Lactobacillus rhamnosus HZBX-05 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus HZBX-05 and application thereof.The lactobacillus rhamnosus HZBX-05 is sent to the China General Microbiological Culture Collection Center on July 27th, 2023 to be preserved in the China General Microbiological Culture Collection Center, the preservation number of the lactobacillus rhamnosus HZBX-05 is CGMCC NO.987, and the preservation number of the lactobacillus rhamnosus HZBX-05 is CGMCC NO.989. The lactobacillus rhamnosus HZBX-05 is CGMCC NO.989. The lactobacillus rhamnosus HZBX-05 is CGMCC NO.989. The preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.28035, and the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, West Beichen Road, Chaoyang District, Beijing; the separated Lactobacillus rhamnosus HZBX-05 is continuously treated for 7 hours in a simulated gastric acid cholate environment, and the result shows that the HZBX-05 still has high survival rate, has the capability of being highly adsorbed to intestinal cells Caco-2 cells, has the capability of inhibiting various pathogenic bacteria, has immunoregulation and anti-inflammation effects, has the effect of resisting helicobacter pylori, and can be used for preparing an anti-inflammatory drug. The symptoms of female vaginitis can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus rhamnosus HZBX-05 and an application thereof. Background Art

[0002] Probiotics refer to live microorganisms that are beneficial to human health when taken in sufficient quantities and meet the relevant national regulations on bacteria used in food. At present, the "List of Bacteria Used in Food" published by the National Food Safety Risk Assessment Center mainly includes bacteria and a small amount of fungi.

[0003] In fact, humans have been using lactobacilli to prepare dairy products since ancient times. The brewing and processing of kumis and yogurt has a history of thousands of years. In recent years, due to the rapid development of genomics and metabolomics, the significant role of intestinal flora in regulating metabolic diseases has been increasingly recognized. In particular, fecal microbiota transplantation technology has been successfully applied in animals and clinical trials, consolidating the theoretical and practical basis for treating metabolic diseases by regulating intestinal flora. Therefore, supplementing with probiotics can treat metabolic diseases by regulating the structure of intestinal flora.

[0004] Among them, Lactobacillus rhamnosus is a type of beneficial intestinal bacteria that plays a positive role in the prevention and treatment of gastrointestinal diseases. It can help prevent and relieve diseases such as intestinal infections, antibiotic-related diarrhea, enteritis, colorectal cancer, and irritable bowel syndrome. Supplementation of Lactobacillus rhamnosus is helpful in maintaining the health of the intestinal microecology and regulating the immune response. It can regulate the immune response of the spleen, intestines, and mesenteric lymph nodes. In addition, the supplementation of Lactobacillus rhamnosus can also reduce the level of intestinal inflammation, thereby reducing the risk of intestinal cancer caused by enteritis, and Lactobacillus rhamnosus can also inhibit other bacteria, therefore, it has the ability to resist Helicobacter pylori and treat vaginitis.

[0005] For example, the prior art CN117384797B discloses a rhamnosus lactobacillus LRPerfectus158 and its use. It has been found that rhamnosus lactobacillus LRPerfectus158 not only has the function of improving exercise endurance and functional recovery, but also has a good preventive and therapeutic effect on gastrointestinal damage and vaginal inflammation caused by low immune function. It has broad application prospects in the preparation of products for preventing or treating gastrointestinal damage caused by exogenous pathogens and opportunistic pathogens; and / or products for preventing or treating vaginal inflammation; and / or antibacterial products; and / or anti-inflammatory products; and / or products for improving exercise endurance and functional recovery.

[0006] Prior art CN 116200306 B discloses a species of Lactobacillus rhamnosus LRa16 and its use and product in the preparation of drugs for treating genital tract infections. Lactobacillus rhamnosus LRa16 can significantly treat vaginal inflammation, with a total cure rate of up to 98% and a recurrence rate of 0 within 3 months. At the same time, it can broadly and efficiently inhibit pathogens such as Candida albicans, Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, and Salmonella.

[0007] However, the application of the above-mentioned prior art records is limited, and the effect needs to be improved. Therefore, it is urgent to discover new strains of Lactobacillus rhamnosus. Summary of the invention

[0008] The present invention first provides a Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) HZBX-05, the rhamnosus Lactobacillus HZBX-05 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 27, 2023, with the deposit number CGMCC NO.28035.

[0009] The present invention also provides a product for preparing the Lactobacillus rhamnosus HZBX-05 for preventing or treating gastrointestinal damage caused by exogenous pathogens and opportunistic pathogens; and / or, in the preparation of an antimicrobial product; and / or, in the preparation of an anti-inflammatory product; and / or, in the preparation of products for enhancing immunity; And / or, use in preparing a medicament for treating reproductive tract infection.

[0010] In certain embodiments, the pathogen is Candida albicans.

[0011] In certain embodiments, the pathogen is Staphylococcus aureus.

[0012] In certain embodiments, the pathogen is Group B Streptococcus (GBS).

[0013] In certain embodiments, the pathogen is drug-resistant Staphylococcus aureus.

[0014] In certain embodiments, the pathogen is drug-resistant Escherichia coli.

[0015] In certain embodiments, the reproductive tract infection is vaginitis.

[0016] In certain embodiments, the vaginitis is selected from one or more of bacterial vaginitis, fungal vaginitis, and Trichomonas vaginitis.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The Lactobacillus rhamnosus isolated by the present invention ( Lacticaseibacillus rhamnosus ) HZBX-05 was treated in a simulated gastric acid and bile salt environment for 7 consecutive hours. The results showed that HZBX-05 still had a high survival rate, had a high ability to adsorb to intestinal cells Caco-2 cells, had the ability to inhibit a variety of pathogens, had immunomodulatory and anti-inflammatory effects, had anti-Helicobacter pylori effects, and could improve the symptoms of female vaginitis.

[0018] Storage Instructions: Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) HZBX-05 was isolated from the reproductive tract of a healthy woman and was sent to the Center for General Microbiology of the China Culture Collection on July 27, 2023 for preservation. It is preserved in the Center for General Microbiology of the China Culture Collection with the preservation number: CGMCCNO.28035. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The production curve results of Lactobacillus rhamnosus HZBX-05; Figure 2 The results of acid production by Lactobacillus rhamnosus HZBX-05; Figure 3 The results of the effect of Lactobacillus rhamnosus HZBX-05 on IL-10; Figure 4 The results of the effect of Lactobacillus rhamnosus HZBX-05 on IFN-g; Figure 5 Results of Lactobacillus rhamnosus HZBX-05 against Helicobacter pylori; compared with the control group NC, *p<0.05, **p<0.01, ***p<0.001.

[0020] Figure 6 Results of Lactobacillus rhamnosus HZBX-05 in the treatment of vaginitis. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0022] The commercial strain Lactobacillus rhamnosus GG is commercially available, ATCC 53103 Lactobacillus rhamnosus (GG), Beijing Bio-Bowei Biotechnology Co., Ltd.

[0023] Example 1: Gastric acid and bile salt tolerance test of Lactobacillus rhamnosus HZBX-05: Commercial artificial gastric juice (pH = 1.5) and artificial intestinal juice (pH 6.8) products were purchased, the pH of the artificial gastric juice was adjusted to 2.5, and then filtered and sterilized with a 0.22 μm filter membrane for use.

[0024] The seed liquid grown to the middle and late logarithmic growth phase was mixed and divided into 3 tubes of equal volume, centrifuged and the supernatant was discarded, washed twice with sterile saline, and then the centrifuged bacteria were resuspended and mixed with equal volumes of saline (blank group), artificial gastric juice (pH 1.5), and artificial intestinal juice (pH 6.8) respectively, and then placed in a 37±1℃ incubator for 3h and taken out, and 10-fold gradient dilution was performed respectively, and 10 -5 -10 -7 The dilutions were spread on 3 plates respectively. After 24 hours, the plates with colony counts in the range of 30-300 were counted and the survival rate was finally calculated. Survival rate (%) = average number of viable bacteria in experimental group / average number of viable bacteria in blank group × 100%.

[0025]

[0026] The calculation results are shown in Table 1. After being treated in a simulated gastric acid and bile salt environment for 7 consecutive hours, Lactobacillus rhamnosus HZBX-05 still had a high survival rate, which was higher than that of Lactobacillus rhamnosus GG (p<0.05).

[0027] Example 2: Production curve and acid production effect of Lactobacillus rhamnosus HZBX-05: After Lactobacillus rhamnosus HZBX-05 was activated and cultured in MRS liquid medium at 37°C overnight, the second generation was inoculated with 5% inoculum for 8 hours, and the third generation was inoculated with 2% inoculum. The OD values ​​at 3h, 5h, 7h, 9h, 11h, 12h, 13h, 15h, 18h, and 24h were measured. 600 A commercial strain (Lactobacillus rhamnosus GG) was used as a control.

[0028] Growth curve Figure 1 As shown, compared with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus HZBX-05 has stronger reproduction ability (p<0.05) and has the potential for industrial production.

[0029] Acid production curve Figure 2 As shown, the acid production ability of Lactobacillus rhamnosus HZBX-05 is stronger than that of Lactobacillus rhamnosus GG (p<0.05), which plays an important role in maintaining the acidic environment of the vagina.

[0030] Example 3: Intestinal adsorption capacity test of Lactobacillus rhamnosus HZBX-05: Preparation of rhamnosus lactobacillus suspension: Lactobacillus rhamnosus (OD600 nm = 1.0) was centrifuged (8 000r / min, 10 min) to collect the bacteria, washed 3 times with sterile PBS, and resuspended in serum-free DMEM medium. LGG was used as a positive control. The cell adhesion study was carried out according to the method of Zhang Rujing et al. (Zhang Rujing, Hu Yafan, Hai Dan, et al. Screening and identification of probiotic lactic acid bacteria with inhibitory effects on intestinal pathogens and adhesion to Caco-2 cells [J]. Food Industry Science and Technology, 2019, 40(20):133-139, 153.). A 8 mm × 8 mm glass slide was pre-placed in a 24-well plate, and the Caco-2 cells were adjusted to 2 × 10 4 / mL, and inoculated into a 24-well plate to allow it to adhere to the wall and grow. After the cells grew into a monolayer, they were washed twice with sterile PBS, and 0.5 mL of DMEM and 0.5 mL of the pre-prepared lactic acid bacteria solution were added to each well and continued to be cultured for 1 hour. Washed three times with sterile PBS to remove the non-adherent lactic acid bacteria, fixed with formaldehyde for 20 minutes, and Gram-stained the slides. 20 fields of view were randomly selected under an optical microscope to observe and count the number of lactic acid bacteria adhered. The results are shown in Table 2.

[0031]

[0032] The results in Table 2 showed that the adhesion number of HZBX-05 was significantly higher than that of LGG (P<0.01).

[0033] Example 4: Antibacterial test of Lactobacillus rhamnosus HZBX-05: Five types of pathogenic strains, Candida albicans, Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus aureus (MRSA), and Escherichia coli (ESBL), were inoculated into nutrient agar medium, cultured overnight at 37°C and 180 rpm in a constant temperature shaker, and then the pathogen suspension was prepared. The high-temperature sterilized MRS solid medium was cooled to about 46±1°C and mixed with the pathogen suspension in a certain proportion before solidification, so that the number of viable pathogens in the test system was 10 6CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium is cooled and solidified, take out the Oxford cup, inject 200 μL of the test sample solution (i.e., the fermentation end-point cell-free supernatant of Lactobacillus rhamnosus HZBX-05 and Lactobacillus rhamnosus GG, and the blank control group is the test solvent MRS broth culture medium) into each well, cover the plate lightly and place it upright at 37°C in a constant temperature incubator, observe after the appropriate incubation time, and measure the diameter of the inhibition zone with a vernier caliper.

[0034]

[0035] As shown in Table 3, the inhibitory effect of Lactobacillus rhamnosus HZBX-05 on pathogens was significantly better than that of Lactobacillus rhamnosus GG (p<0.05).

[0036] Example 5: Lactobacillus rhamnosus HZBX-05 anti-inflammatory: Experimental method: After different cells were infected with pathogens, they were co-cultured with probiotics for 48 hours, and the cytokine content in the culture medium was analyzed using an ELISA kit. The results were as follows: Figure 4 As shown, Lactobacillus rhamnosus HZBX-05 can enhance the expression of anti-inflammatory cytokine IL-10.

[0037] Example 6: Lactobacillus rhamnosus HZBX-05 immunomodulation: Experimental method: Isolate immune cells from human peripheral blood cells (PBMC), co-culture with Lactobacillus rhamnosus HZBX-05 for 48 hours, and use ELISA kit to analyze the cytokine content in the culture medium. Figure 5 As shown, Lactobacillus rhamnosus HZBX-05 can perform immunomodulation.

[0038] Example 7: Results of the anti-Helicobacter pylori effect of Lactobacillus rhamnosus HZBX-05: Pathogenic bacteria culture: Take the frozen glycerol tube (Helicobacter pylori ATCC353909 / ATCC354364), spread it on Columbia blood agar medium (Columbia blood agar is sterilized by high pressure, cooled to about 50°C, 10% sterile defibrinated sheep blood is added, cooled to 45-50°C, poured into a sterile plate, and sealed and stored at 4°C), and cultured at 37°C and 10% CO2 for 72 hours; scrape the bacterial mud under sterile conditions and put it into brain heart infusion liquid culture medium (brain Heart infusion broth was sterilized at 121°C for 15 minutes by high pressure. When cooled to about 50°C, 10% fetal bovine serum was added and mixed, and cooled to room temperature. Incubated at 37°C for 24 hours under microaerobic conditions (oxygen concentration 5%, carbon dioxide concentration 10%, nitrogen concentration 85%, the % is volume %), 5 mL of bacterial solution was taken, centrifuged at 5000 r / min for 5 minutes, and the bacterial cells were resuspended with physiological saline (0.8%, w / v). The concentration of the bacterial solution was adjusted to about 10 6 cfu / mL.

[0039] Inhibition zone test: The Oxford cup double-layer plate method was used. In a clean workbench, take a sterile plate, sterilize the nutrient agar and pour it into the plate, and cover the plate as the lower culture medium. After the agar solidifies, evenly spread 10ml of Columbia blood agar medium on the upper layer as the upper culture medium. After the upper culture medium solidifies, absorb 0.2mL of Helicobacter pylori bacterial liquid on the solid culture medium plate and spread it evenly. After leaving it for 1 hour, make a hole with a diameter of 8mm and a depth of 3mm, and place Lactobacillus rhamnosus HZBX-05 (10 7 cfu / mL) was added into 0.1 mL of sterile water and then injected into the wells. The results were observed and the diameter of the inhibition zone was determined at 37°C for 8 h.

[0040] According to the measurement, the diameter of the inhibition zone of Lactobacillus rhamnosus HZBX-05 against Helicobacter pylori reached 18.42±0.25mm, which indicates that the strain has a significant inhibitory effect on Helicobacter pylori and has achieved unexpected technical results.

[0041] Growth inhibition assay: (1) Experimental group: 1% of the volume ratio of fresh Helicobacter pylori culture was inoculated into a brain heart infusion liquid culture medium supplemented with 2.5% of the fermentation supernatant of Lactobacillus rhamnosus HZBX-05 described in Example 2, Escherichia coli, LGG and Amoxicill (240ug / ml); (2) Control group: Inoculate fresh Helicobacter pylori culture medium at a volume ratio of 1% into brain heart infusion liquid culture medium. Culture at 37°C and 10% CO2 for 24 hours; measure the OD value of the culture medium at a wavelength of 600nm. Calculate the survival rate of Helicobacter pylori by taking the OD600 value of the culture medium of the control group as 100%.

[0042] Survival rate (%) = (1-(control group OD600-experimental group OD600) / control group OD600) × 100%. The calculation results are as follows Figure 5 As shown, Lactobacillus rhamnosus HZBX-05 can inhibit the growth of Helicobacter pylori (HP) and inhibit the urease activity of HP.

[0043] Example 8: Results of treating vaginitis with Lactobacillus rhamnosus HZBX-05: (1) Toxicity test: 10 SPF Kunming mice were gavaged with 0.3 ml of Lactobacillus rhamnosus HZBX-05 suspension (greater than 1×10 9 CFU / mouse), according to the requirements of the 2015 edition of the Chinese Pharmacopoeia, the body weight of each mouse was measured every day, and the behavioral and physiological changes of each mouse before and after injection were observed and recorded.

[0044] The results showed that all animals gained weight within 7 days, no obvious symptoms of poisoning were observed, no abnormal activities or behaviors were observed, and no animals died, indicating that the strain is safe and non-toxic.

[0045] (2) Reproductive tract mucosal irritation test: According to the vaginal mucosal irritation test standards in the "Disinfection Technical Specifications" of the former Ministry of Health, healthy female New Zealand rabbits in their early adulthood and in the non-estrus period were selected. Before the test, the animals' vaginal openings were checked for secretions, congestion, edema and other injuries. Rhamnosus lactobacillus HZBX-05 was used as the test solution, and 3 female New Zealand rabbits were randomly selected for infection, with 2 mL of the test solution injected each time. The control group animals were treated in the same way with saline. Each treatment was performed 24 hours apart for 5 consecutive days. 24 hours after the last infection, the animals were killed by air embolism, the vagina was completely removed and then cut longitudinally, and the mucosa was observed with the naked eye for edema, congestion and other manifestations, and then placed in 10% formaldehyde solution for fixation and histopathological examination.

[0046] According to the vaginal mucosal profiling scoring standard and irritation intensity classification in the Technical Specifications for Disinfection, the vaginal mucosal irritation index of Chinese herbal microecological preparations is 0.71<1, which is considered to be non-irritating to the vaginal mucosa. It can be used as a biological preparation directly for vaginal microecological intervention.

[0047] (3) Clinical trial on volunteers with reproductive tract infection: Trial method: 32 subjects, 20 patients with vaginitis and 12 healthy subjects, statistical analysis of symptom improvement after consuming the compound probiotics containing 05. The remaining 12 healthy patients were followed up and no adverse reactions were found.

[0048] Inclusion criteria for subjects: vaginitis (7 cases of bacterial vaginitis, 7 cases of candidal vaginitis and 6 cases of trichomonas vaginitis), aged 18-65 years, all with a history of sexual life. Non-menstrual period; clinical symptoms combined with clinical vaginitis diagnostic criteria, patients with major mental illness were excluded, sexual life history was prohibited within 1 week; no other drugs or vaginal cleansers were used within 1 week before the trial, or other drugs were ineffective.

[0049] How to eat: Eat HZBX-05 (5X10 9 CFU / g), 1g each morning and evening, for 4 weeks, the results are as follows Figure 6 As shown, both the infection situation and subjective symptoms were significantly improved.

[0050] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )HZBX-05, characterized in that, The rhamnosus Lactobacillus HZBX-05 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 27, 2023, with the deposit number CGMCC NO.28035.

2. A product for preventing or treating gastrointestinal damage caused by exogenous pathogens and opportunistic pathogens using the Lactobacillus rhamnosus HZBX-05 according to claim 1; and / or, in the preparation of an antimicrobial product; and / or, in the preparation of an anti-inflammatory product; and / or, in the preparation of products for enhancing immunity; And / or, use in preparing a medicament for treating reproductive tract infection.

3. The use according to claim 2, characterized in that: The pathogen is Candida albicans.

4. The use according to claim 2, characterized in that: The pathogen is Staphylococcus aureus.

5. The use according to claim 2, characterized in that: The pathogen is Group B Streptococcus (GBS).

6. The use according to claim 2, characterized in that: The pathogen is drug-resistant Staphylococcus aureus.

7. The use according to claim 2, characterized in that: The pathogen is drug-resistant Escherichia coli.

8. The use according to claim 2, characterized in that: The reproductive tract infection is vaginitis.

9. The use according to claim 8, characterized in that The vaginitis is selected from one or more of bacterial vaginitis, fungal vaginitis and Trichomonas vaginitis.

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