Bifidobacterium breve VB316 and use therefor

Abstract

Provided are a bifidobacterium breve VB12 and a use thereof, relating to the field of microorganisms. The preservation number of the bifidobacterium breve is CGMCC No.28160. The bifidobacterium breve has the advantages of strong β-galactosidase activity, strong acid and alkali resistance, and strong cholate resistance etc., has strong bacteriostatic ability to common pathogenic bacteria, can be used for relieving lactose intolerance, regulating intestinal flora, and preventing and treating intestinal diseases caused by pathogenic bacteria infection.

Description

Bifidobacterium breve VB316 and its application Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to Bifidobacterium breve VB316 and applications thereof. Background Art

[0002] Lactose, a disaccharide composed of glucose and galactose, is the primary source of carbohydrates in the milk of humans and most other mammals. Lactose cannot be directly absorbed by the human body and is primarily hydrolyzed into glucose and galactose by lactase (primarily β-galactosidase) at the tips of the villi on the mucosal surface of the small intestine, particularly the jejunum. These are then absorbed through active transport by cells. Lack of lactase or reduced lactase activity in the small intestine can lead to incomplete digestion of lactose in breast milk or other dairy products, resulting in adverse reactions such as abdominal pain, diarrhea, and bloating.

[0003] Current strategies for treating or alleviating lactose intolerance include a low-lactose diet, lactase supplementation, and supplementation with probiotics that exhibit high β-galactosidase activity or a lactose-degrading phenotype. Probiotics, when introduced into the body, can metabolize and produce a variety of enzymes, such as lactase, lipase, protease, and peptidase, which can help treat lactose intolerance. However, bacterial strains that can alleviate lactose intolerance remain underdeveloped.

[0004] Summary of the Invention

[0005] The present invention aims to solve, at least to a certain extent, at least one of the technical problems existing in the prior art. To this end, the present invention provides a Bifidobacterium breve, a fermentation product, a microbial agent, a food, a medicine, a health product, or a feed, and applications thereof. The novel strain of Bifidobacterium breve screened by the present invention has the advantages of strong β-galactosidase activity, strong acid and alkali resistance, and bile salt tolerance, and has strong antibacterial activity against common pathogens. It can be used to alleviate lactose intolerance, regulate intestinal flora, and prevent and treat intestinal diseases caused by pathogenic bacteria infection, and has high application value.

[0006] To this end, in a first aspect of the present invention, the present invention provides a Bifidobacterium breve VB316. According to an embodiment of the present invention, the deposit number of the Bifidobacterium breve is CGMCC No. 28160.

[0007] Collection information:

[0008] Strain name: Bifidobacterium breve VB316

[0009] Deposit date: August 14, 2023

[0010] Depository: General Microbiology Center of China Culture Collection Administration

[0011] Accession number: CGMCC No.28160

[0012] The new strain of Bifidobacterium breve VB316 screened by the present invention has the advantages of strong β-galactosidase activity, strong acid and alkali resistance and bile salt resistance, and has strong antibacterial ability against common pathogens. It can be used to alleviate lactose intolerance, regulate intestinal flora, and prevent and treat intestinal diseases caused by pathogenic bacteria infection, and has high application value.

[0013] In a second aspect of the present invention, a fermentation product is provided. According to an embodiment of the present invention, the fermentation product comprises the aforementioned Bifidobacterium breve VB316.

[0014] In a third aspect, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent comprises at least one of the aforementioned Bifidobacterium breve VB316 and the aforementioned fermentation product. The microbial agent of the present invention can be used to alleviate lactose intolerance, regulate intestinal flora, and prevent and treat intestinal diseases caused by pathogenic bacteria.

[0015] In a fourth aspect, the present invention provides a food, medicine, health product, or feed. According to embodiments of the present invention, the food, medicine, health product, or feed comprises at least one of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product, or the aforementioned microbial agent. The food, health product, or feed of the present invention can be used to alleviate lactose intolerance and regulate intestinal flora; the medicine of the present invention can be used to prevent and / or alleviate lactose intolerance and prevent and / or treat intestinal diseases caused by pathogenic bacteria.

[0016] In a fifth aspect of the present invention, the present invention proposes the use of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product or the aforementioned microbial agent in the preparation of food or health products, wherein the food or health products are used to alleviate lactose intolerance and / or regulate intestinal flora.

[0017] In a sixth aspect of the present invention, the present invention provides the use of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product, or the aforementioned microbial agent in the preparation of a medicine or feed, wherein the medicine or feed has at least one of the following uses:

[0018] Prevent and / or alleviate lactose intolerance;

[0019] Regulate intestinal flora;

[0020] Prevent and / or treat intestinal diseases caused by pathogenic bacteria infection.

[0021] In a seventh aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria in vitro. According to an embodiment of the present invention, the method comprises:

[0022] At least one of the Bifidobacterium breve VB316, the fermentation product, and the microbial agent is co-cultured with a sample containing pathogenic bacteria.

[0023] As mentioned above, the Bifidobacterium breve VB316 strain of the present invention can effectively inhibit the growth of pathogenic bacteria.

[0024] 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

[0025] 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:

[0026] FIG1 is a scanning electron microscopy image of Bifidobacterium breve VB316 in Example 2 of the present invention;

[0027] Figure 2 is a standard curve of o-nitrophenol (ONP) concentration in Example 3 of the present invention, wherein the abscissa is the concentration of the ONP standard and the ordinate is the absorbance of the ONP standard at different concentrations at 405 nm;

[0028] Figure 3 is a graph showing the results of β-galactosidase activity assay of Bifidobacterium breve VB316 and commercial strains in Example 3 of the present invention, wherein VB316 is Bifidobacterium breve VB316 of the present invention, NCFM is Lactobacillus acidophilus of DuPont, USA, BB-12 is Bifidobacterium animalis subsp. lactis of Chr. Hansen, Denmark, CECT5716 is Lactobacillus fermentum of Spain, and LGG is Lactobacillus rhamnosus of Finland. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0032] Herein, the term "BBL solid medium" refers to Bifidobacterium agar medium.

[0033] As used herein, the term "sterile water" refers to water that is free of bacteria by treating water containing bacterial masses through physical methods.

[0034] The present invention provides Bifidobacterium breve, fermentation products, microbial agents, foods, medicines, health products or feeds and uses thereof, which will be described in detail below.

[0035] strain

[0036] The present invention provides Bifidobacterium breve.

[0037] The preservation number of Bifidobacterium breve VB316 according to an embodiment of the present invention is CGMCC No. 28160. This strain is a new isolate and has been deposited in the "General Microbiological Center of China Culture Collection Administration" on August 14, 2023, with the preservation number CGCC No. 28160. The deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0038] The 16S rDNA sequencing result of Bifidobacterium breve VB316 according to an embodiment of the present invention is shown in SEQ ID NO: 1.

[0039] The new strain of Bifidobacterium breve VB316 screened by the present invention has the advantages of strong β-galactosidase activity, strong acid and alkali resistance and bile salt resistance, and has strong antibacterial ability against common pathogens. It can be used to alleviate lactose intolerance, regulate intestinal flora, and prevent and treat intestinal diseases caused by pathogenic bacteria infection, and has high application value.

[0040] As used herein, the terms "Bifidobacterium breve VB316", "Bifidobacterium breve VB316" and "VB316 strain" are used synonymously.

[0041] fermentation products

[0042] The present invention provides a fermentation product. The fermentation product according to an embodiment of the present invention includes the aforementioned Bifidobacterium breve VB316.

[0043] It should be noted that the "fermentation product" of the present invention refers to a solution obtained after culturing Bifidobacterium breve VB316 for a period of time, which solution mainly contains Bifidobacterium breve VB316 and its metabolites; or a supernatant after further treatment by centrifugation, filtration, etc., which supernatant mainly contains metabolites of Bifidobacterium breve VB316; or a bacterial suspension after further treatment by centrifugation, resuspension, etc., which bacterial suspension mainly contains Bifidobacterium breve VB316.

[0044] According to an embodiment of the present invention, the present invention further comprises: a metabolite of the aforementioned Bifidobacterium breve VB316.

[0045] It should be noted that the characteristics and advantages described above for Bifidobacterium breve VB316 are also applicable to this fermentation product and will not be repeated here.

[0046] microbial agents

[0047] The present invention provides a microbial agent. The microbial agent according to an embodiment of the present invention comprises the aforementioned Bifidobacterium breve VB316 and at least one of the aforementioned fermentation products. The microbial agent of the present invention can be used to alleviate lactose intolerance, regulate intestinal flora, and prevent and treat intestinal diseases caused by pathogenic bacteria.

[0048] It should be noted that the microbial agent of the present invention can be a liquid microbial agent, including but not limited to fermentation products, etc.; it can also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0049] It should be noted that, in the microbial agent of the present invention, Bifidobacterium breve VB316 may exist in the form of living cells and / or non-living cells.

[0050] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce, or replicate.

[0051] For example, living cells can be immobilized cells. Herein, "immobilized cells" refer to living cells that are fixed on a carrier and can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0052] 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.

[0053] In some specific embodiments, the aforementioned Bifidobacterium breve VB316 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0054] In some specific embodiments, the dried bacteria are obtained by freeze-drying the Bifidobacterium breve VB316.

[0055] In some specific embodiments, the aforementioned microbial agent may further contain at least one strain acceptable to food, medicine, health care product and feed.

[0056] In some specific embodiments, the aforementioned microbial agent further includes pharmaceutically acceptable excipients or carriers, excipients or carriers acceptable in food or health products, or excipients or carriers acceptable in animal feed.

[0057] 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.

[0058] As used herein, "acceptable in health products" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health product requirements of different countries.

[0059] 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.

[0060] As used herein, "adjuvant or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals, which may be adjusted according to the animal feed requirements of different countries.

[0061] 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.

[0062] 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 Bifidobacterium breve VB316 disclosed herein, 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.

[0063] It should be noted that the characteristics and advantages described above for Bifidobacterium breve are also applicable to this microbial agent and will not be repeated here.

[0064] Food, medicine, health products or feed

[0065] The present invention provides a food, medicine, health product, or feed. The food, medicine, health product, or feed according to embodiments of the present invention comprises at least one of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product, or the aforementioned microbial agent. The food, health product, or feed of the present invention can be used to alleviate lactose intolerance and regulate intestinal flora; the medicine of the present invention can be used to prevent and / or alleviate lactose intolerance and prevent and / or treat intestinal diseases caused by pathogenic bacteria.

[0066] According to an embodiment of the present invention, the invention further comprises auxiliary materials or carriers acceptable in pharmacy, food, health care products or feed.

[0067] In some specific embodiments, the aforementioned Bifidobacterium breve, the aforementioned fermentation product, or the aforementioned microbial agent is added or inoculated into food, health products, or feed, or added to a pharmaceutical composition, thereby further obtaining a food, health product, or feed having the function of alleviating lactose intolerance and / or regulating intestinal flora, or obtaining a medicine that can prevent and / or alleviate pathogenic bacteria infection or intestinal diseases caused by pathogenic bacteria infection.

[0068] Illustratively, the aforementioned foods include but are not limited to: probiotic tablets, fermented dairy products (such as probiotic yogurt), probiotic solid beverages, probiotic milk powder, probiotic cheese, probiotic soy products, probiotic candies, probiotic fermented vegetables, etc.

[0069] For example, the aforementioned medicines include but are not limited to: medicines for human use and medicines for veterinary use. The aforementioned medicines for veterinary use may be for pets, livestock, or wild animals.

[0070] Illustratively, the aforementioned health care products include but are not limited to: health care products for humans and health care products for animals.

[0071] It should be noted that the characteristics and advantages described above for Bifidobacterium breve VB316 are also applicable to the food, medicine, health product or feed, and will not be repeated here.

[0072] use

[0073] The present invention proposes the use of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product or the aforementioned microbial agent in preparing food or health products. The food or health products are used to alleviate lactose intolerance and / or regulate intestinal flora.

[0074] The present invention also proposes the use of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product, or the aforementioned microbial agent in the preparation of a medicine or feed, wherein the medicine or feed has at least one of the following uses:

[0075] Prevent and / or alleviate lactose intolerance;

[0076] Regulate intestinal flora;

[0077] Prevent and / or treat intestinal diseases caused by pathogenic bacteria infection.

[0078] According to an embodiment of the present invention, the aforementioned pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsule perfringens.

[0079] In some specific embodiments, the aforementioned drug further contains an excipient and / or a carrier.

[0080] In some specific embodiments, the aforementioned excipients include at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

[0081] In some specific embodiments, the aforementioned 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.

[0082] In some specific embodiments, the dosage form of the aforementioned drug includes at least one selected from oral solution, powder, granule, capsule, tablet, and pill.

[0083] It should be noted that the characteristics and advantages described above for Bifidobacterium breve VB316 are also applicable to this use and will not be repeated here.

[0084] method

[0085] The present invention provides a method for inhibiting the growth of pathogens in vitro. The method according to an embodiment of the present invention comprises: co-culturing at least one of the aforementioned Bifidobacterium breve VB316, the aforementioned fermentation product, and the aforementioned microbial agent with a sample containing pathogens.

[0086] As mentioned above, Bifidobacterium breve VB316 can effectively inhibit the growth of pathogens.

[0087] According to an embodiment of the present invention, the aforementioned pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Clostridium perfringens.

[0088] It should be noted that the characteristics and advantages described above for Bifidobacterium breve VB316 are also applicable to this method and will not be repeated here.

[0089] 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.

[0090] Example 1: Obtaining Bifidobacterium breve VB316 strain

[0091] The Bifidobacterium breve VB316 strain of the present invention is separated from the feces of healthy children.

[0092] Strain collection and isolation process: 0.5g of feces from a healthy child was added to 5mL of Bifidobacterium BS liquid culture medium and cultured anaerobically at 37°C for 24 hours. The bacterial suspension was diluted and plated onto MRS screening plates (MRS plates containing 50μg / mL mupirocin and 5% serum (v / v)) and incubated anaerobically at 37°C for 48-72 hours. Strains of target morphology were selected and purified by multiple streaking cultures until the colonies on the plates were uniform in morphology. Identification was performed after microscopic examination and the absence of contaminants.

[0093] Strain preservation: The obtained pure culture strains are cultured to a concentration of about 10 7 CFU / mL, 500 μL of 50% glycerol was added to 500 μL of bacterial solution to make the glycerol concentration reach 25%, and then ultra-low temperature storage was carried out at -80℃.

[0094] Example 2: Identification of Bifidobacterium breve VB316 strain

[0095] The culture isolated and purified in Example 1 was further confirmed to be a pure culture by streaking and smear microscopy, and then the bacterial species was identified using experimental methods including Gram staining test, physiological and biochemical tests such as catalase, and 16S rDNA full sequence sequencing.

[0096] The 16S rDNA sequencing results were compared by BLAST, and it was finally identified that the isolated strain was a breve Bifidobacterium, named Bifidobacterium breve VB316 (hereinafter also referred to as "VB316 strain"), which was deposited in the "General Microbiology Center of China Microorganism Culture Collection Administration" on August 14, 2023, with the deposit number CGMCC No. 28160.

[0097] In this embodiment, the main bacterial species identification methods and results are as follows:

[0098] The VB316 bacterial strain was subjected to scanning electron microscopy observation. The specific method was as follows: first, rice-sized colonies were scraped from a flat plate and fixed with glutaraldehyde and osmic acid. Subsequently, the samples were dehydrated using a gradient of ethanol (comprising 30%, 50%, 70%, 80%, 90% and 95% concentrations), with each concentration treated for 15 min and then treated twice with 100% ethanol for 20 min each. The dehydrated samples were dried in a Hitachi HCP-2 critical point drying apparatus and subsequently plated. Finally, the processed samples were observed in a Hitachi SU-8010 scanning electron microscope, and the electron microscope photographs were shown in Figure 1.

[0099] Scanning electron microscopy and Gram staining test results showed that the VB316 strain was Gram-positive, with single colonies of milky white, irregular shape, and a bulge with wrinkles in the middle. After anaerobic culture at 37°C for 4 days, the maximum colony diameter was approximately 2.5 mm, and the cells were polymorphic rods (Figure 1).

[0100] The physiological and biochemical characteristics of the VB316 strain were analyzed, and the specific experiments were carried out according to the Bergey's Bacterial Identification Manual.

[0101] The results of physical and chemical characteristics identification were: catalase negative, oxidase negative, able to utilize glucose, D-ribose, D-cellobiose, lactose, galactose, fructose, xylose, trehalose, and raffinose, unable to hydrolyze starch and gelatin, VP reaction negative, and methyl red negative.

[0102] The 16S rDNA sequencing results of the VB316 strain are shown in SEQ ID NO: 1.

[0103] Example 3: Investigation of β-galactosidase activity of Bifidobacterium breve VB316 strain

[0104] The most important enzyme for alleviating lactose intolerance is β-galactosidase, which breaks down lactose into glucose and galactose. This study used the ONPG method to further determine the β-galactosidase activity of Bifidobacterium breve VB316.

[0105] The principle of β-galactosidase activity detection is as follows: β-galactosidase catalyzes the cleavage of the glycosidic bond of colorless o-nitrophenol β-D-galactopyranoside (ONPG) to produce yellow o-nitrophenol (ONP) and β-D-pyranose. As ONPG decomposes into ONP, the color of the solution gradually deepens. ONP has a maximum absorption peak at 420nm, so the absorbance value can be used to calculate the ONP production and thus the enzyme activity of β-galactosidase.

[0106] The β-galactosidase activity unit (U) was defined as the amount of enzyme required to produce 1 μM ONP per minute under standard conditions.

[0107] The detection method is as follows:

[0108] 1. Preparation of Test Strains

[0109] Thaw the strain collection tube frozen at -80°C on ice, take one loopful of the bacterial suspension and streak it onto a Bifidobacterium solid culture plate, and incubate it in an anaerobic incubator at 37°C for 48 hours. Transfer a single colony to 10mL of Bifidobacterium liquid culture medium and incubate it in an anaerobic environment at 37°C for 24 hours. At a 2% transfer volume, transfer the bacterial suspension to a 250mL Erlenmeyer flask containing 50mL of Bifidobacterium lactose culture medium, place it in an anaerobic workstation at 37°C, and incubate it for 24 hours.

[0110] The Bifidobacterium liquid culture medium is composed of: beef extract 0.5%, tryptone 1%, yeast extract powder 0.5%, glucose 1%, Tween 80 0.1%, K2HPO4 0.2%, sodium acetate 0.5%, ammonium citrate tribasic 0.2%, pH 7.0, sterilized at 115°C for 30 minutes.

[0111] The solid culture medium for Bifidobacterium is: 2% agar is added to the liquid culture medium for Bifidobacterium.

[0112] Bifidobacterium lactose culture medium is: beef extract 0.5%, tryptone 1%, yeast extract powder 0.5%, lactose 3%, Tween 80 0.1%, K2HPO4 0.2%, sodium acetate 0.5%, ammonium citrate tribasic 0.2%, pH 7.0, sterilized at 115℃ for 30 minutes.

[0113] 2. Reagent Preparation

[0114] (1) PBS buffer

[0115] Dissolve 16.1 g of disodium hydrogen phosphate, 5.5 g of sodium dihydrogen phosphate, 0.75 g of potassium chloride, 0.246 g of magnesium sulfate, and 2.7 mL of 2-mercaptoethanol in 800 mL of water. Add 2 mol / L sodium hydroxide solution and adjust the pH to 6.0 ± 0.05. Transfer the solution to a 1 L volumetric flask, dilute to volume with water, and mix thoroughly.

[0116] (2) 10 mM substrate solution

[0117] The o-nitrophenyl-β-D-galactopyranoside (ONPG) substrate solution was prepared by dissolving 301 mg of ONPG in 80 mL of PBS buffer, transferring the solution into a 100 mL volumetric flask, and adjusting the volume with PBS to obtain a 10 mM substrate solution.

[0118] (3) Stop solution

[0119] Dissolve 10 g of sodium carbonate in water and transfer to a 100 mL volumetric flask to volume.

[0120] (4) Test sample preparation

[0121] Centrifuge 8 mL of fermentation broth at 5000 rpm and 4°C for 10 min, discard the supernatant, and wash twice with an equal volume of PBS buffer. Resuspend the cells in 2 mL of PBS buffer, add the sample to the grinding beads, and cryo-grind in a grinder at 65 Hz for 120 s three times. The grinding conditions are: 4°C, grinding time 60 s, and interval time 60 s. Dilute the crude enzyme solution appropriately to provide 0.05–0.25 units of β-galactosidase per mL of the final solution.

[0122] 3. Plotting of the standard curve of o-nitrophenol (ONP)

[0123] Accurately weigh 139 mg of o-nitrophenol (ONP) into a 25 mL beaker and dissolve in 10 mL of 95% ethanol. Transfer the solution to a 1 L volumetric flask and bring to volume with pure water. Use a pipette to pipette 2, 4, 6, 8, and 10 mL of the solution into a 100 mL volumetric flask, bring to volume with 10 wt% sodium carbonate solution, and mix thoroughly. These solutions contain 0.02, 0.04, 0.06, 0.08, and 0.1 μM of ONP per mL, respectively.

[0124] The absorbance was measured at a wavelength of 405 nm, with water as the control, the concentration of ONP as the horizontal axis, and the absorbance of the standard substances at various concentrations as the vertical axis. A standard curve was drawn (Figure 2) to obtain a linear regression equation.

[0125] 1. Determination of β-galactosidase activity

[0126] Take 200 μL of the diluted crude enzyme solution and preheat it in a 30°C metal bath for 5 minutes, add 1 mL of the substrate ONPG concentration solution that has also been preheated at 30°C for 5 minutes, mix well, and react at 400 rpm in a 30°C metal bath for 10 minutes. Then immediately add 400 μL of sodium carbonate solution to terminate the reaction. Pipette 200 μL into the ELISA plate and measure the absorbance at 420 nm within 30 minutes.

[0127] 2. Calculation

[0128] The standard curve obtained by calculation and analysis is: Y=2.5986X-0.0006, R 2 =0.9995

[0129] Wherein, Y is the light absorption value at 420 nm, and X is the concentration of ONP (μmol).

[0130] Enzyme activity (U / mL):

[0131] Where: OD 420 is the absorbance of the sample to be tested at 420 nm; b is the intercept of the standard curve, 0.0006; V is the total volume of the reaction system, 1.6 mL; f is the dilution factor of the enzyme solution; K is the slope of the standard curve, 2.5986; t is the reaction time, 10 min; V1 is the volume of the enzyme solution, 0.2 mL.

[0132] Several commercially available strains of Lactobacillus or Bifidobacterium with excellent lactosidase activity were selected and tested for β-galactosidase activity using the same method to further evaluate the ability of the VB316 strain obtained in Example 1 to alleviate lactose intolerance. The commercial strains NCFM were Lactobacillus acidophilus (DuPont, USA), BB-12 were Bifidobacterium animalis subsp. lactis (Chr. Hansen, Denmark), CECT5716 were Lactobacillus fermentum (Bieser, Spain), and LGG were Lactobacillus rhamnosus (Virio, Finland). The results are shown in Table 1 and Figure 3.

[0133] Table 1 β-galactosidase activity assay results of VB316 strain and control strain

[0134] The above results show that the β-galactosidase activity of the VB316 strain of Example 1 is 3.730 U / mL, which is higher than that of commercial strains, specifically 14.2 times that of the American DuPont Lactobacillus acidophilus NCFM, 10.8 times that of the Spanish Baishi fermentation Lactobacillus CECT5716, and 5.1 times that of the Danish Chr. Hansen Bifidobacterium animalis subspecies lactis BB-12.

[0135] Example 4: Investigation of the antibacterial ability of Bifidobacterium breve VB316 strain

[0136] In this embodiment, the double-layer plate culture method was used to evaluate the antibacterial ability of the VB316 strain of the present invention. Specifically, whether there was an inhibition zone around the single colony in the upper layer and the size of the inhibition zone were used to determine whether the strain of the single colony had the activity of inhibiting indicator bacteria.

[0137] The specific method of the double-layer plate culture method is as follows: the lower culture medium is BBL solid medium, 2 μl of the bacterial suspension of VB316 strain and water are taken and spotted on the BBL solid medium. After anaerobic culture at 37°C for 1 day, mature single colonies are formed. The upper layer is poured with a culture medium containing indicator bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli 8099, Salmonella Paratyphi B CMCC50094, Listeria monocytogenes ATCC19114, Shigella dysenteriae CMCC51252, Clostridium perfringens ATCC 13124), 7-10 mL / plate, and the final concentration of indicator bacteria is 10 6 After solidification, the cells were incubated under appropriate growth conditions for each indicator bacterium for 16–18 h. The presence and size of inhibition zones around individual VB316 colonies were observed and recorded to determine whether the colonies had activity against the indicator bacterium. The results are shown in Table 2.

[0138] BBL solid medium is: peptone 15%, glucose 20%, yeast extract powder 2%, soluble starch 0.5%, sodium chloride 5%, L-cysteine ​​0.5%, tomato extract powder 5%, liver extract powder 2%, Tween 80 1%, 2% agar, pH 7.0, sterilized at 115℃ for 30 minutes.

[0139] Table 2 Evaluation of the antibacterial ability of VB316 strain against six pathogenic bacteria

[0140] The above results show that the VB316 strain of Example 1 has an antibacterial effect on six pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, and Capsula perfringens.

[0141] Example 5: Investigation of gastric acid resistance of Bifidobacterium breve VB316 strain

[0142] This example investigates the tolerance of the VB316 strain to different acidic conditions.

[0143] The test strains were treated at pH 1.0, 2.0, 3.0, and 4.0 for 2 hours, and their survival rates were determined by dilution and plating. The blank control was the VB316 strain treated with sterile water for the same period of time.

[0144] The calculation formula of gastric acid resistance survival rate of the test strain is as follows:

[0145] Among them, cfu represents colony forming unit, the number of viable bacteria in the blank control is represented by N0, and the number of viable bacteria in the test strain is represented by N.

[0146] The results are shown in Table 3. It can be seen that the VB316 strain of Example 1 had a survival rate of 80% when treated at pH 1.0 for 2 hours, and a survival rate greater than 80% when treated at pH 2.0-4.0 for 2 hours.

[0147] The above results show that the VB316 strain of the present invention has good gastric acid resistance.

[0148] Table 3 Gastric acid resistance test data of VB316 strain

[0149] Example 6: Bile Salt Resistance Test of Bifidobacterium breve VB316 Strain

[0150] This example investigates the tolerance of the VB316 strain to different bile salt concentrations.

[0151] The bile salt concentrations were 0.03%, 0.06%, 0.1%, 0.2% and 0.3% respectively, and the treatment time was 3 hours. The survival rate was detected by dilution coating. The blank control was the VB316 strain treated with sterile water (deionized water sterilized at 121℃ for 30 minutes) for the same time.

[0152] The formula for calculating the bile salt tolerance survival rate of the test strains is as follows:

[0153] Among them, cfu represents colony forming unit, the number of viable bacteria in the blank control is represented by N0, and the number of viable bacteria measured under different bile salt concentration treatment conditions is represented by N.

[0154] The results are shown in Table 4. It can be seen that the VB316 strain of Example 1 had little effect on its survival rate when treated at a bile salt concentration of 0.03%-0.3% for 3 hours, especially when treated at a bile salt concentration of 0.3% for 3 hours, the survival rate of the VB316 strain was still as high as 88.3%.

[0155] The above results show that the VB316 strain of the present invention has a certain bile salt resistance.

[0156] Table 4 Bile salt test data of VB316 strain

[0157] Example 8: Study on the intestinal fluid stability of Bifidobacterium breve VB316 strain

[0158] This example investigates the stability of the VB316 strain under simulated intestinal fluid conditions.

[0159] The specific protocol is as follows: Thaw the strain collection tube stored at -80°C on ice, take 10 μL of the sample and add it to 990 μL of sterile intestinal fluid, mix thoroughly, and then apply the sample to the plate for counting at 0 h, 2 h, and 4 h. For the blank control sample, take 10 μL of the sample and add it to 990 μL of sterile water (deionized water sterilized at 121°C for 30 min), mix thoroughly, and then apply the sample to the plate for counting at 0 h.

[0160] The calculation formula for the survival rate of the test strain in the simulated intestinal fluid test is as follows:

[0161] Among them, cfu represents colony forming unit, the number of viable bacteria in the blank control is represented by N0, and the number of viable bacteria measured under simulated intestinal fluid conditions is represented by N.

[0162] The results are shown in Table 5. It can be seen that the VB316 strain of Example 1 has strong survival ability in the simulated intestinal fluid, with a survival rate of 91.2% after 4 hours of treatment with the simulated intestinal fluid. These results demonstrate that the VB316 strain of the present invention has strong intestinal fluid stability and can maintain high activity after passing through the digestive tract.

[0163] Table 5 VB316 strain simulated intestinal fluid test data

[0164] In summary, the VB316 strain of the present invention has strong resistance to acid and bile salts and gastrointestinal fluid, and can maintain high activity after passing through the digestive tract.

[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 Bifidobacterium breve VB316, characterized in that The preservation number of the Bifidobacterium breve is CGMCC No.28160.

2. A fermentation product, characterized in that It comprises the Bifidobacterium breve VB316 described in claim 1.

3. The fermentation product according to claim 2, characterized in that Further including: The metabolite of Bifidobacterium breve VB316 according to claim 1.

4. A microbial agent, characterized in that: The method comprises at least one of the Bifidobacterium breve VB316 according to claim 1 and the fermentation product according to claim 2 or 3.

5. A food, medicine, health product or feed, characterized in that: It comprises at least one of the Bifidobacterium breve VB316 according to claim 1, the fermentation product according to claim 2 or 3, or the microbial agent according to claim 4.

6. The food, medicine, health product or feed according to claim 5, characterized in that: It further includes excipients or carriers acceptable in pharmacy, food, and health care products.

7. Use of the Bifidobacterium breve VB316 according to claim 1, the fermentation product according to claim 2 or 3, or the microbial agent according to claim 4 in the preparation of food or health products, wherein the food or health products are used to alleviate lactose intolerance and / or regulate intestinal flora.

8. Use of the Bifidobacterium breve VB316 according to claim 1, the fermentation product according to claim 2 or 3, or the microbial agent according to claim 4 in the preparation of medicine or feed, wherein the medicine or feed has at least one of the following uses: Prevent and / or relieve lactose intolerance; Regulate intestinal flora; Prevent and / or treat intestinal diseases caused by pathogenic bacteria infection.

9. The use according to claim 8, characterized in that The pathogenic bacteria is selected from at least one of Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsula perfringens.

10. A method for inhibiting the growth of pathogenic bacteria in vitro, characterized in that: include: At least one of the Bifidobacterium breve VB316 according to claim 1, the fermentation product according to claim 2 or 3, and the microbial agent according to claim 4 is co-cultured with a sample containing pathogenic bacteria.

11. The method according to claim 10, characterized in that The pathogenic bacteria is selected from at least one of Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsula perfringens.

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