Ligilactobacillus salivarius VB330 and use thereof

Abstract

Disclosed in the present invention is a Ligilactobacillus salivarius strain VB330, which is preserved in China General Microbiological Culture Collection Center on September 08, 2023, the accession number thereof being CGMCC No. 28406. The Ligilactobacillus salivarius VB330 disclosed by the present invention has broad-spectrum antibacterial characteristics, and has huge application prospects in the preparation of probiotic formulations, oral care products and drugs.

Description

Saliva-combined Lactobacillus VB330 and its application Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a saliva-associated Lactobacillus VB330 and an application thereof. Background Art

[0002] Harmful bacteria pose significant health risks and lead to significant economic losses every year. The traditional method of treating diseases caused by harmful bacteria is to use antibiotics, but their excessive use can lead to the emergence of multi-drug resistant strains, which makes traditional treatments limited in effectiveness. In addition, existing treatments often target a single pathogen, leaving patients vulnerable to infections from other pathogens. Developing effective solutions to combat harmful bacteria is increasingly important, and to address these limitations, researchers have been exploring alternative methods to control harmful microorganisms.

[0003] Probiotics are active bacteria that, when consumed in sufficient amounts, have beneficial effects on the health of the individual. They maintain health by inhibiting the growth of pathogens or competing with them for attachment sites. Lactobacillus salivarius, originally known as Lactobacillus salivarius, is a Gram-positive bacillus that lacks catalase and oxidase and produces lactic acid. Lactobacillus salivarius is one of the strains approved for use in food production and processing by my country's Ministry of Health, according to Announcement No. 65 of 2010. As a probiotic lactobacillus with great potential, Lactobacillus salivarius has become a research hotspot in recent years and is increasingly being used to create probiotic preparations suitable for both humans and animals.

[0004] Currently, traditional Chinese medicine and broad-based antimicrobial drugs are primarily used to kill harmful bacteria in the human body. However, broad-based antimicrobial drugs not only kill harmful bacteria but also affect beneficial bacteria in the human body. Therefore, it is necessary to find an alternative to the existing method of using broad-based antimicrobial drugs to reduce the number of harmful bacteria and maintain human health.

[0005] Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] To this end, the first aspect of the present invention provides a saliva-associated Lactobacillus (Ligilactobacillus salivarius) VB330, which was deposited in the General Microbiology Center of the China Culture Collection Administration on September 8, 2023, with the deposit number CGMCC No.28406.

[0008] The saliva-associated Lactobacillus (Ligilactobacillus salivarius) VB330 provided by the present invention has a broad-spectrum antibacterial property and can effectively inhibit the growth and reproduction of various harmful bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella, Listeria monocytogenes, Enterococcus faecalis, Shigella, Clostridium perfringens, Gardnerella, Malassezia, Candida albicans, Helicobacter pylori, Porphyromonas gingivalis, and Streptococcus mutans.

[0009] The second aspect of the present invention provides a bacterial suspension comprising the salivarius-associated Lactobacillus VB330 described in the first aspect.

[0010] The third aspect of the present invention provides a probiotic preparation comprising the saliva-associated Lactobacillus described in the first aspect and / or the bacterial suspension described in the second aspect.

[0011] The fourth aspect of the present invention provides use of the saliva-associated Lactobacillus VB330 described in the first aspect, the bacterial suspension described in the second aspect, and the probiotic preparation described in the third aspect in the preparation of oral care products.

[0012] According to a specific embodiment of the present invention, the oral care product includes any one of tooth gel, tooth powder, mouthwash, and toothpaste.

[0013] According to a specific embodiment of the present invention, the saliva-associated Lactobacillus VB330 provided by the present invention is a beneficial bacterium with broad-spectrum antibacterial properties. It can replace antibiotics to inhibit some harmful bacteria. Using strain VB330 in oral care products can not only inhibit the growth and reproduction of harmful bacteria in the oral cavity, but also increase the number of saliva-associated Lactobacillus in the oral cavity, that is, the number of beneficial bacteria, providing a new idea for maintaining oral health.

[0014] The fifth aspect of the present invention provides use of the saliva-lactobacillus VB330 described in the first aspect, the bacterial suspension described in the second aspect, and the probiotic preparation described in the third aspect in the preparation of antibacterial drugs.

[0015] According to a specific embodiment of the present invention, the antibacterial drugs include drugs for treating and / or preventing related diseases caused by at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Listeria monocytogenes, Enterococcus faecalis, Shigella, Clostridium perfringens, Gardnerella, Malassezia, Candida albicans, Helicobacter pylori, Porphyromonas gingivalis, and Streptococcus mutans.

[0016] Escherichia coli can cause acute gastroenteritis, Listeria monocytogenes can cause sepsis and meningitis, and Porphyromonas gingivalis can cause periodontitis. The saliva-based Lactobacillus VB330 provided by the present invention can prevent and / or treat these diseases by inhibiting the growth and reproduction of these harmful bacteria.

[0017] According to specific embodiments of the present invention, the saliva-associated Lactobacillus VB330 provided by the present invention exhibits enhanced inhibition against certain bacterial strains, such as Escherichia coli (inhibition zone diameter of approximately 32.5 mm), Staphylococcus aureus (inhibition zone diameter of approximately 27 mm), and Helicobacter pylori (inhibition zone diameter of approximately 17.8 mm). VB330 provided by the present invention can be used to develop treatments specifically targeting these common pathogens.

[0018] The sixth aspect of the present invention provides a medicine comprising at least one of the saliva-associated Lactobacillus VB330 described in the first aspect, the bacterial suspension described in the second aspect, and the probiotic preparation described in the third aspect.

[0019] According to a specific embodiment of the present invention, the medicament further contains an excipient and / or a carrier.

[0020] According to a specific embodiment of the present invention, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

[0021] According to a specific embodiment of the present invention, the carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

[0022] According to a specific embodiment of the present invention, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

[0023] According to an embodiment of the present invention, when saliva combines with Lactobacillus VB330 to exert its effect, it can exist in the form of living cells or in the form of non-living cells.

[0024] According to an embodiment of the present invention, the living cells refer to cells with the ability to metabolize, reproduce or replicate, and the non-living cells refer to cells without the ability to metabolize, reproduce and replicate, including but not limited to dried bacteria (such as freeze-dried powder).

[0025] The saliva-combined Lactobacillus VB330 provided by the present invention can also be used to prepare health-care foods.

[0026] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention.

[0027] Collection information:

[0028] Strain name: VB330

[0029] Deposit date: September 8, 2023

[0030] Depository: China General Microbiological Culture Collection Center (CGMCC)

[0031] Accession number: CGMCC No.28406 BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0033] FIG1 shows a phylogenetic tree comprising Lactobacillus salivarius VB330 constructed in Example 1 of the present invention, wherein PCZQ_s UMNLAv76 and FJ751781_s 325T are both Ligilactobacillus, which has not yet been translated into Chinese. Currently, its Latin name "ligilactobacillus" is commonly used in Chinese literature.

[0034] FIG2 shows a histogram of the diameters of the inhibition zones in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0036] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0037] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0038] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] As used herein, "prevention" and "prevention" are used interchangeably and refer to an approach for obtaining beneficial or desired results, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," Lactobacillus salivarius VB330 or a product containing the same can be administered to a subject at risk for developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0040] As used herein, the terms "treat" and "alleviate" refer to methods used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0041] In this article, "acceptable in health foods" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health food requirements of different countries.

[0042] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0043] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0044] In this article, the term "pharmaceutically acceptable excipients" may include sugars, including monosaccharides or polysaccharides, such as lactose, sucrose, mannitol and sorbitol; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinyl pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic surfactants, cationic surfactants, anionic surfactants; ethylene glycol polymers; fatty alcohols; and cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, etc., which are commonly used excipients in pharmaceutical preparations.

[0045] According to a specific embodiment of the present invention, the present invention proposes a saliva-associated lactobacillus (Ligilactobacillus salivarius) VB330. According to an embodiment of the present invention, the deposit number of the saliva-associated lactobacillus is CGMCC No. 28406. The saliva-associated lactobacillus (Ligilactobacillus salivarius) VB330 of the present invention has a broad-spectrum antibacterial property and can effectively inhibit the growth and reproduction of multiple harmful bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella, Listeria monocytogenes, Enterococcus faecalis, Shigella, Clostridium perfringens, Gardnerella, Malassezia, Candida albicans, Helicobacter pylori, Porphyromonas gingivalis, and Streptococcus mutans. It has great application prospects in the preparation of probiotic preparations, oral care products, and medicines.

[0046] As used herein, “Ligilactobacillus salivarius VB330” and “Lactobacillus salivarius”, “VB330”, or “strain VB330” are synonymous.

[0047] The strain VB330 obtained by screening in the present invention has significant antibacterial ability compared with other saliva-associated Lactobacilli. According to a specific embodiment of the present invention, the strain VB330 of the present invention inhibits far more types of bacteria than other saliva-associated Lactobacilli. In the test of inhibiting harmful bacteria, the strain VB330 of the present invention has the smallest inhibition zone diameter of Gardnerella, about 10.5 mm, and the largest inhibition zone diameter of Escherichia coli, about 32.5 mm. The inhibition zone diameters of almost half of the harmful bacteria are greater than 20 mm, indicating that the strain VB330 has strong and broad-spectrum antibacterial ability.

[0048] According to a specific embodiment of the present invention, the present invention provides a fermentation supernatant, which contains: Lactobacillus salivarius VB330 and / or metabolites of Lactobacillus salivarius VB330. The Lactobacillus salivarius of the present invention has strong antibacterial ability.

[0049] It should be noted that “fermentation supernatant” refers to the supernatant after centrifugation of the fermentation broth, which may contain Lactobacillus salivarius VB330 or its metabolites, or both Lactobacillus salivarius VB330 and its metabolites.

[0050] According to a specific embodiment of the present invention, a probiotic preparation is provided, comprising Lactobacillus salivarius VB330. The probiotic preparation is used to prepare a drug for maintaining intestinal, oral, and vaginal health, and exhibits broad-spectrum, strong antibacterial activity, thus possessing significant application prospects in pharmaceutical preparation.

[0051] It should be noted that the probiotic preparation of the present invention can be a liquid microbial agent, including but not limited to fermentation broth, etc.; it can also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0052] According to an embodiment of the present invention, the saliva-associated Lactobacillus VB330 exists in the form of living cells and / or non-living cells.

[0053] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.

[0054] For example, the living cells may be immobilized cells. Herein, "immobilized cells" refer to saliva-associated Lactobacillus VB330 immobilized on a carrier, which can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0055] In this article, "non-viable cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dried bacteria. Exemplarily, the probiotic preparation is a freeze-dried powder.

[0056] According to a specific embodiment of the present invention, the saliva-associated Lactobacillus VB330 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0057] According to a specific embodiment of the present invention, the dry bacteria are obtained by freeze-drying the saliva-associated Lactobacillus VB330.

[0058] According to an embodiment of the present invention, a medicine or health food is provided, comprising Lactobacillus salivarius VB330. Preferably, the medicine or health food of the present application can be used to prepare a live bacterial preparation for combating harmful bacteria. It should be noted that the medicine or health food of the present application can be various preparations of active Lactobacillus salivarius VB330 alone, or can be used in combination with other active ingredients, as long as they do not affect each other's activity. Furthermore, the best scenario for the medicine or health food is that the active ingredients can complement each other's functions or have a synergistic effect. For example, Lactobacillus salivarius VB330 can be combined with other probiotics to form a composite probiotic tablet to achieve better or more active functions. The specific formulation can be determined based on the activity and components of the composite probiotic tablet and is not limited here. Optionally, the medicine or health food further comprises a carrier or excipient acceptable in health foods, or a pharmaceutically acceptable carrier or excipient. It should be noted that when preparing a live bacterial preparation, it is generally necessary to add a carrier or excipient, as long as the added carrier or excipient does not inhibit or cause adverse side effects with Lactobacillus salivarius VB330. Medicines or health foods can be in the form of powders, tablets, drinks, or capsules.

[0059] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0060] Example 1: Strain collection and identification

[0061] Oral specimens were collected from healthy adults with unstimulated oral cavity. Oral specimens were collected by oropharyngeal swab. The collected oral specimens were added to sterile saline and shaken to obtain a bacterial suspension. A serial dilution was performed, and 100 μL of the undiluted bacterial suspension and the suspensions diluted 10-fold, 100-fold, and 1000-fold, respectively, were applied to MRS plates. The plates were incubated at 37°C for 24-48 hours. A sterile inoculating loop was used to pick white circular colonies of varying sizes and streaked onto MRS solid culture medium plates. The plates were then incubated at 37°C for 24-48 hours. Single colonies of suspected Lactobacillus salivarius strains were again picked and inoculated onto MRS solid culture medium plates. The colonies were observed for appearance, color, morphology, and Gram staining. Strains that met the characteristics of short rod-shaped Gram-positive bacteria were selected.

[0062] Genomic DNA from the isolated strain was extracted using a bacterial genomic DNA extraction kit (purchased from Magen, catalog number D3146-03), and 16s rDNA sequencing was performed. The 16s sequencing results were subjected to BLAST comparison to analyze and evaluate the sequence similarity of the microorganism: a k-mer frequency fingerprint-based method was used. First, a k-mer of length 16 was extracted from each sequence, and its occurrence count was counted to generate a k-mer frequency fingerprint. Next, the cosine distance was used to calculate the similarity between the k-mer frequency fingerprints of the two sequences. Using these distance values, a phylogenetic tree was constructed (as shown in Figure 1), and VB330 was ultimately identified and isolated. VB330 was closely related to 'Ligilactobacillus_salivarius BCRC_14759' and further formed a larger related group with 'Ligilactobacillus_hayakitensis JCM_14209'. Subsequently, the related group was combined with multiple other bacteria and clusters, and the analysis showed that strain VB330 was Ligilactobacillus salivarius.

[0063] The sequence of strain VB330 is:

[0064] Strain VB330 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 8, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.28406.

[0065] Example 2: VB330 inhibition of harmful bacteria test

[0066] The double-layer plate culture method was used to evaluate the antibacterial ability of each probiotic based on the growth of pathogenic bacteria in the upper layer. Preparation of the lower layer of probiotics: Thaw the activated saliva and lactobacillus frozen tubes and spot them on a blank solid plate of MRS at pH 6.9. Culture them anaerobically at 37°C for 1 day. The inoculation volume of the bacteria was 2μl / spot. Preparation of the upper pathogenic bacteria layer: Cool the sterilized upper culture medium to 40-50°C, add different pathogens to the culture medium (the final concentration of pathogens in the culture medium is about 10 6 The culture medium containing pathogenic bacteria was mixed at a rate of 7 ml / dish to each of the above-mentioned probiotic single-bacteria plates that had been grown. After solidification, the probiotics were cultured according to the growth conditions of different pathogens. The culture conditions of each pathogen are shown in Table 1. After the upper plate was grown, whether there was an inhibition zone around the probiotics was observed, and the diameter of the inhibition zone was counted. The statistical results are shown in Table 2, and the bar chart is shown in Figure 2.

[0067] Table 1

[0068] In Table 1, Columbia medium was purchased from BD Difco with the catalog number 294420; Leeming-Notman medium was purchased from Shandong Top Bioengineering Co., Ltd. with the catalog number 20230105; BHI medium was purchased from Qingdao Qingyao Bioengineering Co., Ltd. with the catalog number HB8297-5; the culture medium for Clostridium perfringens assay was purchased from Qingdao Haibo Biological with the catalog number HB8311; the components of 10% fetal bovine serum brucoid broth medium include: fetal bovine serum (purchased from Gibco with the catalog number 12483020) and brucoid broth medium (purchased from Qingdao Haibo Biological with the catalog number HB0241).

[0069] Table 2

[0070] The saliva-associated Lactobacillus strain VB330 provided by the present invention exhibits broad-spectrum inhibitory properties against both Gram-positive and Gram-negative bacteria, and can effectively replace antibiotics to inhibit some harmful bacteria.

[0071] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0072] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0084]

Claims

1. A salivary lactobacillus (Ligilactobacillus salivarius) VB330, characterized in that: The deposit number of the saliva-associated lactobacillus (Ligilactobacillus salivarius) VB330 is CGMCC No.28406.

2. A bacterial suspension, characterized in that: The invention relates to a method for preparing the lactobacillus salivais VB330.

3. A probiotic preparation, characterized in that: It comprises the salivary lactobacillus VB330 described in claim 1 and / or the bacterial suspension described in claim 2.

4. Use of the salivary lactobacillus VB330 according to claim 1, the bacterial suspension according to claim 2, and the probiotic preparation according to claim 3 in the preparation of oral care products.

5. The use according to claim 4, characterized in that: The oral care product includes any one of tooth gel, tooth powder, mouthwash and toothpaste.

6. Use of the salivary lactobacillus VB330 according to claim 1, the bacterial suspension according to claim 2, and the probiotic preparation according to claim 3 in the preparation of antibacterial drugs.

7. The use according to claim 6, characterized in that The antibacterial drugs include drugs for treating and / or preventing related diseases caused by at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Listeria monocytogenes, Enterococcus faecalis, Shigella, Clostridium perfringens, Gardnerella, Malassezia, Candida albicans, Helicobacter pylori, Porphyromonas gingivalis, and Streptococcus mutans.

8. A drug, characterized in that The medicine comprises at least one of the salivarius-associated Lactobacillus VB330 according to claim 1, the bacterial suspension according to claim 2, or the probiotic preparation according to claim 3.

9. The drug according to claim 8, characterized in that The medicament further contains excipients and / or carriers.

10. The drug according to claim 9, characterized in that The excipient comprises at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

11. The drug according to claim 9, characterized in that The carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

12. The drug according to claim 8, characterized in that The dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

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