Lactobacillus rhamnosus VB255 and use thereof

Abstract

Lactobacillus rhamnosus VB255 and a use thereof, the strain being deposited at the China General Microbiological Culture Collection Center on March 31, 2023 under the deposit number CGMCC NO. 26968. Lactobacillus rhamnosus VB255 has good stability, can stably exist in pancreatic juice without affecting the intestinal microecological environment, and has favorable inhibition effects against Candida albicans and / or Shigella.

Description

Lactobacillus rhamnosus VB255 and its application Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to Lactobacillus rhamnosus VB255 and application of Lactobacillus rhamnosus VB255 in inhibiting Candida albicans and / or Shigella. Background Art

[0002] Candida albicans, also known as Candida albicans, is a widely distributed, opportunistic eukaryotic microorganism that can cause superficial or deep infections. It is a human commensal, typically parasitizing various locations within the human body, including the skin, mouth, throat, intestines, and vaginal mucosa. When the immune system is weakened or when there are comorbidities such as diabetes, chronic wasting disease, or long-term use of broad-spectrum antibiotics and immunosuppressants, the body's bacterial flora can become imbalanced, leading to Candida infections and candidiasis.

[0003] Shigella, also known as Shigella dysenteriae, is one of the leading pathogens of intestinal infections. It belongs to the genus Shigella in the family Enterobacteriaceae. It is a Gram-negative bacillus that is either aerobic or facultatively anaerobic. Humans are highly susceptible to Shigella dysenteriae, and it can cause acute poisoning in young children, with a high mortality rate.

[0004] Therefore, for intestinal diseases caused by dual infection of Candida albicans and Shigella, screening out an active probiotic that can simultaneously inhibit these two pathogenic bacteria is of great significance and application value.

[0005] Summary of the Invention

[0006] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides a Lactobacillus rhamnosus strain that exhibits a significant inhibitory effect against Candida albicans and / or Shigella, and can be used to prevent and / or alleviate and / or treat diseases caused by Candida albicans and / or Shigella.

[0007] Therefore, in a first aspect of the present invention, the present invention proposes a Lactobacillus rhamnosus (Lactobacillus rhamnosus). According to an embodiment of the present invention, the Lactobacillus rhamnosus was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on March 31, 2023, with a deposit number of CGMCC NO.26968, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus rhamnosus of the present invention has good stability, can stably exist in pancreatic juice without affecting the intestinal microecological environment, and has a good inhibitory effect on Candida albicans and / or Shigella.

[0008] In a second aspect, the present invention provides a fermentation supernatant. According to an embodiment of the present invention, the fermentation supernatant comprises: the Lactobacillus rhamnosus described in the first aspect; and / or metabolites of the Lactobacillus rhamnosus described in the first aspect. As previously mentioned, the Lactobacillus rhamnosus of the present invention has good stability, can exist stably in pancreatic juice without affecting the intestinal microecological environment, and has a good inhibitory effect on Candida albicans and / or Shigella. Therefore, the fermentation supernatant also has the same advantages.

[0009] In a third aspect, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth contains the Lactobacillus rhamnosus described in the first aspect. As previously described, the Lactobacillus rhamnosus of the present invention has good stability, can be stably present in pancreatic juice without affecting the intestinal microecological environment, and has a good inhibitory effect on Candida albicans and / or Shigella. Therefore, the fermentation broth also has the same advantages.

[0010] In a fourth aspect, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent contains at least one of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, and the fermentation broth described in the third aspect. As previously mentioned, the Lactobacillus rhamnosus of the present invention has good stability, can exist stably in pancreatic juice without affecting the intestinal microecological environment, and has a good inhibitory effect on Candida albicans and / or Shigella. Therefore, the microbial agent also has the same advantages and can be used to prevent and / or alleviate and / or treat diseases caused by Candida albicans and / or Shigella.

[0011] According to an embodiment of the present invention, the microbial agent may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the Lactobacillus rhamnosus exists in the form of living cells and / or non-living cells.

[0013] According to an embodiment of the present invention, the microbial agent may further contain at least one strain acceptable to food, medicine, health food and feed.

[0014] According to an embodiment of the present invention, the microbial agent further includes pharmaceutically, food or health food acceptable excipients or carriers.

[0015] In a fifth aspect of the present invention, the present invention provides a single-dose formulation. According to an embodiment of the present invention, the single-dose formulation comprises 10 9 ~10 11The Lactobacillus rhamnosus described in the first aspect of CFU is used as an active ingredient. The single-dose preparation of the present invention can achieve an inhibitory effect on Candida albicans and / or Shigella through a single administration.

[0016] According to an embodiment of the present invention, the single-dose formulation comprises 10 10 The Lactobacillus rhamnosus described in the first aspect of CFU is used as the active ingredient.

[0017] In a sixth aspect, the present invention provides a food, health food, or feed. According to an embodiment of the present invention, the food, health food, or feed contains at least one of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, and the microbial agent described in the fourth aspect.

[0018] According to an embodiment of the present invention, the food, health food or feed may further include an auxiliary material or carrier acceptable to food or health food.

[0019] In the seventh aspect of the present invention, the present invention proposes the use of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, the microbial agent described in the fourth aspect, or the single-dose preparation described in the fifth aspect in the preparation of a medicament, wherein the medicament has at least one of the following uses: for inhibiting the growth of Candida albicans and / or Shigella; for preventing and / or alleviating and / or treating diseases caused by Candida albicans and / or Shigella.

[0020] According to an embodiment of the present invention, the related disease is at least one selected from the group consisting of oral candidiasis, candidal cheilitis, candidal angular cheilitis, digestive system candidiasis and bacillary dysentery.

[0021] In an eighth aspect of the present invention, a drug is provided, comprising at least one of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, the microbial agent described in the fourth aspect, or the single-dose preparation described in the fifth aspect.

[0022] According to an embodiment of the present invention, the drug may further include at least one of the following additional technical features:

[0023] According to an embodiment of the present invention, the medicine further contains an excipient and / or a carrier.

[0024] According to an 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.

[0025] According to an 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, cationic and anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

[0026] According to an 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.

[0027] In a ninth aspect, the present invention proposes the use of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, the microbial agent described in the fourth aspect, or the single-dose preparation described in the fifth aspect in inhibiting the growth of Candida albicans and / or Shigella.

[0028] In the tenth aspect of the present invention, the present invention proposes the use of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, the microbial agent described in the fourth aspect, or the single-dose preparation described in the fifth aspect in preventing and / or alleviating and / or treating diseases related to Candida albicans and / or Shigella.

[0029] In an eleventh aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria. According to embodiments of the present application, the method comprises contacting the pathogenic bacteria with at least one of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, the microbial agent described in the fourth aspect, or the single-dose preparation described in the fifth aspect.

[0030] In a twelfth aspect, the present invention provides a method for preventing and / or alleviating and / or treating diseases caused by pathogenic bacteria. According to an embodiment of the present application, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the Lactobacillus rhamnosus described in the first aspect, the fermentation supernatant described in the second aspect, the fermentation broth described in the third aspect, the microbial agent described in the fourth aspect, or the single-dose formulation described in the fifth aspect.

[0031] According to an embodiment of the present application, the pathogenic bacteria include Candida albicans and / or Shigella.

[0032] According to an embodiment of the present application, the related disease is at least one selected from the group consisting of oral candidiasis, candidal cheilitis, candidal angular cheilitis, digestive system candidiasis, and bacillary dysentery.

[0033] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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:

[0035] FIG1 is a scanning electron microscope photograph of Lactobacillus rhamnosus VB255 according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," Lactobacillus muciniphila ferments 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.

[0041] 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.

[0042] The present invention provides a Lactobacillus rhamnosus, a fermentation supernatant, a fermentation broth, a microbial agent, a single-dose preparation, and uses thereof, which will be described in detail below.

[0043] strains

[0044] In one aspect of the present invention, the present invention proposes a Lactobacillus rhamnosus (Lactobacillus rhamnosus). According to an embodiment of the present invention, the Lactobacillus rhamnosus was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on March 31, 2023, with a deposit number of CGMCC NO.26968. The Lactobacillus rhamnosus of the present invention has good stability, can exist stably in pancreatic juice without affecting the intestinal microecological environment, and can significantly inhibit the growth of Candida albicans and / or Shigella.

[0045] As used herein, "Lactobacillus rhamnosus" and "Lactobacillus rhamnosus" are synonymous.

[0046] Fermentation supernatant

[0047] In another aspect, the present invention provides a fermentation supernatant. According to an embodiment of the present invention, the fermentation supernatant comprises: the aforementioned Lactobacillus rhamnosus; and / or a metabolite of the aforementioned Lactobacillus rhamnosus.

[0048] It should be noted that the “fermentation supernatant” refers to the supernatant after the fermentation broth is centrifuged and filtered, which mainly contains the metabolites of Lactobacillus rhamnosus.

[0049] fermentation broth

[0050] In another aspect of the present invention, a fermentation broth is provided. According to an embodiment of the present invention, the fermentation broth comprises the aforementioned Lactobacillus rhamnosus.

[0051] It should be noted that the "fermentation broth" refers to the process in which Lactobacillus rhamnosus is cultured in a culture medium for a period of time, and Lactobacillus rhamnosus utilizes nutrients in the culture medium to synthesize bacteria and secrete products.

[0052] microbial agents

[0053] In yet another aspect, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent comprises the aforementioned Lactobacillus rhamnosus, and / or the aforementioned fermentation supernatant, and / or the aforementioned fermentation broth. The microbial agent of the present invention is used to prevent and / or treat diseases caused by Candida albicans and / or Shigella.

[0054] It should be noted that the microbial agent 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.

[0055] According to an embodiment of the present invention, the Lactobacillus rhamnosus exists in the form of living cells and / or non-living cells.

[0056] In this context, "living cells" refer to Lactobacillus rhamnosus cells that have the ability to metabolize, reproduce or replicate.

[0057] For example, the living cells may be immobilized cells. Herein, "immobilized cells" refer to Lactobacillus rhamnosus fixed on a carrier, which can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0058] 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 microbial agent is a freeze-dried powder.

[0059] As a specific embodiment, the Lactobacillus rhamnosus exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0060] As a specific embodiment, the dry bacteria are obtained by freeze-drying the Lactobacillus rhamnosus.

[0061] As a specific embodiment, the microbial agent may further contain at least one strain acceptable to food, medicine, health food and feed.

[0062] As a specific embodiment, the microbial agent further includes pharmaceutically acceptable excipients or carriers, or excipients or carriers acceptable to food or health food.

[0063] As used herein, "acceptable in food" refers to substances or compositions that can be consumed by humans, which may be adjusted according to food requirements in different countries.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the Lactobacillus rhamnosus of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present disclosure.

[0068] Single-dose preparations

[0069] In another aspect of the present invention, the present invention provides a single-dose formulation. According to an embodiment of the present invention, the single-dose formulation comprises 10 9 ~10 11 The single-dose preparation of the present invention can achieve an inhibitory effect on Candida albicans and / or Shigella through a single administration.

[0070] According to an embodiment of the present invention, the single-dose formulation comprises 10 10 CFU of the aforementioned Lactobacillus rhamnosus served as the active ingredient.

[0071] use

[0072] In another aspect of the present invention, the present invention provides a use of the aforementioned Lactobacillus rhamnosus, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent or the aforementioned single-dose preparation in the preparation of a medicament, wherein the medicament has at least one of the following uses: for inhibiting the growth of Candida albicans and / or Shigella; for preventing and / or alleviating and / or treating diseases caused by Candida albicans and / or Shigella.

[0073] As a specific embodiment, the related disease is at least one selected from the group consisting of oral candidiasis, candidal cheilitis, candidal angular cheilitis, digestive system candidiasis and bacillary dysentery.

[0074] In another aspect of the present invention, the present invention provides a use of the aforementioned Lactobacillus rhamnosus, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent or the aforementioned single-dose preparation in inhibiting the growth of Candida albicans and / or Shigella.

[0075] In another aspect of the present invention, the present invention provides a use of the aforementioned Lactobacillus rhamnosus, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent or the aforementioned single-dose preparation in preventing and / or alleviating and / or treating diseases related to Candida albicans and / or Shigella.

[0076] method

[0077] In another aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria. According to an embodiment of the present application, the method comprises: contacting at least one of the aforementioned Lactobacillus rhamnosus, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned single-dose preparation with the pathogenic bacteria.

[0078] According to an embodiment of the present application, the pathogenic bacteria include Candida albicans and / or Shigella.

[0079] In another aspect, the present invention provides a method for preventing and / or alleviating and / or treating diseases caused by pathogenic bacteria. According to an embodiment of the present application, the method comprises administering to a patient a pharmaceutically acceptable dose of at least one of the aforementioned Lactobacillus rhamnosus, the aforementioned fermentation supernatant, the aforementioned fermentation broth, the aforementioned microbial agent, or the aforementioned single-dose formulation.

[0080] According to an embodiment of the present application, the pathogenic bacteria include Candida albicans and / or Shigella.

[0081] According to an embodiment of the present application, the related disease is at least one selected from the group consisting of oral candidiasis, candidal cheilitis, candidal angular cheilitis, digestive system candidiasis, and bacillary dysentery.

[0082] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0083] Example 1 Isolation and identification of strains

[0084] 1. The isolation and purification method of the strain is as follows:

[0085] (1) Take 0.1 g of feces from a one-month-old infant and place it in a sterile test tube, add 0.9 mL of sterile water, and shake thoroughly;

[0086] (2) Dilute the bacterial solution by the doubling dilution method, with a total of 7 gradients: 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ;

[0087] (3) 100 μl of bacterial solution was taken from each gradient and spread on a sterilized MRS solid plate, and then placed in a 37°C incubator for anaerobically incubated for 48 h;

[0088] (4) Pick a single colony and streak it onto an MRS solid plate. Place it in a 37°C incubator for anaerobically incubation for 48 h and subculture it five times until the colony morphology is uniform.

[0089] (5) The obtained single colonies were identified by 16S rRNA, and the strains identified as Lactobacillus rhamnosus were inoculated into MRS liquid culture medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and cultured anaerobically at 37°C for 48 h;

[0090] (6) Take 100 μl of culture medium and dilute to 10 -3 Then, the cells were spread on MRS plates and cultured anaerobically at 37°C for 24 h;

[0091] (7) Pick a single colony and culture it in MRS liquid medium at 37°C for 24 h to obtain the target strain. Then, store the bacteria in 20% glycerol and store at -80°C for future use.

[0092] 2. Molecular Biological Identification of the Strains

[0093] The 16S rRNA sequence was compared with the strains in GenBank for homology. The strain of the present invention had the highest homology of 99% with Lactobacillus rhamnosus 6551 and was identified as Lactobacillus rhamnosus and named Lactobacillus rhamnosus VB255. It was deposited on March 31, 2023 at the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.26968. The scanning electron micrograph of Lactobacillus rhamnosus VB255 is shown in Figure 1. It can be seen that the bacteria are rod-shaped. The 16S rRNA sequence of the strain is as follows:

[0094] Example 2 Detection of physiological and biochemical characteristics of strains

[0095] The culture medium required for detecting the physiological and biochemical characteristics of Lactobacillus rhamnosus VB255, namely, MRS agar medium, was prepared as follows: peptone 10.0 (g / L), beef extract powder 8.0 (g / L), yeast extract powder 4.0 (g / L), glucose 20.0 (g / L), dipotassium hydrogen phosphate 2.0 (g / L), diammonium hydrogen citrate 2.0 (g / L), sodium acetate 5.0 (g / L), magnesium sulfate 0.2 (g / L), manganese sulfate 0.04 (g / L), agar 20 (g / L), Tween 80 1.0 (g / L), pH 6.7, and high-pressure sterilization at 121°C for 15 min.

[0096] Physiological and biochemical characteristics analysis was performed according to the Bergey's Bacterial Identification Manual. The test contents are described in detail as follows:

[0097] (1) Catalase

[0098] After culturing for 24 hours, take a small loop of Lactobacillus rhamnosus VB255 with a sterile inoculating loop and smear it on a glass slide with 3% hydrogen peroxide. Observe immediately. If bubbles are generated, it is positive; if no bubbles are generated, it is negative.

[0099] (2) Oxidase

[0100] Soak the oxidase test paper with distilled water, pick up a single Lactobacillus rhamnosus VB255 colony with a thin glass rod, and apply it on the test paper. If it turns blue or blue-purple within 30 seconds, it is a strong positive; if it does not change color within 2 minutes, it is a negative.

[0101] (3) Milk decomposition

[0102] In a test tube containing 2ml of sterile water, use an inoculating needle to pick 1-3 Lactobacillus rhamnosus VB255 colonies from the plate. Place them in sterile water and carefully grind them to create a bacterial suspension. Add 100μl of the bacterial suspension to the milk tube. Incubate at 37°C for 24-48 hours. Observe the tube after the incubation period. If the layers turn pink and solidify, the test is positive; if they do not change color, the test is negative.

[0103] (4) Hydrolysis of hippuric acid

[0104] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 4 h. After incubation, slowly add 0.2 ml of ninhydrin solution along the tube wall without shaking. Incubate at 37°C for 10 min and observe. A dark purple color indicates a positive result; a light purple color or no color change indicates a negative result.

[0105] (5) Indole test

[0106] Add 100 μl of the bacterial suspension in step (3) to the peptone water, incubate at 37°C for 24 h, and then add 0.2 ml to 0.3 ml of Kovacs indigo reagent. If red appears on the upper layer, it is an indigo-positive reaction; otherwise, it is a negative reaction.

[0107] (6) Urease

[0108] Add 100 μl of the bacterial suspension from step (3) to the biochemical tube and incubate at 37°C for 24 h. If a rose-red color appears on the upper layer, it is positive; otherwise, it is negative.

[0109] (7) Hydrogen sulfide production

[0110] Add 100 μl of the bacterial suspension from step (3) to the biochemical tube and incubate at 37°C for 18-24 hours. If a black color appears, it is positive; otherwise, it is negative.

[0111] (8) Glucose semisolid

[0112] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18-24 hours. If a yellow color appears, it is positive; if a gray-purple, purple, or purple-red color appears, it is negative.

[0113] (9) Arginine dihydrolase

[0114] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18-24 hours. After inoculation, add sterile liquid paraffin to cover the surface of the culture medium. If a purple or purple-red color appears, it is positive; if a yellow color appears, it is negative.

[0115] (10) Amygdalin / Rhamnose

[0116] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18-24 hours. If a yellow color appears, it is positive; if a light red or red color appears, it is negative.

[0117] (11) Glucose / Xylose / Melibiose / Turanose / Trehalose / Galactose / Arabose / Sorbitol

[0118] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18-24 hours. If a yellow color appears, it is positive; if a blue or blue-green color appears, it is negative.

[0119] (12) D-ribose / melezitose / mannose / fructose

[0120] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 24-48 hours. If a yellow color appears, it is positive; if a gray-purple, purple, or purple-red color appears, it is negative.

[0121] (13) Cellobiose / maltose / mannitol / salicin / sucrose / raffinose / inulin / lactose

[0122] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 24-48 hours. If yellow appears, it is positive; if purple or purple-gray appears, it is negative.

[0123] (14) Nitrate reduction

[0124] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 18-24 hours. After incubation, add 2-3 drops each of nitrate reduction reagents A and B and observe immediately. If the cell turns red, it is positive; if it does not change color, it is negative.

[0125] (15) Gluconate

[0126] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 24-48 hours. After incubation, add Benedict's reagent in a 1:1 volume ratio, mix well, and boil for 10 minutes. After cooling, observe the results. If a yellow, orange, or orange-red precipitate appears, it is positive; if a blue or green precipitate appears, it is negative.

[0127] (16) VP determination

[0128] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 2-4 days. Add the VP kit and shake the tube thoroughly to observe the results. A positive reaction will result in a red color appearing immediately or within a few minutes. If negative, continue incubation at 37°C for another 4 hours and observe again.

[0129] (17) Methyl red (MR)

[0130] Add 100 μl of the bacterial suspension from step (3) to a biochemical tube and incubate at 37°C for 2-4 days. Add one drop of methyl red reagent and observe immediately. If bright red appears, it is positive; if yellow appears, it is negative.

[0131] (18) Gelatin liquefaction

[0132] Add 100 μl of the bacterial suspension from step (3) to the biochemical tube and incubate at 37°C for 24-72 hours. Remove the tube and place it in a refrigerator at 2-8°C for 30 minutes. Observe the tube and see if it is still liquid. If not, the test result is positive. Otherwise, it is negative.

[0133] (19) Starch hydrolysis

[0134] Add 100 μl of the bacterial suspension from step (3) to the biochemical tube and incubate at 37°C for 48-96 hours. After the incubation period, add iodine solution. If the bacteria do not turn blue, it is positive; if they turn blue, it is negative.

[0135] (20) Esculin

[0136] Add 100 μl of the bacterial suspension from step (3) to the biochemical tube and incubate at 37°C for 24-48 hours. After inoculation, add sterile liquid paraffin to cover the surface of the culture medium. If black appears, it is positive; if purple or purple-gray appears, it is negative.

[0137] Table 1 Physiological and biochemical test results of Lactobacillus rhamnosus VB255 Note: + indicates positive; - indicates negative.

[0138] Example 3 Strain stability

[0139] The experimental method for the choline resistance stability of Lactobacillus rhamnosus VB255 is as follows:

[0140] Preparation of bacterial suspension: Take the counted bacterial suspension (the viable bacteria count should reach about 10 9 -10 10 / ml), and after thawing, divided into three groups (0h group, 2h group and 4h group), and added into the packaged simulated pancreatic juice, gastric juice and intestinal juice respectively, wherein 10μl bacterial suspension and 990μl simulated liquid were added and mixed. The number of viable bacteria in the 0h group was counted at time 0, and the bacterial suspensions of the 2h group and 4h group were placed in anaerobic bags and cultured in a 37℃ incubator. The viable bacteria were taken out and counted after culturing for 2h and 4h.

[0141] (1) Preparation of artificial pancreatic juice: Normal saline (sodium chloride: 0.9%) was first prepared into a 1% sodium chloride solution, and then taurocholine was added to different 1% sodium chloride solutions at concentrations of 0.033%, 0.066%, 0.11%, 0.22%, and 0.33%, respectively. The solution was sterilized at 121°C for 30 min. After the temperature dropped to room temperature, 0.1% trypsin solution was added to the sterilized solution (0.1% trypsin solution was prepared by filtering the 1% trypsin solution with a sterile membrane and adding it to normal saline at a ratio of 1:9).

[0142] The results of the choline resistance stability experiment are shown in Table 2. Within 4 hours, the survival rate of Lactobacillus rhamnosus VB255 in 0.033-0.33% choline solution was as high as over 95%, indicating that Lactobacillus rhamnosus VB255 can be stably present in 0.033-0.33% choline solution, that is, Lactobacillus rhamnosus VB255 can exist stably in pancreatic juice, further indicating that the strain can adapt to the intestinal environment.

[0143] Table 2 Choline survival rate of Lactobacillus rhamnosus VB255

[0144] (2) Preparation of artificial simulated gastric juice: physiological saline (sodium chloride: 0.9%) was first prepared into a 1% sodium chloride solution, and then adjusted to different pH values ​​(pH = 1, pH = 2, pH = 3, pH = 4) and packaged. The solution was sterilized at 121°C for 30 min, and after the temperature dropped to room temperature, 0.3% pepsin solution was added to the sterilized solution (0.3% pepsin solution was prepared by filtering the solution with a sterile membrane and adding it to physiological saline at a ratio of 1:9).

[0145] The gastric juice stability results are shown in Table 3. Lactobacillus rhamnosus VB255 died instantly in gastric juice with a pH of 1-2, had a 100% mortality rate within 2 hours in gastric juice with a pH of 3, had a 99% mortality rate within 2 hours in gastric juice with a pH of 4, and had a 100% mortality rate within 4 hours. Therefore, the instability of Lactobacillus rhamnosus VB255 in gastric juice needs to be considered in the development of subsequent dosage forms.

[0146] Table 3 Survival rate of Lactobacillus rhamnosus VB255 in gastric juice

[0147] (3) Preparation of artificial simulated intestinal fluid: Artificial simulated intestinal fluid was purchased from Beijing Coolbo Technology Co., Ltd., product number SL66102. The intestinal fluid simulation fluid was prepared according to the product instructions.

[0148] The results of intestinal fluid stability are shown in Table 4. After being treated in the intestinal fluid at pH 8.0 for 8 hours, the survival rate of the strain was 100%, indicating that the strain was able to exist stably in the intestinal fluid.

[0149] Table 4 Survival rate of Lactobacillus rhamnosus VB255 in intestinal fluid

[0150] Example 4 Antibacterial Test

[0151] The culture medium for Lactobacillus rhamnosus VB255 was MRS agar, prepared as follows: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 20 g / L, Tween 80 1.0 g / L, pH 6.7, and autoclaved at 121°C for 15 min. Culture was performed anaerobically at 37°C for 1 day.

[0152] The antibacterial experiment used a double-layer plate culture method. After optimization and evaluation, the specific antibacterial model preparation method is as follows:

[0153] Preparation of probiotics on the lower plate: Thaw the activated Lactobacillus rhamnosus VB255 cryovial and take 2 μl (the amount of viable bacteria is 10 6 ) were spotted on MRS solid plates, with two spots for each strain, and the culture conditions were anaerobic overnight culture at 37°C.

[0154] Preparation of the upper pathogenic bacteria layer: After high temperature sterilization, the upper culture medium is cooled to 40-50℃, and different pathogens are added as required. The final concentration of pathogens is 10 8 / ml, add 7ml of culture medium containing pathogenic bacteria to the above-mentioned plate on which Lactobacillus rhamnosus VB255 has grown. After the plate solidifies, culture it under appropriate culture conditions according to the growth conditions of different pathogens. After the upper plate grows well, observe whether an inhibition zone is formed around the colony of Lactobacillus rhamnosus VB255, and measure the size of the inhibition zone to determine the antibacterial ability of Lactobacillus rhamnosus VB255. For all antibacterial experiments, the diameter of the inhibition zone of the strain is measured and the average value of three groups of data is taken.

[0155] The control strains are Lactobacillus rhamnosus VB162 (deposit number is ATCC NO.7469), Lactobacillus rhamnosus LGG (derived from Kangcuile children's probiotics, obtained by screening) and Lactobacillus rhamnosus LRa05 (purchased from Weikang Probiotics Co., Ltd.). The antibacterial ability of the four strains of Lactobacillus rhamnosus is shown in Table 5. Compared with Lactobacillus rhamnosus VB162, Lactobacillus rhamnosus LGG and Lactobacillus rhamnosus LRa05, Lactobacillus rhamnosus VB255 has an antibacterial ability against Candida albicans and Shigella that is significantly improved, indicating that the Lactobacillus rhamnosus VB255 of the present invention can simultaneously inhibit the growth of Candida albicans and Shigella, and its inhibitory ability is significantly better than that of the control strain.

[0156] Table 5 Antibacterial test results (diameter of inhibition zone in mm)

[0157] Example 5 Lactobacillus rhamnosus inhibits the formation of Candida albicans and Shigella biofilms

[0158] 96-well microplate quantitative detection method: adjust the indicator bacteria concentration to 10 with PBS buffer. 8 CFU / ml, 8000r / min, 4℃ centrifugation for 5min to collect the bacteria, and re-dissolve with fresh LB medium for later use. Use PBS buffer to adjust the concentration of different Lactobacillus rhamnosus bacterial suspensions to 10 9 CFU / ml. Add 200 μl of LB medium to the wells along the edge of a 96-well microtiter plate. Add 100 μl of indicator bacteria solution and 100 μl of Lactobacillus rhamnosus culture to each of the remaining wells. A well containing 100 μl of indicator bacteria and 100 μl of PBS buffer serves as a control. Place the 96-well microtiter plate in a 37°C incubator for 24 hours, then discard the culture medium. Wash each well three times with 200 μl of PBS buffer to remove adherent bacteria. Remove the PBS buffer and dry at room temperature. Add 200 μl of 95% methanol to each well for 15 minutes, remove the methanol, and dry at room temperature. Stain each well with 200 μl of 1% crystal violet for 5 minutes, rinse with deionized water to remove excess dye, and dry at room temperature. Dissolve the dye in 160 μl of 33% glacial acetic acid to each well for 30 minutes. Transfer 125 μl to a new 96-well microtiter plate and measure absorbance at 590 nm using a microplate reader. Biofilm inhibition rate / %=1-OD experimental group / OD control group.

[0159] The inhibition rates of different Lactobacillus rhamnosus on the biofilm formation of Shigella and Candida albicans are shown in Table 6. Compared with Lactobacillus rhamnosus VB162 (deposit number ATCC NO.7469), Lactobacillus rhamnosus LGG, and Lactobacillus rhamnosus LRa05, Lactobacillus rhamnosus VB255 has a better inhibitory effect on the biofilm formation of Shigella and Candida albicans, with an inhibition rate of 47.1% on Shigella biofilm and 54.24% on Candida albicans.

[0160] Table 6 Inhibition of biofilm formation of Shigella and Candida albicans by different Lactobacillus rhamnosus Note: The letters in the upper right corner of the same row of data indicate significant differences (P<0.05)

[0161] Example 6 Intestinal cell adhesion experiment

[0162] Caco-2 cells were inoculated with 2 mL of incomplete DMEM and 1 mL of Lactobacillus rhamnosus VB255 culture medium at various growth stages (delayed growth phase, logarithmic growth phase, stationary phase, and apoptosis phase). An equal volume of sterile PBS buffer was used instead of the Lactobacillus rhamnosus VB255 culture medium as a control group. Three replicate wells were cultured in a 37°C, 5% CO2 incubator for 2 hours. The cells were then rinsed five times with PBS, air-dried, and fixed with methanol for 20 minutes. After Gram staining, 25 randomly selected fields of view were used under an oil immersion lens. The number of Lactobacillus rhamnosus adherent to 100 cells was counted. The percentage of cell-adherent bacteria to the total number of added bacteria was defined as the Lactobacillus rhamnosus VB255 adhesion rate. The results of Lactobacillus rhamnosus VB255 adhesion to Caco-2 cells are shown in Table 7. The adhesion rates of Lactobacillus rhamnosus VB255 to Caco-2 cells at different growth stages differed significantly (P < 0.05). The adhesion performance of Lactobacillus rhamnosus VB255 in the logarithmic phase was the best, with an adhesion rate of 70.05%, indicating that this strain can effectively adhere to intestinal cells.

[0163] Table 7 Lactobacillus rhamnosus intestinal adhesion efficiency

[0164] 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.

[0165] 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.

Claims

1. A Lactobacillus rhamnosus, characterized in that It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 31, 2023, with the deposit number CGMCC NO.26968.

2. A fermentation supernatant, characterized in that: contain: The Lactobacillus rhamnosus according to claim 1; and / or The metabolite of Lactobacillus rhamnosus according to claim 1.

3. A fermentation broth, characterized in that: Contains the Lactobacillus rhamnosus according to claim 1.

4. A microbial agent, characterized in that: Contains at least one of the Lactobacillus rhamnosus according to claim 1, the fermentation supernatant according to claim 2, and the fermentation broth according to claim 3.

5. The microbial agent according to claim 4, characterized in that: The Lactobacillus rhamnosus exists in the form of living cells and / or non-living cells.

6. The microbial agent according to claim 4, characterized in that: The microbial agent further includes pharmaceutically, food or health food acceptable auxiliary materials or carriers.

7. A single-dose preparation, characterized in that Contains 10 9 ~10 11 CFU Lactobacillus rhamnosus described in claim 1 is used as an active ingredient.

8. A food, health food or feed, characterized in that: The method comprises at least one of the Lactobacillus rhamnosus according to claim 1, the fermentation supernatant according to claim 2, the fermentation liquid according to claim 3 and the microbial agent according to any one of claims 4 to 6.

9. Use of the Lactobacillus rhamnosus according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, or the single-dose preparation according to claim 7 in the preparation of a medicament, wherein the medicament has at least one of the following uses: inhibiting the growth of Candida albicans and / or Shigella; Prevent and / or alleviate and / or treat diseases caused by Candida albicans and / or Shigella.

10. The use according to claim 9, characterized in that The related diseases are selected from at least one of oral candidiasis, candidal cheilitis, candidal angular cheilitis, digestive system candidiasis and bacillary dysentery.

11. Use of the Lactobacillus rhamnosus according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, or the single-dose preparation according to claim 7 in inhibiting the growth of Candida albicans and / or Shigella.

12. Use of the Lactobacillus rhamnosus according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, or the single-dose preparation according to claim 7 in preventing and / or alleviating and / or treating diseases related to Candida albicans and / or Shigella.

13. A method for inhibiting the growth of pathogenic bacteria, characterized in that: include: The pathogenic bacteria are contacted with at least one of the Lactobacillus rhamnosus according to claim 1, the fermentation supernatant according to claim 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, and the single-dose preparation according to claim 7.

14. The method according to claim 13, characterized in that The pathogenic bacteria include Candida albicans and / or Shigella.

Images