CN110777087B

CN110777087B - Lactobacillus johnsonii and application thereof

Info

Publication number: CN110777087B
Application number: CN201910732805.8A
Authority: CN
Inventors: 承磊; 刘瑶; 王琼; 曾婉秋
Original assignee: Sichuan Anaerobic Biotechnology Co ltd
Current assignee: Sichuan Anaerobic Biotechnology Co ltd
Priority date: 2019-08-09
Filing date: 2019-08-09
Publication date: 2020-08-07
Anticipated expiration: 2039-08-09
Also published as: WO2021027741A1; CN110777087A

Abstract

The invention discloses lactobacillus johnsonii L john-1, which belongs to an advantageous strain of female vaginas in China, and an application technology of the advantageous strain capable of being applied to vaginal microecological preparations, and is beneficial to improving the cure rate of bacterial vaginitis and reducing the recurrence rate of the bacterial vaginitis, wherein the strain is preserved in a China center for type culture collection in 2019 in 6 months, and the preservation number of the strain is CCTCC No. M2019426.

Description

Lactobacillus johnsonii and application thereof

Technical Field

The invention relates to the field of microorganisms, and particularly relates to lactobacillus johnsonii and application thereof.

Background

From research reports of various aspects such as a document published by a journal practical gynecological journal, namely research progress and clinical significance of vaginal microecology, a document published by a journal Chinese microecology, namely research progress of survival state of lactobacillus in vaginal microecology, and the like, the situation that more than 300 microorganisms coexist in human vagina can be known, and the microorganisms are restricted and balanced with each other to form dynamic balance, so that the occurrence of various vaginitis is related to the unbalance of vaginal microecological environment.

The vagina of healthy women of reproductive age is a microenvironment with lactobacilli as the dominant flora, and the bacteria can produce H₂O₂Lactic acid, lactein, and competitively adhere to vaginal epithelium, occupy binding sites, consume nutrition in vagina, and obtain their predominant position in vagina, and inhibit hyperproliferation of other bacteria. At present, more than 20 kinds of lactobacillus are separated from the microenvironment of the vagina, and the most common dominant lactobacillus in the vagina of healthy women in China comprises lactobacillus crispatus, lactobacillus gasseri, lactobacillus jensenii, lactobacillus johnsonii, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus acidophilus, lactobacillus inerticus and the like.

When the vaginal microecological balance is broken by environmental factors or external intervention, the vaginal microecological environment enters a fragile state, and the propagation and invasion of pathogenic bacteria are not easy to resist, so that various vaginal inflammations are generated. Bacterial Vaginosis (BV) is a common gynecological disease, the infection rate is 15-52%, the bacterial vaginosis and other anaerobic bacteria are mainly used for over-breeding to replace lactobacillus to cause vaginal dysbacteriosis, and the BV is reported to be a risk factor for causing tissue chorionitis, amniotic fluid infection, post-cesarean section endometritis and other pregnancy and pregnancy complications.

Aiming at BV, the clinical treatment method is to adopt metronidazole or clindamycin, the metronidazole is a precursor drug, in an anaerobic environment, the intracellular enzymatic reduction of bacteria reduces the nitro group of the metronidazole into amino group, so that the antibiotic is converted into an active form, and then the helix structure of the antibiotic is damaged through covalent binding with DNA, single-strand and double-strand break are caused, and the DNA is degraded and the pathogen dies; clindamycin can bind to 50S ribosome subunit on bacterial ribosome, prevent the extension of peptide chain, inhibit the protein synthesis of bacterial cell and result in bacterial death. Although the treatment by antibiotics has quick response, the method also has great defects, and has the following two aspects: (1) all antibiotic-sensitive microorganisms in the vaginal microenvironment are inhibited, so that the vaginal microecology is not recovered to a healthy balance state capable of resisting pathogenic bacteria after treatment, and the inhibited or killed pathogenic microorganisms or external pathogenic microorganisms can reproduce again and even cause diseases to cause relapse or new vaginal inflammation; (2) microorganisms develop drug resistance and antibiotics cannot balance the vaginal micro-ecological environment, resulting in refractory BV. Therefore, although the antibiotic treatment has quick response, the relapse rate is high, and the relapse rate is as high as 30% within 3 months.

The treatment of vaginal microecological imbalance comprises three steps of sterilization, mucosa repair and vaginal microecological balance restoration. Sterilization is the first step in the treatment of vaginal inflammation, inhibiting or killing pathogenic microorganisms, including hyperproliferative aerobic and anaerobic bacteria, blastospores or hyphae, trichomonas, and the like. After the pathogenic microorganisms are inhibited or killed, the immune repair of the vaginal mucosa and the recovery of the dominant lactobacillus are the ultimate targets for treating the vaginal inflammation. During this period, if the repair of vaginal mucosa, the recovery process of lactobacillus are affected, and the physicochemical environment in vagina is not restored to normal, the inhibited pathogenic microorganisms or foreign pathogenic microorganisms may reproduce again and even cause disease, and relapse or new vaginal inflammation occurs. The probiotics can quickly occupy receptors of vaginal epithelium in the vagina to generate a protective effect on the vagina, so that the vagina is promoted to restore to a normal microenvironment, and the recurrence of vagina inflammation is reduced. Therefore, the use of probiotic probiotics in the treatment of BV is the most preferred way in view of the current technology.

The vaginal micro-ecological preparation currently on the market at home only has 2 kinds, one is a commercial medicine with the trade name of "Yanhua" produced by the biological pharmacy limited company of Xian Zhenghao, and comprises a streptococcus enterocolis (R) ((R))Streptococcus faecalis) The species is not the vaginal dominant species, and the bacteria of this species are conditionally pathogenic; the other is a commercial drug with the trade name of Vanzyzqi produced by the company of double-drug industry Limited of inner Mongolia, which comprises 1 species of lactobacillus (Lactobacillus delbrueckii)Lactobacillus delbrueckii) The strain does not belong to the dominant vaginal strain in China.

Therefore, the dominant bacterial strain of the vagina of women in China with strong probiotic capability is searched, and the vaginal microecological preparation prepared by the dominant lactobacillus strain is more beneficial to treating the bacterial vaginitis.

Disclosure of Invention

The invention aims to provide a Lactobacillus johnsonii L john-1, which belongs to a dominant strain of female vagina in China and an application technology of applying the dominant strain in a vaginal microecological preparation, wherein the strain is preserved in China Center for Type Culture Collection (CCTCC for short) in 2019 and has a preservation number of CCTCC No. M2019426, and the address of the strain is Wuhan university in Wuhan city, Hubei province, the postal code of the strain is 430072, and the telephone of the strain is 027-once 68754052.

The inventor obtains a strain of lactobacillus from the vagina of a healthy female in China, and proves that the strain of lactobacillus has superior probiotic capability. This strain constitutes a first aspect of the invention.

As the strain is the dominant strain of the vagina of women in China, the strain can be applied to women health care products or medicines for treating vaginitis, which forms the second aspect of the invention. The female sanitary health care product refers to an external health care product.

The third aspect of the invention is to provide a microbial inoculum which takes lactobacillus johnsonii L john-1 as an active ingredient, and the microbial inoculum can be a bacterial suspension or freeze-dried bacterial powder.

Lactobacillus johnsonii L john-1 can also be used in the preparation of external genital hygiene products such as sanitary napkins, tampons or sanitary care solutions for external genitalia.

Lactobacillus johnsonii L john-1 can also be used in the preparation of a medicament or a health care product for regulating the balance of vaginal flora.

The Lactobacillus johnsonii L john-1 can also be used in the preparation of a medicine or a health care product with the function of adhering vaginal epithelial cells.

The Lactobacillus johnsonii L john-1 can also be used in the preparation of drugs or health care products for preventing and treating vaginal pathogenic bacteria, wherein the pathogenic bacteria include any one or more of but are not limited to Gardnerella vaginalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli ability, Salmonella paratyphi B ability and Shigella dysenteriae.

The lactobacillus johnsonii L john-1 can also be applied to the external care product for the infants delivered by caesarean section, as the infants delivered by caesarean section do not pass through the vagina of women in the production process, no exogenous probiotics are obtained, and therefore, the probiotics screened from the vagina of women can be made into the external care product to be smeared on the bodies of the infants.

The whole genome gene sequence of the lactobacillus johnsonii L john-1 is shown in a sequence table.

After the whole genome sequence of Lactobacillus johnsonii L john-1 was uploaded to EzBiocloud, and compared with the whole genome sequence that can be linked to all the same species, the Average Nucleotide Identity (ANI) ratio obtained was as shown in Table 1 below, confirming that it is a novel Lactobacillus strain.

TABLE 1 comparison of L john-1 with different Lactobacillus johnsonii whole genome ANIs

Sequence file name	Comparison of document species	Comparing file names	ANI（%）
				Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_000498675.1_ASM49867v1_ genomic.fna.gz	95.5668
Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002253275.1_ASM225327v1_ genomic.fna.gz	95.4233
				Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002253205.1_ASM225320v1_ genomic.fna.gz	95.3823
Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002253165.1_ASM225316v1_ genomic.fna.gz	95.3732
				Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002253185.1_ASM225318v1_ genomic.fna.gz	95.3683
Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002253245.1_ASM225324v1_ genomic.fna.gz	95.3515
				Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002253285.1_ASM225328v1_ genomic.fna.gz	95.3379
Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_001270785.1_ASM127078v1_ genomic.fna.gz	95.3079
				Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_001572665.1_ASM157266v1_ genomic.fna.gz	95.1633
Ljohn- 1.genomic.fasta	Lactobacillus johnsonii (A)Lactobacillus johnsonii）	GCA_002803395.1_ASM280339v1_ genomic.fna.gz	95.0963

The invention has the beneficial effects that:

the screened lactobacillus johnsonii has genetic stability, shows excellent bacteriostatic ability to gardnerella vaginalis, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, salmonella paratyphi b and shigella dysenteriae, and has good lactic acid production ability and Hela cell adhesion ability.

Drawings

FIG. 1 is a photograph showing the morphology of a colony of Lactobacillus johnsonii L john-1.

FIG. 2 is a gram-stained image of Lactobacillus johnsonii L john-1.

FIG. 3 is a scanning electron micrograph of Lactobacillus johnsonii L john-1.

Detailed Description

Reference will now be made in detail to specific embodiments of the present invention, including but not limited to the following examples. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified.

The bacterial culture medium components and preparation methods used in the following examples were as follows:

MRS broth is prepared by weighing 52.0 g of MRS finished culture medium powder, dissolving in 1L distilled water, heating to boil, cooling to room temperature, adding 0.55 g of cysteine hydrochloride, stirring to dissolve, adjusting pH to 6.5, loading quantitative liquid distributor, introducing N2, heating to boil, boiling for 20 min under slightly boiling state, cooling, packaging into 10 m L anaerobic tubes, sterilizing at 118 deg.C for 20 min under humid heat, and storing in shade and in dark place.

The preparation of MRS solid culture medium comprises weighing 52.0 g of MRS finished product culture medium powder and 15.0g of agar powder, dissolving in 1L distilled water, boiling, adding 0.55 g of cysteine hydrochloride after boiling, adjusting pH to 6.5, performing wet heat sterilization at 118 deg.C for 20 min, and storing in shade and in dark place.

The preparation of the hydrogen peroxide semi-quantitative culture medium comprises the steps of weighing 52.0 g of MRS finished product culture medium powder and 15.0g of agar powder, dissolving the MRS finished product culture medium powder and the agar powder into 1L distilled water, adjusting the pH value to 6.5, carrying out high-temperature moist heat sterilization at 118 ℃ for 20 min, putting the mixture into a 50 ℃ water bath kettle after sterilization, keeping the temperature for 30 min, adding TMB (so that the final concentration of the TMB is 0.25 mg/m L) and HRP (so that the final concentration of the HRP is 0.01 mg/m L), mixing uniformly, cooling and solidifying, marking the name of the culture medium and the preparation date, and putting the culture medium in.

The preparation of oxygen-free PBS comprises weighing potassium dihydrogen phosphate 0.27 g, disodium hydrogen phosphate 1.42 g, sodium chloride 8 g, potassium chloride 0.2 g, dissolving in distilled water 1L, heating to boil, cooling to room temperature, adding cysteine hydrochloride 0.55 g, stirring to dissolve, adjusting pH to 6.5, loading quantitative liquid distributor, introducing N₂Heating to boil, boiling for 30 min under slightly boiling state, cooling, packaging into 10 m L anaerobic tubes, sterilizing at 121 deg.C for 30 min, and storing in shade and in dark place.

Preparing oxygen-free BHI liquid culture medium by weighing BHI finished product culture medium powder 37.0 g, dissolving in 1L distilled water, heating to boil, cooling to room temperature, adding cysteine hydrochloride 0.55 g, stirring to dissolve, adjusting pH to 6.5, loading quantitative liquid separator, and introducing N₂Heating to boil, boiling for 20 min, cooling, and packaging₂And CO₂(ratio of 1: 1), subpackaging into 10 m L anaerobic tubes, performing wet heat sterilization at 118 ℃ for 20 min, and storing in shade and in the dark for later use.

Preparing oxygen-free BHI semisolid culture medium by weighing BHI finished product culture medium powder 37.0 g, dissolving in 1L distilled water, heating to boil, cooling to room temperature, adding agar powder 6 g and cysteine hydrochloride 0.55 g, stirring to dissolve, adjusting pH to 6.5, loading quantitative liquid distributor, and introducing N₂Heating to boil, boiling for 20 min under slightly boiling state, slightly cooling, and introducing N during cooling and packaging₂And CO₂(ratio of 1: 1), packaging into 10 m L anaerobic tubes, sterilizing at 118 deg.C for 20 min, and storing in shade and dark.

The nutrient broth liquid culture medium is prepared by weighing 10 g of peptone, 3 g of beef powder and 5 g of sodium chloride, dissolving into 1L distilled water, adjusting pH to 7.2, heating to boil, cooling to room temperature, subpackaging, sterilizing each 10 m L, carrying out damp-heat sterilization at 121 ℃ for 15min, and storing in shade and in dark place for later use.

The nutrient broth solid culture medium is prepared by dissolving 10 g of peptone, 3 g of beef powder, 5 g of sodium chloride and 6 g of agar powder in 1L distilled water, adjusting the pH to 7.2, slightly cooling, timely subpackaging into 10 m L anaerobic tubes, performing moist heat sterilization at 121 ℃ for 15min, and storing in shade and in the dark for later use.

Example 1

The method for separating the lactobacillus johnsonii comprises the following steps:

collecting vaginal secretion samples of 20-40 year old Chinese females who pass through health examination by using a vaginal cotton swab, putting 2 m L sterile and anaerobic PBS buffer solution into an anaerobic tube filled with the cotton swab, fully shaking and uniformly mixing the samples, continuously diluting the samples in a ten-fold gradient manner by using the buffer solution as a stock solution, coating liquid diluted by 10000 times by 100 mu L on an MRS solid culture medium, placing the culture medium in an anaerobic incubator at 37 ℃ for culturing for 48 hours, picking out a suspected lactobacillus single colony in the MRS broth culture medium for culturing for 24 hours, transferring one part of the cultured bacterium solution to be cultured continuously, extracting bacterial DNA of the other part of the bacterium solution, amplifying and sequencing the bacteria by 16S, comparing sequencing results B L, analyzing the strains according to the comparison results and respectively preserving to obtain a total 1336 strains of lactobacillus, screening the 16S rRNA genotype, inhibiting the Gardner bacterium test, the lactic acid production test, the Hela cell test, and adhering the lactobacillus strain to the best screened L-john AST.

The screening method of lactobacillus johnsonii comprises the following steps:

1. viability assay in Low pH culture environments

1.1 activation: the lactobacillus is preserved and activated in MRS broth with pH value of 6.5, and cultured at 37 ℃ overnight;

1.2, switching: transferring the activated bacteria liquid into MRS broth culture with pH value of 4-5, and measuring OD once every 2-3 h₆₀₀A value;

1.3 comparative OD₆₀₀Value, screening for ability to proliferate rapidly or OD₆₀₀Higher values of strains from different samples.

2.16S rRNA genotype Screen

If the 16S rRNA sequences of the lactobacilli of the same species separated from the same sample are different, which indicates that the genotypes of the lactobacilli are different, the physiological characteristics of the lactobacilli can also be different, so that the 16S rRNA genotypes of the lactobacilli screened by the low-pH culture activity comparison are screened.

3. Gardnerella vaginalis inhibition assay

Activating lactobacillus, mixing 0.1 m L bacteria solution with MRS solid culture medium, pouring into 6 cm dish, completely solidifying, culturing at 37 deg.C for 48 hr, taking out the dish, perforating on agar culture medium with 6 mm inner diameter perforator to obtain bacterial cake, activating and transferring Gardnerella vaginalis, diluting Gardnerella vaginalis with anaerobic sterile PBS buffer solution at 10 times gradient to 100 times, and respectively taking 10 times of bacteria solution^-1And 10^-20.5 m L diluent and 5.25 m L BHI solid culture medium containing 5% horse serum are mixed evenly, poured into a 9cm plate, after complete solidification, lactobacillus cakes are lightly placed on the surface of BHI agar, 4 cakes are symmetrically placed in each plate, 2 bacteria are parallel, the plate is placed in an anaerobic sealed tank with an anaerobic gas producing bag, the culture is carried out for 48 h at 37 ℃, and the size of the inhibition zone is measured by a vernier caliper.

4. Lactic acid production test

Activating lactobacillus, transferring to MRS broth, culturing at 37 deg.C for 48 h, measuring pH with pH0.5-5.0 test paper, recording pH value of lactobacillus liquid cultured for 48 h, and selecting strains for liquid chromatography under the following two conditions: performing liquid chromatography on a strain with a pH value of 2.5 under the first condition; selecting a strain with a lower pH value from lactobacillus of the same species to perform liquid chromatography; after determining the liquid chromatography sample, diluting the supernatant by 5 times, adding concentrated sulfuric acid for pretreatment, and filtering by using a 0.22 um needle filter before loading. The relevant parameters of liquid chromatography are as follows:

the instrument model is as follows: agilent, analytical liquid chromatography 1200

The type of the chromatographic column: berle, Aminex HPX-87H

Mobile phase: 0.005M H₂SO₄At a speed of 0.6 m L/min

Detector and detection wavelength: DAD,207 nm; RID, differential refractive signal

The sample size is 20 mu L.

5. Capacity of producing hydrogen peroxide

After lactobacillus is activated, 2 mu L bacterial liquid is pipetted into MRS agar containing 0.25 mg/m L of 3,3',5,5' -tetramethylbenzidine solution and 0.01 mg/m L of horse radish peroxidase, 2 plates are arranged in parallel at observation time points of 24 h, 48 h and 72 h, the plates at the same observation time points are placed in the same anaerobic sealed tank with an anaerobic gas generating bag and cultured at 37 ℃, after the corresponding time, the corresponding plates are taken out and exposed in the air, and after 30 min, the coloration reaction is observed and recorded by taking pictures, wherein the lactobacillus delbrueckii is taken as a positive control, the blue color produced by the lactobacillus delbrueckii is 4 minutes in depth, the blue color produced by the lactobacillus delbrueckii is 3 minutes in equivalent to the blue color produced by the lactobacillus delbrueckii, the blue color produced by the lactobacillus delbrueckii is 2 minutes in light color, the blue color produced by the lactobacillus delbrueckii is 1 minute (slight coloration reaction), and the unchanged color is 0 minute.

6. Adhesion Hela cell assay

Activating lactobacillus, centrifugally washing the lactobacillus for 2 times, resuspending by PBS, sucking 100 mu L of lactobacillus suspension into a 96-hole cell culture plate containing Hela cells, standing and incubating at 37 ℃ for 30 min, washing for 2 times by sterile PBS after 30 min to wash the lactobacillus adhered without washing, adding 25 mu L of pancreatin solution into each hole of the 96-hole cell culture plate containing the Hela cells, placing the cell in a 37 ℃ incubator for digesting the cells, adding 75 mu L of complete culture medium into each hole after the Hela cells are digested into spheres, repeatedly blowing and beating uniformly, sucking 20 mu l of bacterium suspension after complete digestion, diluting by 10 times of sterile PBS in a gradient manner, selecting a proper dilution gradient for a pouring counting experiment, culturing at 37 ℃ for 48 h, and counting.

The properties of the Lactobacillus johnsonii L john-1 of the present invention obtained from the above screening are shown in Table 2 below.

TABLE 2 probiotic Properties of Lactobacillus johnsonii L john-1

	Diameter of Gardner bacterium inhibition zone (mm) for vagina	Lactic acid content (mg/L)	Score for hydrogen peroxide production	Number of single cell adhesion
					Lactobacillus johnsonii L john-1	17.77	16064.53	1	6
Standard strains	14.29	12964.12	3	7.7

Example 2

The biochemical identification of Lactobacillus johnsonii L john-1 was as follows:

the bacterial strain is biochemically identified by adopting an API 50 CH L lactobacillus identification system produced by French Merrier company to carry out a methyl red test (MR test), an acetyl methyl carbinol test (VP test), an indigo matrix test, an esculin hydrolysis test, a trisaccharide iron test, a Krebs iron test, a urease test, a phenylalanine deaminase test, an amino acid decarboxylase test, a gelatin liquefaction test, a sodium malonate test, a citrate test (citrate test), a nitrate reduction test, a litmus milk test and a bacterial dynamic test, and the specific results are as follows:

lactobacillus johnsonii L john-1 is capable of hydrolyzing esculin to glucose and escin, MR test is negative to indicate that organic acid produced by metabolizing glucose is insufficient to discolor the developer, VP test is negative to indicate that metabolizing glucose does not produce pyruvic acid, indigo substrate test results are that the bacterium does not decompose tryptophan in peptone to produce indole, ferric trisaccharide test indicates that metabolizing glucose lactose sucrose and no hydrogen sulfide, and ferric chloride test indicates that metabolizing fermented glucose lactose and no hydrogen sulfide, urease test, phenylalanine deaminase test, amino acid decarboxylase test, gelatin liquefaction test are negative to indicate that the bacterium does not produce urease, phenylalanine deaminase, amino acid decarboxylase, gelatinase, sodium malonate test, citrate test, nitrate reduction test is negative to indicate that the bacterium does not use sodium malonate as a carbon source, citrate as a nitrogen source and carbon source, does not reduce to nitrite, and milk pistil test finds that the bacterium can make milk ferment milk sugar to produce glucose, xylose, mannose.

Example 3

Antibiotic susceptibility testing of Lactobacillus johnsonii L john-1.

According to the requirement of an antibiotic susceptibility test in the third general theory of live microbial products of the pharmacopoeia of the 2015 edition, the sensitivity of the strain to the antibiotic is determined by adopting an agar diffusion paper sheet method, and the sensitivity level of the strain to the antibiotic is judged according to the size of an inhibition zone.

Activating and streaking lactobacillus johnsonii L john-1 in MRS broth culture medium, activating Escherichia coli in nutrient broth, culturing at 37 deg.C, activating lactobacillus, selecting a ring of bacteria liquid, streaking on MRS solid culture medium, placing the plate in an anaerobic sealed tank with anaerobic gas generating bag, culturing at 37 deg.C, activating Escherichia coli, selecting a ring of bacteria liquid, streaking on nutrient broth solid culture medium, and placing the plate in a sealed tank for culturing at 37 deg.C.

Selecting a plurality of single bacterial colonies from the cultured agar plate, directly inoculating the single bacterial colonies into physiological saline to prepare bacterial suspension, adjusting the turbidity of the suspension to enable the turbidity to reach 0.5 McLeod concentration, immersing a sterile cotton swab into the suspension within 15min preferably after the turbidity of the suspension is adjusted, and rotating the swab above the liquid tightly close to the inner wall of the test tube for a plurality of times, so that redundant liquid on the cotton swab can be removed.

The whole agar surface is marked by a swab, the whole agar surface is inoculated on a plate with a dry surface, lactobacillus johnsonii L john-1 bacterial suspension is inoculated on an MRS agar plate, escherichia coli bacterial suspension is respectively inoculated on an MH agar plate and an MRS agar plate, after inoculation is finished, a drug sensitive paper sheet distributor is used for distributing and placing drug sensitive paper sheets on the plate, 6 drug sensitive paper sheets are placed on each plate, 3 drug sensitive paper sheets are parallel, 12 drug sensitive paper sheets (all OXOID) are counted in the experiment, and the information is respectively as follows:

TABLE 3 inhibitory Capacity of antibiotics to Lactobacillus johnsonii L john-1

Name of antibiotic

Content (wt.)

Diameter of bacteriostatic circle (mm)

Drug resistance

Intermediary agent

Sensitivity of

Reference bacterium

Ampicillin

10 µg

32.43

≤18

19-21

≥22

Haemophilus sp

Erythromycin

15 µg

31.6

≤15

16-20

≥21

Streptococcus pneumoniae

Imipenem

10 µg

34.31

–

≥16

Haemophilus sp

Vancomycin

30 µg

19.33

–

≥17

Streptococcus pneumoniae

Clindamycin

2 µg

12.05

≤15

16-18

≥19

Streptococcus pneumoniae

Nystatin

100 units

6

–

Antifungal, reference-free bacteria

Metronidazole

5 µg

6

–

Sterile reference bacterium

Piperacillin/tazobactam

110 µg

39.81

≤17

18-20

≥21

Enterobacteriaceae

Ceftizoxime

30 µg

31.25

≤14

15-19

≥20

Genus staphylococcus

Fluconazole

25 µg

6

–

Antifungal, reference-free bacteria

Norfloxacin hydrochloride

10 µg

6

≤12

13-16

≥17

Enterobacteriaceae

Gentamicin

10 µg

7.97

≤12

13-15

≥15

Enterobacteriaceae

As a result:

(1) lactobacillus johnsonii L john-1 is sensitive to five antibiotics ampicillin, erythromycin, imipenem, piperacillin/tazobactam and ceftizoxime;

(2) lactobacillus johnsonii L john-1 is resistant to four antibiotics, nystatin, metronidazole, fluconazole and gentamicin;

(3) lactobacillus johnsonii L john-1 is sensitive to vancomycin;

(4) lactobacillus johnsonii L john-1 is resistant to clindamycin;

(5) lactobacillus johnsonii L john-1 was resistant to norfloxacin.

Example 4

Lactobacillus johnsonii L john-1 has the ability to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae.

Activating Lactobacillus johnsonii L john-1, mixing 0.1 m L bacterial liquid with MRS solid culture medium, pouring into 6 cm plate, completely solidifying, culturing at 37 deg.C for 48 h, taking out the plate, perforating on agar culture medium with 6 mm inner diameter perforator to obtain bacterial cake, activating and transferring Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae, diluting with 0.9% physiological saline at 10 times gradient to 100 times, and respectively taking 10 times of bacterial liquid^-1And 10^-2Mixing the diluted solution 0.5 m L with 5m L nutrient agar solid culture medium, and pouring into a 9cm flatAnd (3) slightly placing the lactobacillus cakes on the surface of nutrient agar after complete solidification in a dish, symmetrically placing 4 cakes in each dish, enabling 2 bacteria in each strain to be parallel, placing the flat dish into a sealed tank, culturing for 24 hours at 37 ℃, and measuring the size of a bacteriostatic zone by using a vernier caliper.

The results obtained are shown in the following table:

TABLE 4 inhibitory Activity of Lactobacillus johnsonii L john-1 against aerobic pathogens

Diameter of bacteriostatic circle (mm)	Staphylococcus aureus	Escherichia coli	Shigella dysenteriae	Pseudomonas aeruginosa	Salmonella paratyphi type B
						Lactobacillus johnsonii L john-1	12.39	9.65	12.41	13.28	12.45
Standard strains	11.28	9.32	11.93	13.09	12

Example 5

Bacterial genomic DNA was extracted from Lactobacillus johnsonii L john-1 at 0 th generation (T0) and 30 th generation (T30), PCR was performed using primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3'), and PCR amplification products of T0 and T30 were sequenced, and the sequencing results of T0 and T30 were identical, indicating genetic stability.

Example 6 toxicity test

The test sample is Lactobacillus johnsonii L john-1 viable bacteria capsule with low dose of 2.0 x 10⁹CFU/grain; high dose: 1.0*10¹⁰CFU/pellet.

The administration route is as follows: vaginal administration

Frequency and duration of administration: DAY1 was administered 1 time

The administration method comprises the following steps: the blank control group is given with blank capsules, the low and high dosage groups of the test sample are respectively given with test sample capsules with corresponding specifications, and 1 capsule is given to the vagina.

Reasons for the choice of route of administration: the clinical planned administration route is simulated, and vaginal administration is selected.

Observation frequency and time: after the 1-3 groups of animals are dosed, cage-side observation is carried out on the acute toxic reaction for at least 4 hours, and 4 hours later, the observation is finished on the animals without obvious abnormal reaction; for animals with obvious abnormal manifestations, detailed clinical observations should be made, and the end time of the day of observation is determined by the person in charge of the subject.

Detailed clinical observations: when 1-3 groups of animals receive the feed, the animals are taken out of the rearing cage 1 time per week before administration and after administration for detailed clinical observation, and the observation frequency can be increased as required. The detailed clinical observation period may not be recorded for general clinical observations. Observations include, but are not limited to, behavioral activity, skin, quilt hair, eyes, ears, nose, abdomen, external genitalia, anus, limbs, feet, respiration.

Weight: animals were received, before grouping, weighed 1 time per week after dosing, and all animals were weighed before they found dead or euthanized.

Food intake: after administration, 1-3 groups of animals were subjected to 1-time food intake (addition/remainder) measurement every week, and the results were expressed in "g/animal/day". The remaining feed volume of the cage animals was determined before the animals were fasted overnight.

As a result, no obvious abnormality is observed in clinical observation, body weight and food intake, and the result proves that the medicine is nontoxic.

The invention is well implemented in accordance with the above-described embodiments. It should be noted that, based on the above structural design, in order to solve the same technical problems, even if some insubstantial modifications or colorings are made on the present invention, the adopted technical solution is still the same as the present invention, and therefore, the technical solution should be within the protection scope of the present invention.

Sequence listing

<110> Szechwan anaerobic Biotechnology Limited liability company

<120> Lactobacillus johnsonii and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1437

<212>DNA

<213> Lactobacillus johnsonii (L Acobacter johnsonii)

<400>1

gctgatccta taaaggttat cccaccggct ttgggtgtta cagactctca tggtgtgacg 60

ggcggtgtgt acaaggcccg ggaacgtatt caccgcggcg tgctgatccg cgattactag 120

cgattccagc ttcgtgtagg cgagttgcag cctacagtcc gaactgagaa cggctttaag 180

agatccgctt gccttcgcag gttcgcttct cgttgtaccg tccattgtag cacgtgtgta 240

gcccaggtca taaggggcat gatgacttga cgtcatcccc accttcctcc ggtttgtcac 300

cggcagtctc attagagtgc ccaacttaat gatggcaact aatgacaagg gttgcgctcg 360

ttgcgggact taacccaaca tctcacgaca cgagctgacg acagccatgc accacctgtc 420

tcagcgtccc cgaagggaac acctaatctc ttaggtttgc actggatgtc aagacctggt 480

aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540

caattccttt gagtttcaac cttgcggtcg tactccccag gcggagtgct taatgcgtta 600

gctgcagcac tgagaggcgg aaacctccca acacttagca ctcatcgttt acggcatgga 660

ctaccagggt atctaatcct gttcgctacc catgctttcg agcctcagcg tcagttgcag 720

accagagagc cgccttcgcc actggtgttc ttccatatat ctacgcattc caccgctaca 780

catggagttc cactctcctc ttctgcactc aagttcaaca gtttctgatg caattctccg 840

gttgagccga aggctttcac atcagactta ttgaaccgcc tgcactcgct ttacgcccaa 900

taaatccgga caacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg 960

tgactttcta agtaattacc gtcaaataaa ggccagttac tacctctatc tttcttcact 1020

accaacagag ctttacgagc cgaaaccctt cttcactcac gcggcgttgc tccatcagac 1080

tttcgtccat tgtggaagat tccctactgc tgcctcccgt aggagtttgg gccgtgtctc 1140

agtcccaatg tggccgatca gtctctcaac tcggctatgc atcattgcct tggtaagccg 1200

ttaccttacc aactagctaa tgcaccgcag gtccatccaa gagtgatagc agaaccatct 1260

ttcaaactct agacatgcgt ctagtgttgt tatccggtat tagcatctgt ttccaggtgt 1320

tatcccagtc tcttgggcag gttacccacg tgttactcac ccgtccgccg ctcgcttgta 1380

tctagtttca tttagtgcaa gcactaaaat catctaggca agctcgctcg actgcag 1437

Claims

1. Lactobacillus johnsonii is named as Lactobacillus johnsonii (L actinobacillus johnsonii L) L john-1, and the preservation number is CCTCC No. M2019426.

2. Use of a lactobacillus johnsonii according to claim 1 in the manufacture of a medicament for the prevention or treatment of vaginitis.

3. Use of a lactobacillus johnsonii strain according to claim 1 in the manufacture of a feminine hygiene product for the prevention of vaginitis.

4. Use of a lactobacillus johnsonii strain according to claim 1 in the manufacture of an external genital hygiene product.

5. Use of a lactobacillus johnsonii according to claim 1 for the preparation of a medicament or health care product for modulating vaginal flora balance.

6. The use of a Lactobacillus johnsonii strain according to claim 1 for the preparation of a medicament or a health care product with vaginal epithelial cell adhesion.

7. Use of a lactobacillus johnsonii according to claim 1 for the preparation of a medicament or health care product for the prevention or treatment of vaginal pathogenic bacteria.

8. Use according to claim 7, wherein the pathogenic bacteria include, but are not limited to, any one or more of Gardnerella vaginalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae.

9. Use of lactobacillus johnsonii according to claim 1 in a topical infant care product delivered via caesarean section.