MRS broth medium, inoculated into 200mL MRS broth medium at a ratio of 3%, and incubated at 37°C for 4h. The two strains of Lactobacillus rhamnosus after standing were divided into centrifuge tubes, 1-10mM H2O2 solution was added respectively, mixed quickly, and incubated at 37°C for 1h. After centrifugation at 4000r / min for 10min, the supernatant was discarded, and physiological saline was added to each tube to resuspend the bacteria. Centrifugation at 4000r / min for 10min. The supernatant was discarded, and physiological saline was added to each tube to resuspend the bacteria. After gradient dilution, the colony count was performed using the clock method, and the survival rate was calculated according to formula (1).

[0083] The results are as follows Figure 7 As shown in Figure 2, the viable count and survival rate of L. rhamnosus SAL2 were higher in 1-9 mM H2O2, with a gentle downward trend and a small amplitude, and the viable count was always maintained at 10 7CFU / mL level, the survival rate was higher than 96%; when the H2O2 concentration was 10mM, the number of viable bacteria dropped to 10 6 CFU / mL order of magnitude, the survival rate dropped to 87.88%. The number of viable bacteria of L. rhamnosus GG in 1-10mMH2O2 ranged from 10 8 The CFU / mL level was reduced to 10 3 CFU / mL order of magnitude; the survival rate of the strain was 101.10% at 1mM H2O2, and then the survival rate of the strain continued to decrease with the increase of concentration until the survival rate of the strain was only 48.44% at 10mM H2O2. Starting from 2mM H2O2, the survival rate of L.rhamnosus GG was significantly lower than that of L.rhamnosus SAL2, indicating that L.rhamnosus SAL2 has a stronger tolerance to H2O2.

[0084] 2.12.2 DPPH free radical scavenging ability test

[0085] 3 mL of sample was mixed with 3 mL of 0.2 mM DPPH anhydrous ethanol solution and reacted at room temperature in the dark for 30 min. The control group was replaced by sterile saline solution; the blank group was replaced by anhydrous ethanol sample instead of DPPH anhydrous ethanol solution. Centrifuge at 4000 r / min for 10 min, take the supernatant and measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging ability according to formula (4):

[0086]

[0087] like Figure 8 As shown in the figure, at different culture times, the DPPH free radical scavenging ability of the fermentation supernatant of the two strains was significantly stronger than that of the bacterial suspension. The scavenging rate of the fermentation supernatant was higher than 95%; the scavenging rate of the bacterial suspension of L.rhamnosus SAL2 was 12.93%-17.35%, and the scavenging rate of the bacterial suspension of L.rhamnosus GG was 6.43-8.91%. After 16-48h of culture, the scavenging rate of the fermentation supernatant of L.rhamnosus SAL2 was slightly higher than that of L.rhamnosus GG; after 72h of culture, the scavenging rate of the fermentation supernatant of L.rhamnosus GG was higher than that of L.rhamnosus SAL2, but the DPPH free radical scavenging rate of the two strains was relatively stable, and did not show a significant upward or downward trend with the extension of culture time.

[0088] At different culture times, the clearance rate of L.rhamnosus GG suspension was significantly lower than that of L.rhamnosus SAL2. After 16 hours of culture, the clearance rate of L.rhamnosus GG suspension was as high as 8.91%, while the clearance rate of L.rhamnosus SAL2 suspension showed a fluctuating increase, and the clearance rate of L.rhamnosus SAL2 suspension reached a maximum of 17.35% after 72 hours of culture. The above results show that the fermentation supernatants of the two strains showed excellent scavenging ability of DPPH free radicals, and the suspension of L.rhamnosus SAL2 was better than that of L.rhamnosus GG, and most of the active substances that scavenged DPPH free radicals were present in the fermentation supernatant.

[0089] 2.12.3 Hydroxyl free radical scavenging ability test

[0090] Add 1 mL of o-phenanthroline solution (2.5 mmol / L), 1 mL of PBS (pH 7.4), and 1 mL of ferrous sulfate solution (2.5 mmol / L) to the test tube, mix thoroughly, then add 1 mL of sample and 1 mL of H2O2 solution (20 mmol / L), and place in a 37°C water bath for 1.5 h. The control group reacts with 1 mL of distilled water instead of 1 mL of H2O2; the blank group reacts with 1 mL of distilled water instead of 1 mL of sample. Centrifuge at 4000 r / min for 10 min, take the supernatant and measure the absorbance at 536 nm. Calculate the hydroxyl radical scavenging ability according to formula (5):

[0091]

[0092] like Fig. 9 As shown in the figure, the hydroxyl radical scavenging rate of the fermentation supernatant of the two strains of Lactobacillus rhamnosus was higher than that of the bacterial suspension, which was consistent with the DPPH free radical scavenging rate. The scavenging rate of the fermentation supernatant of L. rhamnosus SAL2 was 25.29-41.13%, and the scavenging rate of the fermentation supernatant of L. rhamnosus GG was 27.80-40.71%. After 16-48 hours of culture, the scavenging rate of the fermentation supernatant of the two strains increased with time. After 72 hours of culture, the scavenging rate of L. rhamnosus SAL2 decreased slightly, while that of L. rhamnosus GG maintained an upward trend. The scavenging rates of the fermentation supernatant of L. rhamnosus SAL2 after 48 hours of culture and L. rhamnosus GG after 72 hours of culture were the highest, at 41.13% and 38.58%, respectively.

[0093] After 24 hours of culture, the removal rates of L.rhamnosus SAL2 and L.rhamnosus GG suspensions were the highest, 17.87% and 17.78% respectively; the removal rates after 16 hours of culture were the lowest, 9.32% and 2.14% respectively. The ability of L.rhamnosus SAL2 suspension to scavenge hydroxyl free radicals was slightly better than that of L.rhamnosus GG. The above results also show that the active substances for scavenging hydroxyl free radicals produced by the two strains are mostly present in the fermentation supernatant, and the scavenging abilities of the two strains are comparable.

[0094] 2.12.4 Superoxide anion free radical scavenging ability test

[0095] Add 4.5mL Tris-HCl (50mmol / L, pH 8.2) to 0.1mL sample, incubate in a water bath at 25℃ for 20min, add 0.4mL pyrogallol (25mmol / L, preheated to 25℃), incubate in a water bath at 25℃ for 4min again, and immediately drop 0.1mL HCl (8mol / L) to terminate the reaction. The control group is pure supernatant or pure bacterial suspension instead of sample, and distilled water is used instead of reagent; the blank group is 0.1mL distilled water instead of 0.1mL sample. Centrifuge at 4000r / min for 10min, take the supernatant and measure the absorbance at 325nm. Calculate the superoxide anion radical scavenging ability according to formula (6):

[0096]

[0097] like Fig.10 As shown in the results, the superoxide anion scavenging ability of the fermentation supernatants of the two strains of Lactobacillus rhamnosus was better than that of the bacterial suspension, which was consistent with the results of DPPH free radical scavenging rate and hydroxyl free radical scavenging rate. The scavenging rate of the fermentation supernatant of L. rhamnosus SAL2 gradually increased with the extension of the culture time, reaching a maximum value of 24.45% at 72h; the scavenging rate of L. rhamnosus GG showed a fluctuating pattern, reaching a maximum value of 19.93% at 24h. The scavenging rate of the fermentation supernatant of L. rhamnosus SAL was better than that of L. rhamnosus GG at any culture time. The scavenging ability of the two bacterial suspensions was poor overall, with the scavenging rate of L. rhamnosus SAL2 being 0.52-1.51% and the scavenging rate of L. rhamnosus GG being 0.41-4.78%.

[0098] The above results once again showed that the active substances for scavenging superoxide anion radicals produced by the two strains were mostly present in the fermentation supernatant, and L. rhamnosus SAL2 was superior to L. rhamnosus GG.

[0099] 3. Whole genome sequencing and sequence analysis of Lactobacillus rhamnosus SAL2

[0100] In this study, high-throughput sequencing and bioinformatics techniques were used to obtain the framework map and complete map of the L. rhamnosus SAL2 genome, and a comprehensive analysis was conducted on the functional genes, virulence genes, and drug-resistant genes. At the same time, EPS-related genes were analyzed in order to reveal its potential value in the application of probiotics.

[0101] 3.1 Materials: L. rhamnosus SAL2 strain, MRS broth medium: refer to 2.2

[0102] 3.2 DNA extraction and detection

[0103] After activation, L. rhamnosus SAL2 was inoculated into MRS broth medium, and the bacterial precipitate was collected by centrifugation after culture for whole genome sequencing. DNA was extracted from the sample, and the total amount of DNA was detected using fluorescent dye (Quant-iT PicoGreen dsDNAAssay Kit), and the integrity of the DNA was detected by 1% agarose gel electrophoresis, and stored at -20°C for future use.

[0104] 3.3 Gene library construction

[0105] Whole genome shotgun (WGS) sequencing was used to construct libraries of L. rhamnosus SAL2 with different insert fragments. The constructed libraries were sequenced using the second-generation sequencing technology (NGS) based on the Illumina MiSeq sequencing platform and the third-generation single-molecule sequencing technology based on the PacBio sequencing platform. The specific information of the libraries is shown in Table 3.

[0106] Table 3 Gene library

[0107]

[0108] 3.4 Data quality control and genome sequence assembly

[0109] The second-generation sequencing data of L. rhamnosus SAL2 based on the Illumina MiSeq sequencing platform contains 8,300,320 reads, a total base count of 1,253,348,320bp, and a GC content of 47.02%. Statistics based on the third-generation offline data of the Pac Bio sequencing platform include 499,152 reads, a total genome length of up to 4,901,468,452bp, and no uncertain bases. The L. rhamnosus SAL2 genome contains a circular chromosome with a fragment length of 2,989,570bp, no plasmid, and a GC content of 46.76%. The chromosome genome circle map is shown in Fig.11 .

[0110] 3.5 Antibiotic resistance gene analysis

[0111] Using the Comprehensive Antibiotic Resistance Database (CARD) and BLAST software, 20 antibiotic resistance genes were annotated in the chromosome of L. rhamnosus SAL2, accounting for 0.723% of the total protein-coding genes, as shown in Table 4.

[0112] Table 4 L. rhamnosus SAL2 antibiotic resistance genes

[0113]

[0114]

[0115] Because L. rhamnosus SAL2 has no plasmid and its inherent drug resistance genes do not have the ability to be horizontally transferred to other bacteria, this strain can be used in parallel with certain drugs in order to achieve better therapeutic effects.

[0116] 3.6 Functional Annotation

[0117] The coding genes were compared with the gene database using BLASTP and Diamond comparison tools to obtain gene function annotation results.

[0118] (1) NR annotation results: The species classification of L. rhamnosus SAL2 is highly concentrated in the genus Lactobacillus, so the taxonomic affiliation of L. rhamnosus SAL2 was determined.

[0119] (2) COG annotation results: A total of 2340 genes in L. rhamnosus SAL2 were annotated to 18 COG categories, accounting for 84.57% of the entire gene sequence. Excluding 565 genes of unknown function, the largest proportion of coding genes were annotated to carbohydrate transport and metabolism, with a total of 285 genes, followed by translation (195 genes), amino acid transport and metabolism (174 genes), and transcription, ribosome structure and biosynthesis (155 genes).

[0120] (3) TCDB annotation results: 615 genes were annotated in the genome of L. rhamnosus SAL2, accounting for 22.23% of the total gene sequence. In the first-level classification statistics, the genes related to primary active transport and electrochemical potential driven transporter function were the largest, with 260 and 116 genes respectively; the genes related to group transfer and incompletely characterized transport system were the second largest, with 100 and 69 genes respectively; the genes related to channels and pores, transport auxiliary factors, and transmembrane electron carriers were the least, with 28, 27, and 15 genes respectively. In the second-level classification statistics, the genes related to PP phosphorylation driven transporter were the largest, reaching 249; the genes related to uniporters, cotransporters, antiporters, and phosphate transfer driven transporters (PTS) were the second largest, with 116 and 87 genes respectively.

[0121] (4) GO annotation results: 2005 genes in the genome of L. rhamnosus SAL2 were annotated in the GO database, accounting for 72.46% of the entire gene sequence. Among them, 1911 genes were annotated to the biological process ontology, among which 730, 617, and 518 genes were highly annotated to the cellular nitrogen metabolism process, biosynthesis process, and small molecule metabolism process, respectively. There were 709 genes annotated to the cell composition ontology, among which 494, 489, and 389 genes were highly annotated to the intracellular, cell component, and cytoplasmic processes, respectively. There were 1854 genes annotated to the molecular function ontology, among which 635, 299, and 198 genes were highly annotated to the ion binding, DNA binding, and oxidoreductase activity processes, respectively.

[0122] (5) KEGG annotation results: In the KEGG database, 1403 genes in the genome of L. rhamnosus SAL2 were annotated, accounting for 50.71% of the entire gene sequence.

[0123] KEGG Pathway divides all pathways involved in genes into 6 primary categories, of which most genes are annotated into three primary categories: metabolism, environmental information processing, and genetic information processing, with 880, 235, and 194 genes, respectively. Metabolism-related genes account for the highest proportion, with 310 genes in carbohydrate metabolism, followed by 148 in amino acid metabolism, and 80 in energy metabolism. Among environmental information processing genes, membrane transport genes have the highest abundance of 168. Among genetic information processing genes, translation genes have the highest abundance of 82.

[0124] (6) antiSMASH annotation results: The antiSMASH software was used to mine the possible secondary metabolite synthesis gene clusters in the whole genome of L. rhamnosus SAL2, and a total of two secondary metabolite synthesis gene clusters were found, namely type III polyketide synthase (T3PKS) and ribosome synthesis and post-translational modification polypeptide (RiPP-like). Using the MiBIG database and ClusterBlast algorithm to analyze similar gene clusters, we found that the T3PK3 gene cluster of L. rhamnosus SAL2 had more than 90% homology with Lactobacillus rhamnosus BPL5, Lactobacillus rhamnosus QC, Lactobacillusrhamnosus FAM 20558, Lactobacillus rhamnosus LR5, Lactobacillus rhamnosus GG, and Lactobacillus rhamnosus IDCC 3201; the RiPP-like gene cluster had more than 90% homology with Lactobacillus rhamnosus ATCC 8530, Lactobacillus rhamnosus BPL5, and Lactobacillusrhamnosus FAM 20558.

[0125] Through comparative analysis with the COG and KEGG databases, L.rhamnosus SAL2 had the largest number of annotated genes related to carbohydrate transport and metabolism, and the high abundance of translation, transcription, amino acid transport and metabolism, ribosome structure and biosynthesis-related genes also indicated that L.rhamnosus SAL2 had an efficient mechanism in transporting and metabolizing two major nutrients, carbohydrates and amino acids, and in cell growth, which played an important role in its survival and competition in the complex intestinal environment of cancer patients. It also preliminarily confirmed from the genetic level that L.rhamnosus SAL2 may have the potential to synthesize EPS.

[0126] The annotation results of L.rhamnosus SAL2 in TCDB and KEGG databases show that its material transport mode is mainly active transport. The original habitat of L.rhamnosus SAL2 is the intestinal environment of colon cancer patients with relatively scarce nutrients. Through the active transport mechanism, it can efficiently absorb nutrients and eliminate metabolic waste, thereby maintaining its survival and reproduction. EPS synthesis is a complex process. Active transport not only helps L.rhamnosus SAL2 to absorb the sugar substances required for growth, but also provides the necessary energy support for its synthesis of EPS. In addition, active transport may also be involved in the transport of key substances in the EPS synthesis process, such as transporting monosaccharides from outside the cell to inside the cell to provide substrates for the extension of polysaccharide chains. The 87 PTS genes annotated in the TCDB database can mediate sugar transport and phosphorylation and regulate carbon metabolism, which means that L.rhamnosus SAL2 has the ability to finely regulate sugar metabolism.

[0127] The genome of L.rhamnosus SAL2 was annotated with gntZ, pepN, zwf, btuE, speC, and gor antioxidant genes in the KEGG database; and tuf, srtA, and epsB adhesion genes, indicating that L.rhamnosus SAL2 has strong adhesion and antioxidant abilities, which is consistent with the study of probiotic properties. Oxidoreductases are enzymes that catalyze redox reactions between two molecules and participate in energy metabolism, substance generation and decomposition, and signal transduction. The genome of L.rhamnosus SAL2 was annotated with a large number of oxidoreductase activity genes in the molecular functions of the GO database, which plays an important role in L.rhamnosus SAL2's adaptation to the intestinal environment, resistance to oxidative stress, and maintenance of bacterial homeostasis. In the antioxidant test, the fermentation supernatant of L.rhamnosus SAL2 had a strong free radical scavenging ability, which was mainly due to the fact that most antioxidants existed in the fermentation supernatant in the form of extracellular secretions. Exopolysaccharides are secondary metabolites synthesized and secreted outside the cell by Lactobacillus during its growth and metabolism. Studies have shown that exopolysaccharides are related to the strain's tolerance to environmental stress and are also a key component of Lactobacillus' antioxidant function. Functional gene analysis showed that L. rhamnosus SAL2 has a strong ability to synthesize and metabolize carbohydrates, and it is speculated that exopolysaccharides may be the antioxidant material basis of L. rhamnosus SAL2.

[0128] T3PKS is one of the most abundant biosynthetic gene clusters in lactic acid bacteria. It has a simple structure and complex products. The synthesized flavonoids, diphenylethylene and pyrone have antibacterial, antitumor and immunosuppressive activities. RiPPs-like, as a bacteriocin synthesis gene cluster, has the ability to inhibit pathogenic bacteria, enhance probiotic colonization and reduce inflammation. As a potential postbiotic preparation, L. rhamnosus SAL2 secondary metabolites are expected to play an important role in improving human health, such as regulating intestinal flora, anti-oxidation, and immunomodulation.

[0129] 3.7 Analysis of genes related to sugar metabolism

[0130] (1) Sugar transport system

[0131] Polysaccharide synthesis requires the bacteria to transport sugar substances from the extracellular to the intracellular, thereby synthesizing the active precursor sugar nucleotides necessary for EPS and generating energy in the process. Phosphoenolpyruvate-sugar phosphotransferase system (PEP-PTS) is the main mode of transport of sugar substances. In the L. rhamnosus SAL2 genome, it is composed of PEP-dependent phosphotransferase (Enzyme I, EI, chr_1711 encoding), histidine phosphate carrier protein (chr_1712 encoding) and membrane-bound sugar-specific permease (Enzyme II, EII). As shown in Table 5, L. rhamnosus SAL2 contains 32 sugar-specific permease (EII) genes.

[0132] Table 5 L. rhamnosus SAL2 PEP-PTS related genes

[0133]

[0134]

[0135] (2) Key enzymes for sugar nucleotide synthesis

[0136] Sugar nucleotides are precursors for the synthesis of EPS. As shown in Table 6, enzymes that synthesize different sugar nucleotides were annotated by L. rhamnosus SAL2, which can use different carbon sources as substrates to synthesize a variety of nucleotides through a series of reactions.

[0137] Table 6 Some key enzymes in sugar nucleotide synthesis of L. rhamnosus SAL2

[0138]

[0139]

[0140] (2) CAZy annotation results: A total of 112 genes encoding carbohydrate-active enzymes in the L. rhamnosus SAL2 genome were annotated in the CAZy database.

[0141] The diversity of the sugar transport system of L.rhamnosus SAL2 indicates its ability to utilize different carbon sources. The synthesis of EPS requires a variety of sugar substances as precursors, and the acquisition of these precursors depends on an effective sugar transport system. In the process of synthesizing sugar nucleotides, L.rhamnosus SAL2 uses multiple key enzymes to effectively convert sugar sources into sugar nucleotides, providing L.rhamnosus SAL2 with the ability to synthesize EPS and reflecting its high adaptability and diversity in sugar metabolism.

[0142] GHs are key enzymes in carbohydrate metabolism, which can hydrolyze glycosidic bonds between two or more carbohydrates and carbohydrates and non-carbohydrates. Among them, GH 1 and GH13 have the largest number of annotations. GH 1 mainly encodes β-glucosidase and β-galactosidase; β-glucosidase can hydrolyze the β-D-glucosidic bond at the non-reducing end of the substrate to release glucose and the corresponding ligand; β-galactosidase can hydrolyze lactose into glucose and galactose. GH 13 mainly encodes α-amylase, which mainly degrades starch or glycogen. The abundant sugar hydrolases in the genome of L. rhamnosus SAL2 can efficiently utilize and metabolize different carbon sources, which provides the basis for the formation of sugar nucleotides. L. rhamnosus SAL2 has obtained more GTs gene annotations. GTs are responsible for transferring sugar groups from activated sugar donors to specific receptors such as polysaccharides, lipids or proteins to form glycosidic bonds. By transferring various sugar nucleotides, the repeating units of EPS are constructed. CBMs can recognize and bind to specific carbohydrate structures to improve the catalytic efficiency of enzymes. Four genes of L. rhamnosus SAL2 were predicted to belong to CBM36 and CBM50, respectively. The CBM36 functional module was related to xylanase; CBM50 mainly encoded modules connected to various enzymes of the GH18, GH19, GH23, GH24, GH25 and GH73 families, i.e. chitin or peptidoglycan-related enzymes. Comprehensive carbohydrate transport metabolism and active enzyme analysis revealed that L. rhamnosus SAL2 had the ability to produce and secrete EPS.

[0143] Conclusion: Whole genome sequencing analysis of L. rhamnosus SAL2 showed that it has strong carbohydrate anabolism, antioxidant and exopolysaccharide secretion capabilities.

[0144] It should be noted that, although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present invention specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. Lactobacillus rhamnosus SAL2 deposited in CGMCC with the deposit number of CGMCC No.32935.

2. Application of Lactobacillus rhamnosus SAL2, which is deposited in CGMCC with the deposit number of CGMCC No.32935, in inhibiting bacterial growth.

3. The application according to claim 2, characterized in that: The bacteria include Staphylococcus aureus, Enterococcus faecium, Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella typhimurium, and Shigella flexneri.

4. Application of Lactobacillus rhamnosus SAL2, which is deposited in CGMCC with the deposit number CGMCC No.32935, in antioxidant aspects.

5. An antibacterial agent, characterized in that The antibacterial agent contains the Lactobacillus rhamnosus SAL2 according to claim 1 as its effective ingredient.

6. An antioxidant, characterized in that The antioxidant contains the Lactobacillus rhamnosus SAL2 according to claim 1 as its effective ingredient.

7. Application of Lactobacillus rhamnosus SAL2, which is deposited in CGMCC with the deposit number of CGMCC No. 32935, in the preparation of drugs, foods and health products.

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