TWI412371B - A novel strain of lactobacillus and its use in inhibition of vaginitis - Google Patents

Abstract

A lactic acid bacterium that inhibit vaginitis is selected from a group consisted of Lactobacillus acidophilus (BCRC 910494), Lactobacillus delbrueckii subsp. Lactis (BCRC910518) and Lactobacillus plantarum (BCRC910519), and the use of the lactic acid bacterium mentioned above is to manufacture a food composition or medicine composition to inhibit vaginitis.

Description

Lactobacillus inhibiting vaginitis pathogen and use thereof

The present invention relates to a lactobacillus, and more particularly to a Lactobacillus lactis having the ability to inhibit vaginal pathogens.

According to the statistics of obstetrics and gynecology in China, about 75% of women have at least one chance of vaginal infection in their lifetime, and the countries with subtropical climate in China are more likely to cause bacteria and molds to breed, and the vagina will produce different symptoms according to different pathogens. Infection. The current causes of female vaginal infections are as follows:

(1) mold infection

The more common pathogen of mold infection is Candida albicans , and the Candida albicans loves to grow in a warm and humid environment. Therefore, when the female genitals are not clean or wear tight-fitting underwear, the genitals are caused. When it is stuffy or wet, it is easy to cause the growth of Candida albicans, causing symptoms such as itching, burning, edematous swelling, urinary pain, etc., and insufficient personal resistance (for example: lack of sleep, before and after menstruation, during pregnancy... When it is), it is more likely to cause recurrent infections. Currently used for treatment by oral administration (for example: Fluconazole...etc.) or anti-fungal vaginal suppository (for example: Fenticonazole nitrate..., etc.). However, these treatment methods are prone to cause adverse side effects (eg, nausea, dizziness, etc.), especially for patients with insufficient resistance (eg, pregnant women, etc.), which are more likely to have side effects.

(two) bacterial infection

The more common bacterial pathogens are mixed pathogens (eg, Gardnerella vaginalis , Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , and chains). Streptococcus , etc., and the mixed pathogens prefer to grow in an alkaline environment (pH>4.5). Therefore, too frequent sexual intercourse or too frequent flushing of the vagina can easily lead to an increase in vaginal pH. It causes bacteria to grow, which in turn produces a large amount of grayish-white or yellow vaginal secretions that cause a smell similar to fishy smell. The current method of treatment is oral administration (for example, penicillin, tetracycline, etc.). However, these oral medications are prone to cause undesirable side effects (eg, allergies, nausea, dizziness, etc.), and even cause a decrease in the original beneficial bacteria in the female vagina to destroy the balance of the bacterial phase.

(3) Trichomonas infection

Trichomonas infections are mostly transmitted by sexually transmitted infections or indirectly in warm and humid environments (eg, warmth, swimming pools, etc.). The vaginal infection caused by the trichomoniasis is prone to severe itching, genital redness, and the like. Symptoms, even the production of pus thick secretions, have a stench. The current method for treatment is taking antibiotics (for example, Metronidazole, etc.), however, the use of antibiotics is likely to cause adverse side effects such as nausea, abdominal discomfort, difficulty in urinating, and the like.

It is known from the above that the current methods for treating vaginitis are mostly taken orally, and the oral medications are mostly antibiotics or steroids, and therefore, the drugs kill the pathogens and also harm the probiotics in the body. (eg Lactobacillus, etc.), making the vaginal environment of patients in the early stages of drug treatment It is in a state of sterility, and gradually produces a situation in which probiotics and pathogens compete with each other for growth. However, the growth of probiotics in the body tends to be slower than that of the pathogens. Therefore, it is easy to cause a decrease in host immunity at the same time as drug treatment, and in the case of repeated infections, it is necessary to repeatedly treat antibiotics or steroid drugs, and even to cause resistance to pathogenic bacteria. The drug damages the host's immune system.

In recent years, there have been reports on the use of probiotics for the treatment of vaginal infections. It has been found that certain Lactobacillus strains have the function of inhibiting the growth of pathogenic bacteria of vaginal infection. For example, the Patent No. 200806303 of the Republic of China indicates that it is known. Lactobacillus acidophilus , Lactobacillus bifidus , Lactobacillus brevis , Lactobacillus bulgaricus , etc., used to inhibit infection of vaginal pathogens, etc. The bacillus secretes various metabolites, such as lactic acid, hydrogen peroxide, bacteriocin or antimicrobial peptide, which maintains a constant vaginal pH (pH 3.8 to 4.2) to inhibit the growth of vaginal pathogens; or directly attached to the vagina The cells compete for the growth of vaginal pathogens to maintain the balance of the bacteria in the vagina.

However, the above-mentioned conventional Lactobacillus can only produce a bacteriostatic action against a specific pathogen, and the F. faecalis can only inhibit Candida albicans but cannot improve the vaginal bacterial infection. Therefore, the bacteriostatic effect is poor, especially when When a patient cross-infects a plurality of vaginal pathogens, the bacteriostatic effect of the conventional Lactobacillus is limited, and it cannot be widely applied to the prevention or treatment of various vaginal pathogens.

Furthermore, the conventional lactobacillus can secrete a variety of metabolites, but Lactic acid is the main antibacterial means. However, the lactic acid is a weak acid, and its bactericidal power is low, especially when the patient is seriously infected, the conventional lactobacillus often cannot effectively control the spread of vaginal pathogens; The vaginal cell has insufficient adsorption capacity and cannot stay in the vaginal environment for a long time, so that the antibacterial ability of the conventional lactobacillus cannot be effectively exerted; therefore, it is known that the bacteriostatic effect of the conventional lactobacillus is limited and can only be exerted on specific pathogenic bacteria. And its antibacterial ability is poor, can not effectively inhibit the growth of pathogenic bacteria.

In view of the high threats and high infection rates caused by vaginitis to the Taiwan population in recent years, and the possible cross-infection of different types of pathogens, novel strains with simultaneous inhibition of different types of pathogens should be developed to effectively improve clinical care. The prevention and treatment effect of the pathogenic bacteria against vaginitis.

The main object of the present invention is to improve the above disadvantages and to provide a lactobacillus which inhibits vaginitis pathogens, which is capable of simultaneously inhibiting the growth of different types of vaginitis pathogens.

A second object of the present invention is to provide a lactic acid bacterium capable of inhibiting vaginal pathogens, which has good bactericidal ability against vaginal pathogenic bacteria.

Still another object of the present invention is to provide a lactobacillus which inhibits a vaginitis pathogen, which has a good adsorption capacity for a host vaginal cell to prolong the bactericidal ability of the lactobacillus.

Another object of the present invention is to provide a lactobacillus which inhibits a vaginitis pathogen, which is capable of using a lactobacillus which inhibits a vaginitis pathogen. A food or pharmaceutical composition for inhibiting a vaginitis pathogen is prepared.

In order to achieve the above object, the lactobacillus of the present invention for inhibiting vaginitis pathogenic bacteria is deposited in the Republic of China Food Industry Development Research Institute, the lactobacillus lactis having the accession number BCRC910518, and the Lactobacillus lactis strain has SEQ ID No: 1 The 16S rDNA sequence; and the use of a lactobacillus for preparing a food composition or a pharmaceutical composition for inhibiting infection of a vaginitis pathogen.

The above and other objects, features, and advantages of the present invention will become more apparent from the aspects of the invention. Lactobacillus is a Lactobacillus delbrueckii subsp.lactis strain obtained from a dairy product; the lactobacillus has the bactericidal ability of the fungal vaginitis pathogen, the bactericidal ability of the bacterial vaginitis pathogen, and the secretion of peroxidation The ability of hydrogen and the adsorption characteristics of the host vaginal cells, and compared with the conventional lactic acid bacteria have better antibacterial ability and bacteriostatic effect, can be applied to the prevention or treatment of commercial preparation of vaginitis.

Referring to Fig. 1, the lactobacillus of the present invention is obtained by the following treatments, and includes a culture step S1, an identification step S2, and a preservation step S3.

This culturing step S1 separates the lactic acid bacteria contained in the dairy product and cultures them. The present invention is sampled from a dairy product (for example, cheese, etc.), placed in a culture solution for enrichment culture, to be properly cultured. After the time, the culture solution was serially diluted, and further inoculated to a medium to select a growable strain. In the proliferation culture system of the present embodiment, the dairy product was inoculated in a liquid MRS medium (MRS Broth) at a volume ratio of 1:50, and cultured at 37 ° C for 20 hours to obtain a proliferation culture bacterial solution, and the proliferation culture was serially diluted. In the bacterial solution, the diluted proliferation culture broth was inoculated with a solid MRS medium (MRS Agar) by smearing, and the growable strain was selected. The formulations of the liquid MRS medium and the solid MRS medium are shown in Tables 1 and 2.

The identification step S2 determines the classification of the strain according to the physical form, physiological characteristics or molecular characteristics of the Lactobacillus. In this embodiment, the Lactobacillus is subjected to Gram staining, catalase reaction, and molecular identification of 16S rDNA, and the staining pattern of the Lactobacillus (resulting as Gram-positive bacteria) and the 16S rDNA partial sequence (such as SEQ ID No: 1)) The strain with a similarity of 99% in the sequence alignment database (BLAST) of the National Center for Biotechnology (NCBI) is Lactobacillus delbrueckii subsp. Lactis and the catalase reaction test. The result of the identification (negative reaction with catalase) confirmed that the lactic acid bacteria of the present invention was Lactobacillus delbrueckii subsp. Lactis , and the accession number was BCRC910518, which was named Lactobacillus lactis LDL114.

The preservation step S3 is such that the Lactobacillus lactis can be stored for a long time under a low temperature environment for subsequent use. The Lactobacillus lactis of the present invention was inoculated to the solid MRS medium, and cultured at 37 ° C for 48 hours, and then a single colony was selected and transferred to an MRS culture solution (as shown in the formula of Table 1) for cold. Frozen storage. Preferably, the MRS culture solution contains 30% antifreeze (for example, glycerin), and is temporarily stored in a constant temperature storage chamber at -80 ° C for later use.

In order to confirm that the Lactobacillus strain of the present invention has better antibacterial ability and bacteriostatic effect, the ability of the Lactobacillus lactis to secrete hydrogen peroxide and cell adsorption, and the inhibition of the Lactobacillus lactis to the pathogenic bacteria are respectively tested. Observe its bactericidal effect on vaginitis pathogens.

Some Lactobacillus strains can secrete lactic acid, hydrogen peroxide, bacteriocin and other substances to fight against pathogenic bacteria. Among them, hydrogen peroxide can react with halogen ions (such as chloride ions) to form a toxic oxide against pathogens (such as oxidized hydroxide). Chlorine), while increasing the ability of lactobacilli to fight pathogens. Therefore, in order to understand the ability of the lactobacillus of the present invention to secrete hydrogen peroxide, the present embodiment is to culture the Lactobacillus of the present invention and Lactobacillus reuteri and Lactobacillus rhamnosus in the liquid MRS medium, respectively. After growth, the detection of hydrogen peroxide is carried out by using a commercial kit for detecting hydrogen peroxide (Bioxytech ^® .H ₂ O ₂ -560 ^TM , DXIS), wherein the appropriate time means that the number of growth bacteria of the Lactobacillus of the present invention reaches 10 ^{At 9} o'clock, the cells were centrifuged to obtain a supernatant to test the concentration of hydrogen peroxide against the supernatants. The case of the secretion of hydrogen peroxide by the lactobacillus of the present invention is recorded in Table 3:

From the above results, it is known that the Lactobacillus lactis LDL114 of the present invention has a similar secretion capacity to hydrogen peroxide as the L. lactis strain. From this, it can be confirmed that the Lactobacillus lactis LDL114 of the present invention has the ability to secrete hydrogen peroxide, and the hydrogen peroxide in the host is easily reacted with a halogen ion to form the toxic oxide, and the toxic oxide is The vaginal pathogen produces a poisoning action, and therefore, the Lactobacillus lactis LDL114 of the present invention can exert the preferred bactericidal ability.

In order to further understand the adsorption capacity of the Lactobacillus of the present invention on vaginal cells, in this embodiment, the vaginal epithelial cells are cultured stably, and then 5×10 ⁴ vaginal epithelial cells per ml are planted in a 24-well tray, to be cultured 24 After the hour, the final concentration of 10 ⁸ CFU/mL of Lactobacillus was added to the 24-well plate, and then co-cultured for 2 hours at 37 ° C with 5% carbon dioxide, followed by slow rinsing with PBS buffer to remove Lactobacillus not adsorbed to vaginal epithelial cells, followed by breaking the vaginal epithelial cells, and measuring the number of Lactobacillus bacteria adsorbed on the vaginal epithelial cells in solid MRS medium. Among them, the Lactobacillus strain includes the Lactobacillus lactis LDL114 of the present invention, and the conventional Lactobacillus lactis and Lactobacillus rhamnosus. The vaginal epithelial cell line selected in this example, a human cervical cancer cell line Hela cell, was purchased from Taiwan Food Industry Development Research Institute BCRC 60005, and the detailed adsorption situation of the Lactobacillus of the present invention is recorded in Table 4.

As is apparent from the results of the fourth table, the Lactobacillus lactis LDL114 of the present invention produces a better adsorption effect on the vaginal epithelial cell line. It can be confirmed that the Lactobacillus lactis LDL114 of the present invention has the ability to adsorb human vaginal epithelial cells, and has better adsorption power than other lactobacilli, since the vaginal pathogens (including Candida albicans and V. vaginalis) are only adsorbed to the host. The vaginal cells, and the Lactobacillus lactis LDL114 of the present invention can stay on the vaginal cells of the host for a long time, so that it can exert a better bacteriostatic ability in the host. Moreover, the Lactobacillus lactis IDL114 of the present invention has a better adsorption capacity, thereby making up for the deficiency of the amount of hydrogen peroxide secretion, and can also exert a better antibacterial effect in the host body.

From the above, the Lactobacillus of the present invention can be adsorbed to the host vaginal cells, staying in the host for a long time to produce a better bacteriostatic ability, and the Lactobacillus strain can secrete a large amount of hydrogen peroxide to produce toxicity to vaginal pathogens. The oxide has a better antibacterial effect against pathogenic bacteria of fungal vaginitis (such as Candida albicans) and bacterial vaginitis (such as Lactobacillus vaginalis); the lactobacillus can be widely used in commercial treatment or prevention of vagina Preparation of products infected with inflammatory pathogens, such as health foods, medical preparations or processed foods; the lactic acid bacteria can be processed into various product forms, such as tablets, alone or in combination with other desired ingredients (such as carriers, nutrients, etc.). Capsules, drinks, powders or drops, etc., can be administered to individuals in a variety of ways Control the infection of vaginal pathogens and the related diseases caused by them.

Furthermore, the bacteriostatic ability and the bacteriostatic effect of the Lactobacillus of the present invention are further confirmed, and the inhibition of the Lactobacillus lactis LDL114 against the pathogenic bacteria is separately tested, thereby observing the bactericidal effect against the vaginitis pathogenic bacteria, as detailed in Figures 2 to 4 and Tables 6-1 to 6-3.

Taking the Candida albicans as an example (such as Tables 6-1 and 6-2), the liquid medium containing the Lactobacillus lactis LDL114 is contained in a co-culture for a long time of 0 to 24 hours. The number of pathogenic bacteria has not increased with time. From this, it was found that the Lactobacillus lactis LDL114 has a long-term bacteriostatic effect.

Furthermore, taking the vaginal vaginal bacterium as an example (such as Table 6-3), the Lactobacillus lactis LDL114 is simultaneously cultured for 0 to 24 hours. In the liquid medium, the number of pathogenic bacteria contained in the liquid medium maintains a decreasing trend with the increase of the normal growth rate of the pathogen over time, and even rises to 24 hours with time, the number of bacteria of the vaginal vaginal bacillus is compared with that of The number of bacteria measured by the control group decreased significantly. Therefore, it was found that the Lactobacillus lactis LDL114 could not completely inhibit the growth of the vaginal vaginal bacterium, but it could have the effect of slowing down the growth rate of the pathogenic bacteria under the long-term culture, even in the case of 24 hours. The number of bacteria that reduce the pathogen is slightly reduced to achieve additional sterilization.

The lactobacillus of the present invention for inhibiting vaginitis pathogens is capable of acting on host vaginal cells to simultaneously achieve the effect of inhibiting the growth of different types of vaginitis pathogens.

The lactobacillus which inhibits the vaginitis pathogenic bacteria of the present invention can utilize the poisoning action of hydrogen peroxide on the pathogenic bacteria to fully exert the bactericidal effect of the lactobacillus.

The Lactobacillus which inhibits the vaginitis pathogenic bacteria of the present invention can adhere to vaginal cells by utilizing cell adsorption characteristics, and can limit the growth of vaginitis pathogenic bacteria in the host vagina for a long time, thereby achieving better antibacterial effect.

The use of the lactobacillus of the present invention is to inhibit the vagina as described above. Controlling the infection of vaginal pathogens is an effect of the present invention.

The complex of the Lactobacillus of the present invention is a combination of the Lactobacillus which inhibits the vaginal pathogen as described above with other compatible strains to enhance the antibacterial ability of the Lactobacillus, thereby achieving the effect of inhibiting the pathogenic bacteria of the vaginitis.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

S1‧‧‧ cultivation steps

S2‧‧‧ Identification steps

S3‧‧‧Save step

Fig. 1 is a flow chart showing the treatment of the lactobacillus of the present invention.

Fig. 2: One of the cases in which LDL114 of the present invention inhibits Candida albicans.

Figure 3: The second aspect of the present invention LDL114 inhibits Candida albicans.

Figure 4: The LDL114 of the present invention inhibits the vaginal bacillus case.

<110> Shenghe Biotechnology Co., Ltd.

<120> Lactobacillus which inhibits vaginitis pathogens and uses thereof

<160> 1

<210> 1

<211> 1466

<212> DNA

<213> Lactobacillus lactis Lactobacillus delbrueckii subsp.lactis

<400> 1

S1‧‧‧ cultivation steps

S2‧‧‧ Identification steps

S3‧‧‧Save step

Claims (3)

A lactobacillus which inhibits a vaginitis pathogen is deposited in the Republic of China Food Industry Development Research Institute, and has a lactation number of BCRC910518, and the Lactobacillus strain has the 16S rDNA sequence as shown in SEQ ID No: 2. The lactobacillus which inhibits the pathogen of vaginitis according to the first aspect of the patent application has the ability to secrete hydrogen peroxide and adsorb to cells. A use of a lactobacillus for inhibiting a vaginitis pathogen as described in claim 1 is for the preparation of a food composition or a pharmaceutical composition for inhibiting infection of a vaginitis pathogen.