CN116515686B - Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof - Google Patents

Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof Download PDF

Info

Publication number
CN116515686B
CN116515686B CN202310337582.1A CN202310337582A CN116515686B CN 116515686 B CN116515686 B CN 116515686B CN 202310337582 A CN202310337582 A CN 202310337582A CN 116515686 B CN116515686 B CN 116515686B
Authority
CN
China
Prior art keywords
lactobacillus crispatus
streptococcus
roea
premenstrual
lactobacillus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202310337582.1A
Other languages
Chinese (zh)
Other versions
CN116515686A (en
Inventor
贾长生
施凌艳
卞徐磊
胡鸿飞
赵培天
吴贵珍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Kerong Biopharmaceutical Co ltd
Original Assignee
Jiangsu Kerong Biopharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Kerong Biopharmaceutical Co ltd filed Critical Jiangsu Kerong Biopharmaceutical Co ltd
Priority to CN202310337582.1A priority Critical patent/CN116515686B/en
Publication of CN116515686A publication Critical patent/CN116515686A/en
Application granted granted Critical
Publication of CN116515686B publication Critical patent/CN116515686B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a lactobacillus crispatus, which is lactobacillus crispatus Roea (Lactobacillus crispatus Roea), and is preserved in China center for type culture Collection, the preservation date is 2022, 12 months and 23 days, and the preservation number is CCTCC NO: m20222049. The invention also discloses application of the lactobacillus crispatus in preparing products for treating female reproductive infection and regulating premenstrual psychological symptoms. Compared with the prior art, the invention provides a brand new lactobacillus crispatus Roea which has high acid production and acid resistance and can inhibit various fungi including streptococcus pyogenes, streptococcus pharyngeal inflammation and the like; the vaginal mucosa is nonirritating, can be used as a biological agent for directly carrying out vaginal microecological intervention, and can effectively treat female reproductive infections such as colpitis; in addition, the lactobacillus crispatus roua can be used for improving female premenstrual psychological symptoms, and has remarkable effect.

Description

Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof
Technical Field
The invention belongs to the technical field of lactobacillus crispatus, and particularly relates to lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof.
Background
The internal vaginal environment of a female is a microecological environment formed by colonised microbiota in the vaginal cavity of the female. Normally, a large number of microorganisms are lodged in the vaginal cavity of women, which microorganisms play an important role in the local area, maintaining the vaginal health of women. The female vagina microorganism group takes lactobacillus as main dominant bacteria, and simultaneously has a small amount of other anaerobic bacteria, and the lactobacillus can produce acid and hydrogen peroxide (H) 2 O 2 ) The weak acidic environment of the vagina is maintained so as to inhibit the growth of pathogenic bacteria and maintain the normal internal environment of the vagina. When the abundance of lactobacillus in vagina is reduced or the function of lactobacillus is reduced, other anaerobic bacteria in vagina are excessively proliferated, so that the microecology of the vagina is unbalanced, the internal environment is disturbed, and the infection of pathogens can be promoted, thereby causing local inflammation and pathological changes; meanwhile, local pathogen infection of vagina can increase the risks of pelvic inflammatory disease, premature birth, flow and the like. For example, group B streptococcus is an important cause of puerperal sepsis and neonatal meningitis in pregnant women, and parasitic infection in the maternal genital tract can cause infant infection, and also can cause puerperal infection, bacteremia, endocarditis, skin and soft tissue infection, and osteomyelitis; infection of the female genital tract by streptococcus pyogenes causes fever and erythema, and causes abscess disease; infection of the female reproductive system by streptococcus buccinis can cause hypoimmunity of postpartum females, puerperal infection, which is manifested by infection of uterine cavity, pelvic cavity, abdominal pain, lumbago, leucorrhea increase, fever and other uncomfortable symptoms. In recent years, there have been studies on the use of lactobacillus for improving and remodelling the intravaginal environment by administering it to a patient, such as lactobacillus crispatus, which can inhibit various bacteria such as common escherichia coli, candida albicans, staphylococcus aureus, etc., but has limited the use of less bacteria that can be inhibited against group B streptococcus, streptococcus pyogenes, streptococcus pharyngeri, etc.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the invention provides lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof.
The technical scheme is as follows: in order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a lactobacillus crispatus, which is a lactobacillus crispatus Roea (Lactobacillus crispatus Roea) preserved in China center for type culture Collection, with a preservation date of 2022, 12 months and 23 days, and a preservation number of cctccc NO: m20222049.
In a second aspect, the invention provides the use of a lactobacillus crispatus as described above, comprising the following:
the application of the lactobacillus crispatus in preparing a bacteriostatic agent.
Further, the bacteriostatic agent can inhibit escherichia coli, candida albicans, staphylococcus aureus, group B streptococcus, streptococcus pyogenes and/or streptococcus pharyngolaryngitis.
Or the application of the lactobacillus crispatus in preparing a vaginal microecological intervention agent or a product for treating female reproductive infection; preferably, the female reproductive infection is vaginitis; the Lactobacillus crispatus is effective in treating vaginitis and also preventing recurrence thereof.
Alternatively, the use of said Lactobacillus crispatus for the preparation of a product for improving premenstrual psychological symptoms in women. Further, the female is a healthy female aged 20-35 years.
In a third aspect, the present invention provides a composition comprising said Lactobacillus crispatus.
In a fourth aspect, the invention provides the use of said composition comprising the following:
the application of the composition in preparing a bacteriostatic agent.
Further, the bacteriostatic agent can inhibit escherichia coli, candida albicans, staphylococcus aureus, group B streptococcus, streptococcus pyogenes and/or streptococcus pharyngolaryngitis.
Or, the composition is applied to the preparation of a vaginal microecological intervention agent or a product for treating female reproductive infection; preferably, the female reproductive infection is vaginitis; the Lactobacillus crispatus is effective in treating vaginitis and also preventing recurrence thereof.
Or, the composition is applied to the preparation of medicines for improving female premenstrual psychological symptoms. Further, the female is a healthy female aged 20-35 years.
The product comprises a bacteriostatic agent or a medicine and the like.
The beneficial effects are that: compared with the prior art, the invention provides a brand new lactobacillus crispatus Roea which has high acid production and acid resistance and can inhibit various fungi including streptococcus pyogenes, streptococcus pharyngeal inflammation and the like; the vaginal mucosa is nonirritating, can be used as a biological agent for directly carrying out vaginal microecological intervention, and can effectively treat female reproductive infections such as colpitis; in addition, the lactobacillus crispatus roua can be used for improving female premenstrual psychological symptoms, and has remarkable effect.
Drawings
FIG. 1 shows the result of gram staining.
FIG. 2 is a Lactobacillus crispatus Rose biological evolutionary tree.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and the examples.
Example 1
In this example, lactobacillus selected from healthy human body was subjected to screening for genital tract flora-regulated growth, and samples were collected and stored
To obtain lactobacillus crispatus in the genital tract of healthy females, 36 samples of genital tract secretions of healthy females were collected, the standard of healthy samples is: no antibacterial medicine is orally taken or locally applied to vagina within 1 month, no irregular menstruation is caused, menstrual period is avoided, no intrauterine device is arranged in uterus, sexual life and vaginal lavage exist within 3 days, the PH value of the vagina is less than or equal to 4.5, and the cleanliness is I-II degrees. Three samples of each female genital tract sample are collected by using a sterile cotton swab, immediately put into an anaerobic bag, then put into an ice box for preservation, and returned to a laboratory in time for freezing and preservation, sequencing, accounting and extraction. 1 part of healthy genital tract sample is selected, 15-20% of sterile glycerin is added, the mixture is fully vortex-vibrated and stored in a freezing tube, the freezing tube is pre-cooled for 2 hours at 4 ℃ and then pre-frozen for 8 hours at-20 ℃ by adopting a step pre-cooling method, and finally the mixture is stored at-80 ℃ or liquid nitrogen for screening dominant strains.
EXAMPLE 2 screening of Lactobacillus crispatus strains with high acid resistance
After the samples collected and stored in the example 1 are quickly dissolved, 100 mu L of genital secretion suspension is taken and diluted with a gradient of 10 times, 3 to 5 MRS agar culture mediums are respectively coated, the anaerobic culture tanks are filled with the culture tanks, and ternary mixed gas (80% of nitrogen, 10% of hydrogen and 10% of carbon dioxide) is used for replacing the gas in the tank, so that the final oxygen concentration in the culture tanks is less than or equal to 5%, and the culture tanks are cultured in a 37 ℃ incubator for 48 hours. And (3) selecting independent colonies with bright colors and plump shapes, and performing high-flux strain preliminary identification by adopting a Matrix-assisted laser desorption ionization time-of-flight massspectrometry (MALDI-TOF MS, short for flight mass spectrometry) technology.
After single colonies initially identified as Lactobacillus crispatus by MALDI-TOF MS equipment were picked up in 15ml of MRS medium and cultured at 37℃for 8 hours, a fermentation broth was obtained.
The bacterial amount of the fermentation liquor is observed by comparing the measured fermentation OD600 with a microscope, the bacterial strains with poor liquid fermentation effect or no obvious bacterial strain are discarded, 0.5ml of the rest 45 parts of fermentation liquor are respectively taken for sequencing and identifying the bacterial strains for standby, 2ml of glycerol solution with 40 percent of 2ml is taken for precooling and then frozen to-80 ℃ for standby bacterial strain preservation, the rest fermentation liquor is continuously placed in a shaking table for fermentation culture at 37 ℃ for measuring the pH value every 6 hours, and the pH value is continuously measured for 4 times. After the secondary fermentation for 24 hours, the pH of the fermentation liquor can reach 3.0 at the lowest. The pH value of the fermentation liquid in the fermentation process is selected to drop fast and OD is selected 600 The samples with higher final fermentation pH value of 3.0-3.8 are taken as candidate strains, and the total number of the strains is 5, and the number is 1-5.
Example 3 in vitro bacteriostasis experiment
Study of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on intervention against genital floraThe method has the advantages of more results and better effect, the method is used as a control strain, the control strain and 5 selected candidate strains are used for evaluating the antibacterial capacity, the selected candidate strains are respectively inoculated into 50ml of MRS liquid culture medium, after the culture is carried out for 16 hours at 37 ℃, the fermentation broth is centrifuged for 10 minutes at 10000r/min, and the supernatant and thalli are respectively collected. The cells were washed twice with 0.9% sterile saline and then OD was prepared with 0.9% sterile saline 600 The bacterial suspension is 0.5-0.8, and the bacterial suspension is preserved at 4 ℃ for standby.
And (3) taking escherichia coli 8099, candida albicans ATCC10231 and staphylococcus aureus ATCC 6538 as in-vitro bacteriostasis test strains, and performing bacteriostasis circle test on fermentation supernatant and bacterial suspension of each strain by adopting an agar perforation method. The MRS culture medium is regulated by lactic acid to have the same PH value with the supernatant liquid as a blank control, the metronidazole solution with the concentration of 0.05mg/mL is used as a positive control for inhibiting escherichia coli and staphylococcus aureus, and the clotrimazole solution with the concentration of 0.05mg/mL is used as a positive control for inhibiting candida albicans. And measuring the size of the antibacterial ring, wherein the antibacterial ring radius generated by the sample to be measured is larger than that of the MRS control liquid, so that the antibacterial ring has antibacterial activity. The results of bacterial strain evaluation with significant bacteriostatic effect are shown in table 1, except for a part of bacterial strain with insignificant bacteriostatic effect.
The bacterial suspension and supernatant of the No. 2 strain have obvious inhibition effects on escherichia coli 8099, candida albicans ATCC10231 and staphylococcus aureus ATCC 6538, and the antibacterial capacity is obviously superior to that of lactobacillus rhamnosus GR-1 and lactobacillus reuteri RC-14.
Table 1 evaluation of antibacterial effect
Note that: a has significance compared with GR-1, b has significance compared with RC-14
EXAMPLE 4 preservation, identification of Strain and classical storage of Strain
The evaluation results in the screening process of the strains in the comprehensive examples 1-3 can not only produce acid/resist acid at high level and inhibit pathogenic bacteria causing gynecological diseases, but also have the mucosa high adhesion capability, namely the No. 2 strain, which is the target strain.
(1) Preservation of strains
After streaking the screened Lactobacillus crispatus Roea on MRS agar medium for 48 hours, single colonies were selected and placed in 10ml of liquid medium, which is MRS liquid medium containing 4.5% soybean lecithin and 2% calcium carbonate. Placing the culture medium in 37 ℃ for constant temperature culture for 12-16 h, adding glycerol solution with the concentration of 30-40% into the culture medium according to the volume of 1:1 when the OD600 value of the culture medium is not less than 1.2, blowing and mixing uniformly by a liquid suction device, sub-packaging into 2ml sterile freezing pipes, pre-cooling for 2h at 4 ℃, pre-freezing for 4h at-20 ℃, and finally transferring to a refrigerator at-80 ℃ or liquid nitrogen for preservation.
(2) Further morphological characterization
The purified bacterial liquid is taken and observed under a microscope, and the gram positive, slender rod and micro-bending chain appear, no flagella, no spore, white colony, irregular edge, flaky shape and raised center.
(3) Gram staining
The result of the gram staining was gram positive, as shown in figure 1.
(4) 16SrRNA molecular biological identification
The published 16S universal primer (primer sequence: 8F:5'-AGAFTTTGATCCTGGCTCA-3'; 460R: 5 '-GGTTACCTTGTTACGACTT-3') is used for amplifying and sequencing the 16S rDNA gene sequence of the No. 5 strain, and the PCR amplified product is sent to Shanghai Biotechnology Co., ltd for sequencing identification. The nucleotide sequence of the 16SrDNA of the No. 2 strain is a sequence 1 in a sequence table; through 16SrDNA gene comparison, the similarity rate of the gene is 99% compared with Lactobacillus crispatus in Genebank; in combination with the identification of a microbial system, the strain No. 2 is identified as a Lactobacillus crispatus, named Lactobacillus crispatus Roea, the 16S rDNA of which is shown as SEQ ID NO.1, and the biological evolutionary tree is shown as FIG. 2.
The biological identification of the Lactobacillus crispatus Roea is subjected to classical collection, and the strain is preserved in China Center for Type Culture Collection (CCTCC) for type culture collection (abbreviated as CCTCC) in 2022, and the preservation number is CCTCC NO: m20222049, the preservation address is university of Wuhan in Wuhan City of Hubei province of the people's republic of China.
EXAMPLE 5 Roea inhibition of group B Streptococcus
Bacterial suspensions and supernatants of roua, GR-1 and RC14 were prepared as described in example 3 as experimental ingredients for inhibiting group B streptococcus. And (3) performing a bacteriostasis zone test on fermentation supernatant and bacterial suspension of each strain by adopting an agar perforation method. The obtained 4 positive reproduction samples of the group B streptococcus are subjected to coating culture by adopting a group B streptococcus chromogenic medium to observe a bacteriostasis zone, and statistical results show that the bacterial suspension and supernatant of the Roea strain have stronger inhibition capability on the group B streptococcus.
Note that: the "-" is that no obvious inhibition zone appears, the "+" is that the diameter of the inhibition zone is 5mm-15mm, the "++" is that the diameter of the inhibition zone is 15mm-25mm, and the "++" is that the diameter of the inhibition zone is more than 25mm.
EXAMPLE 6 inhibition of Roea on group A Streptococcus (Streptococcus pyogenes) and Streptococcus pharyngeal and buccal
Bacterial suspensions and supernatants of roua, GR-1 and RC14 were prepared as in example 3, and streptococcus pyogenes ATCC19615 and streptococcus strainophyllus ATCC 9895 were selected as test strains for in vitro bacteriostasis experiments, and fermentation supernatants and bacterial suspensions of each strain were subjected to bacteriostasis circle experiments by an agar perforation method. The MRS culture medium is regulated by lactic acid to have the same PH value with the supernatant liquid as a blank control, and penicillin solution with the concentration of 0.05mg/mL is used as a positive control for inhibiting streptococcus pyogenes and streptococcus angina. The results of statistics of the size of the inhibition zone show that the bacterial suspension and the supernatant of the Roea strain have stronger inhibition capability on streptococcus pyogenes and streptococcus strainois.
Note that: the "-" is that no obvious inhibition zone appears, the "+" is that the diameter of the inhibition zone is 5mm-15mm, the "++" is that the diameter of the inhibition zone is 15mm-25mm, and the "++" is that the diameter of the inhibition zone is more than 25mm.
Example 7 stimulation experiments of genital tract mucosal cells
According to the vaginal mucosa stimulation experimental standard in the national ministry of health, female New Zealand rabbits which are healthy as early as possible and in the non-estrus period are selected, and whether secretion, congestion, edema and other damage conditions of the vaginal orifice of the animal are detected before the experiment. Lactobacillus crispatus Roea lyophilized powder 0.8g (2.7X10) 11 CFU/g) was dissolved in 100ml of sterile physiological saline as a test solution, and 3 female new zealand rabbits were randomly selected to be contaminated with 2ml of the test solution each time. Animals in the control group were treated similarly with physiological saline. Each time interval is 24 hours, and the time is 5 days. The animals were sacrificed 24h after the last contamination by air embolism, longitudinal incision was made after complete vaginal cutting, visual observation of the presence or absence of oedema, congestion, etc. of the mucosa, and then fixed in 10% formaldehyde solution for histopathological examination.
According to the grading standard of vaginal mucosa side mark and the grading of the irritation intensity in the disinfection technical specification, the vaginal mucosa irritation index of the lactobacillus crispatus Roea freeze-dried powder is 0.89 < 1, and the vaginal mucosa irritation index is judged to be non-irritating. Can be used as a biological agent for directly applying to vaginal microecology intervention.
EXAMPLE 8 treatment of vaginitis
98 patients with vaginitis collected and treated in a certain hospital are selected, and are divided into an observation group and a control group by adopting a random digital table method, 47 patients are observed in the observation group, the course of the disease is 6-60 days, and the average time is 17.62+/-0.71 days; 31 cases of recurrence and 16 cases of onset; the pH value of the vagina is 5.42+/-1.52; of these, 38 cases of bacterial vaginitis and 9 cases of trichomonas vaginitis. 51 cases of control group, the course of disease is 6-60 days, and the average time is 17.68+/-0.78 days; 34 cases of recurrence and 17 cases of onset; the pH value of the vagina is 5.41+/-1.56; of these, bacterial vaginitis is 39 cases and trichomonas vaginitis is 12 cases. The clinical data of group 2 were compared without significant differences (P > 0.05).
Patients with bacterial vaginitis in the control group are treated by adopting 1 piece of metronidazole vaginal effervescent tablet for 1 piece of vaginal placement treatment each time, and 7d of treatment are carried out each time; metronidazole tablet 400mg is administered orally to patients with trichomonas vaginitis, 2 times daily, for 7d. Patients in the observation group were given live Lactobacillus crispatus Roea preparation (containing Lactobacillus crispatus Roea 5.8X10) for vagina based on the control group treatment 9 CFU/tablet), 1 vaginal insert at a time, 1 time per night, 7 days of treatment.
After 7 days of contrast dry prognosis, the contrast group has 15 cases of significant effect, 21 cases of effect and 14 cases of no effect, and the total effective rate is 72%; the observation groups show 43 cases, 7 cases and 0 cases, and the total effective rate is 100%. The clinical effect of the observation group is obviously higher than that of the control group.
Vaginal cleaning grade comparison. The comparative difference in vaginal cleaning levels was statistically significant (P > 0.05) for the pre-treatment two groups of patients, and after treatment, both groups were reduced in vaginal cleaning level and the observed group (1.01.+ -. 0.13 cleaner) was lower than the control group (2.3.+ -. 0.47 cleaner) (P < 0.05).
Example 9 intervention in vaginitis and recurrent prevention
In this example, 9 control groups treated with metronidazole still had no clinical cure after the control group of example 8 was treated, and only 2 patients in the observation group had no clinical cure after the next menstrual cycle. Meanwhile, 5 cases (7/50) clinically cured of metronidazole recurred in the second follow-up period, while only 1 case of BV patients clinically cured of the active bacterial formulation of Lactobacillus crispatus (1/50) had recurred. The results show that the treatment of gynecological inflammation by the Roea intervention can effectively improve the cure rate and prevent recurrence.
Example 10 improvement of premenstrual psychological symptoms in healthy young women
The intestinal-brain axis plays a vital role in maintaining intestinal homeostasis and brain function through bi-directional communication. Women of childbearing age may also experience physiological and/or emotional discomfort several weeks before menstruation, with over 90% of young women suffering from premenstrual symptoms to some extent, with symptoms varying from person to person. Menstrual cycle-dependent physiological or psychological symptoms are associated with periodic changes in the levels of the reproductive hormones estradiol (E2) and progesterone (P4), pituitary hormones (gonadotrophins), prostaglandins and neurotransmitters in the brain. E2 and P4 may also affect the mood and sleep patterns of females. E2 modulates various neurotransmitter systems, including serotonin and dopamine, affecting emotion. Genital hormones have been shown to regulate the sleep pattern in women throughout adulthood. These hormones also affect anxiety-like behavior in rodent models. The Lactobacillus crispatus observed in the present invention can affect changes in premenstrual E2 and P4 levels, thereby alleviating premenstrual endocardial symptoms.
Women between 20-35 years old, unused medications, menstrual cycles typically shorter than 25 days or longer than 38 days, 83 subjects scored more than 60 on the self-assessed depression scale (SDS), were selected and randomly assigned to either the control group (38) or the observation group (45). Lactobacillus crispatus Roea (2 g/bag, containing Lactobacillus crispatus Roea 1.5X10) was administered to the panel 10 CFU/bag, maltodextrin as auxiliary material), the control group was given the same dose of maltodextrin (2 g/bag, maltodextrin as the ingredient) and 1 bag was consumed each morning and evening every day, while participants were required to not consume live lactobacillus-containing foods such as fermented milk or other probiotic-containing or probiotic-containing products, and the intervention period was about 180 days. Saliva was collected between 14 hours and 16 hours from menstrual period 1 and 3 to 6 days (luteal phase) and 3 to 6 days (follicular phase) after menstruation, control interventions were started 7 days after the end of menstrual period 1, saliva was collected 3 to 6 days (luteal phase) before menstrual period 5 and 3 to 6 days (follicular phase) after menstruation from the start of interventions, and saliva samples were stored at-80 ℃ until detection analysis. Estradiol (E2) and progesterone (P4) in the saliva sample were detected using salivary progesterone ELISA kit (saliretrics) and salivary 17 β -estradiol ELISA detection kit (saliretrics), respectively. After the initial stem, the premenstrual root fills out a premenstrual tension syndrome (PMTS) -Visual Analog Scale (VAS) questionnaire for each menstrual cycle, and the premenstrual symptoms are assessed.
* The differences are statistically significant (p < 0.05)
Of the 12 premenstrual symptoms listed in the PMTS-VAS questionnaire, the Roea observations group scored significantly lower than the placebo group in terms of "mood down", "anxiety", "reduced interest in activity", "easy fatigue" and "more sleep". The data indicate that daily intake of Roea improves psychological and sleep related premenstrual symptoms.
The levels of follicular saliva E2 and P4 after menstruation were not significantly affected by the administration of Roea before and after intervention. The luteal phase saliva P4 levels were significantly increased before the roua menses and E2 remained relatively stable compared to the control group. In combination with premenstrual symptoms, lactobacillus crispatus roua can affect E2 and P4 secretion during luteal phase, thereby improving premenstrual symptoms.
Example 11 Industrial fermentation of Lactobacillus crispatus Roea and preparation of lyophilized powder
(1) And (5) configuring a culture medium.
The preparation scheme of the industrialized high-density fermentation lactobacillus crispatus roua culture medium is as follows: 10g/L of soybean peptone, 8g/L of yeast extract powder, 28g/L of anhydrous glucose, 3g/L of galacto-oligosaccharide, 2g/L of dipotassium hydrogen phosphate, 2g/L of diamine hydrogen citrate, 5g/L of sodium acetate, 0.5g/L of manganese sulfate, 1g/L of L-cysteamine hydrochloride and 1ml/L of tween 80 are prepared, and the raw materials are prepared by adopting food-grade raw materials.
(2) Liquid culture
After the culture medium is prepared, the pH value is regulated to 6.5, sterilization is carried out for 30min at 115 ℃, when the temperature is cooled to 38-39 ℃, the fermentation tank inoculation culture is carried out according to 3-10 percent of inoculation quantity, preferably 8 percent, the culture temperature is 35-37 ℃, the pH value is 6.2-6.5, the gas is not introduced in a sealing way in the fermentation process, the pressure of the fermentation tank is kept by nitrogen, and the fermentation tank is stirred slowly. Continuously culturing for 8-16 hr to obtain fermentation broth with viable bacteria content of 5.8X10 at 8 hr 8 CFU/mL, and the viable count of the fermentation broth measured at 16h is 3.1X10 9 CFU/mL, the level of the viable bacteria of the fermentation is higher, and the industrial high-density fermentation production requirement is met.
(3) Centrifuging
In order to avoid strain aging caused by excessively long fermentation time and influence the activity of the freeze-dried powder strain, the live strain amount reaches 10 after 8-12 hours of culture 8 CFU/mL-10 9 CFU/mL according to OD 600 And (3) after the growth curve enters a platform period, rapidly cooling the fermentation liquor to below 15 ℃, and centrifuging at 5000rpm by using a tubular centrifuge to obtain bacterial sludge.
(4) Protective agent emulsification and lyophilization
Diluting the obtained bacterial mud to have the water content of 60-80%, and adding a bacterial mud protective agent in an equal volume for emulsification; the bacterial mud protective agent comprises the following components: 70% of water, 10% of skim milk powder, 5% of sucrose, 5% of mannitol, 1% of tween-80, 3% of betaine, 0.5% of sodium glutamate and 5% of soluble starch. And (5) uniformly stirring, and then loading into a vacuum freeze dryer for freeze vacuum drying, wherein the thickness of the freeze-dried material is less than or equal to 1cm. The freeze dryer temperature profile is: keeping the temperature at 50 ℃ for 4 hours, increasing the temperature by 5 ℃ every 2 hours until the temperature reaches 0 ℃ at 24 hours, then increasing the temperature by 3 ℃ every 2 hours, and keeping the final maximum temperature at 30 ℃ unchanged until the materials are dried, wherein the materials are dried and characterized by having the moisture content lower than 5% and the water activity of 0.08-0.20aw.
The activity of the freeze-dried bacterial powder can reach 2.7X10 11 CFU/g, can be stored conveniently. It is also convenient for adding lactobacillus powder or preparing microecological intervention preparation.

Claims (12)

1. The lactobacillus crispatus is characterized in that the lactobacillus crispatus is lactobacillus crispatus Roea (Lactobacillus crispatus Roea), and is preserved in China center for type culture Collection, the preservation date is 2022, 12 months and 23 days, and the preservation number is CCTCC NO: m20222049.
2. Use of lactobacillus crispatus according to claim 1 in the preparation of a bacteriostatic agent.
3. The use according to claim 2, wherein the bacteriostatic agent is capable of inhibiting escherichia coli, candida albicans, staphylococcus aureus, group B streptococcus, streptococcus pyogenes and/or streptococcus pharyngeri.
4. Use of lactobacillus crispatus according to claim 1 for the manufacture of a vaginal microecological intervention agent or a product for the treatment of female genital infections.
5. The use according to claim 4, wherein the female reproductive infection is vaginitis.
6. Use of lactobacillus crispatus according to claim 1 for the preparation of a product for improving premenstrual psychological symptoms in females.
7. A composition comprising the lactobacillus crispatus of claim 1.
8. Use of the composition of claim 7 in the preparation of a bacteriostatic agent.
9. The use according to claim 8, wherein the bacteriostatic agent is capable of inhibiting escherichia coli, candida albicans, staphylococcus aureus, group B streptococcus, streptococcus pyogenes and/or streptococcus pharyngeri.
10. Use of the composition of claim 7 for the preparation of a vaginal microecological intervention agent or a product for the treatment of female genital infections.
11. The use according to claim 10, wherein the female reproductive infection is vaginitis.
12. Use of the composition of claim 7 for the preparation of a product for improving premenstrual psychological symptoms in women.
CN202310337582.1A 2023-03-31 2023-03-31 Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof Active CN116515686B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310337582.1A CN116515686B (en) 2023-03-31 2023-03-31 Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310337582.1A CN116515686B (en) 2023-03-31 2023-03-31 Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof

Publications (2)

Publication Number Publication Date
CN116515686A CN116515686A (en) 2023-08-01
CN116515686B true CN116515686B (en) 2023-10-31

Family

ID=87394924

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310337582.1A Active CN116515686B (en) 2023-03-31 2023-03-31 Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof

Country Status (1)

Country Link
CN (1) CN116515686B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104178437A (en) * 2013-11-08 2014-12-03 苏州欧赛微科生物医药科技有限公司 Lactobacillus crispatus and its application in gynecological diseases
CN111893057A (en) * 2020-06-29 2020-11-06 哈尔滨美华生物技术股份有限公司 Lactobacillus crispatus for preventing and treating female urogenital infection and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2595089B (en) * 2018-12-21 2023-02-15 Reid Gregor Compositions for use in methods for preventing or treating incontinence, overactive bladder, or menstrual cramping

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104178437A (en) * 2013-11-08 2014-12-03 苏州欧赛微科生物医药科技有限公司 Lactobacillus crispatus and its application in gynecological diseases
CN111893057A (en) * 2020-06-29 2020-11-06 哈尔滨美华生物技术股份有限公司 Lactobacillus crispatus for preventing and treating female urogenital infection and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Health benefits of probiotics and prebiotics in women;Michael de Vrese;《Menopause Int》;第15卷(第1期);第35-40页全文 *
女性终末期肾病月经异常患者性激素周期性改变与 子宫内膜乳杆菌相关性分析;雷湫宇等;《中国微生态学杂志》;第31卷(第1期);第97-100页全文 *

Also Published As

Publication number Publication date
CN116515686A (en) 2023-08-01

Similar Documents

Publication Publication Date Title
TWI652343B (en) Lactobacillus crimper (LACTOBACILLUS Crispatus) and application thereof
ES2305313T3 (en) BACTERIA THAT PRODUCE LACTIC ACID FOR USE AS PROBIOTIC ORGANISMS IN THE HUMAN VAGINE.
CN107299065B (en) Lactobacillus plantarum and application thereof in preparation of vagina bacteriostatic drugs
TWI627276B (en) Novel lactobacillus crispatus strain
US11083761B2 (en) High potency stable formulations of vaginal Lactobacillus
CN108004188B (en) Lactobacillus rhamnosus and application thereof in preparing vagina bacteriostatic medicine
ES2573729T3 (en) Use of Streptococcus salivarius in the treatment of chronic respiratory tract infections
CN116200306B (en) Lactobacillus rhamnosus LRa16, and application and product thereof in preparation of medicines for treating genital tract infection
CN110540945B (en) Lactobacillus jensenii and application thereof in preparing vaginal antibacterial drugs
CN105663172A (en) Ecological gynaecological externally-applied medicine composition and preparation method and application thereof
CN110831610A (en) Use of vaginal lactobacillus for improving success rate of in vitro fertilization
US20040022775A1 (en) Methods of treating viral infections in mammals
CN113278548B (en) Lactobacillus crispatus and application thereof in producing products for improving human vaginal environment
TWI412371B (en) A novel strain of lactobacillus and its use in inhibition of vaginitis
CN116515686B (en) Lactobacillus crispatus for treating female reproductive infection and regulating premenstrual psychological symptoms and application thereof
CN111471623A (en) Composition of three lactobacilli and application thereof
JPH02503800A (en) Bacterial preparations for the prevention and treatment of inflammatory processes and allergic diseases
CN116004457A (en) Humanized lactobacillus plantarum and application thereof
KR102014810B1 (en) Composition for prevention and treatment of vaginitis
CN114717169B (en) Lactobacillus helveticus and application thereof in preparing products for regulating microecology of genital tract and relieving dryness of genital tract
WO2020165919A1 (en) A composition for competitive inhibition of pathogens and restoration of microbial ecological balance
CN117327628B (en) Lactobacillus gasseri Mia capable of fermenting Chinese herbal medicine and resisting HPV virus and application thereof
RU2151606C1 (en) Strain of bacterium bifidobacterium longum z4 for preparing bacterial preparations normalizing microflora in vagina microbiocenosis disorder
CN110538201A (en) Lactobacillus composition and application thereof
RU2247148C2 (en) Strain of microorganism lactobacillus plantarum p4, strain of microorganism lactobacillus buchneri p0 and probiotic preparation based on thereof for correction of dysbacteriosis of different etiology in humans and animals

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant