Lactobacillus reuteri VB319, and culture apparatus and use therefor

Abstract

Provided are a lactobacillus reuteri VB319 and a culture apparatus and use therefor, relating to the field of microorganisms. The preservation number of the lactobacillus reuteri is CGMCC No.28158. The lactobacillus reuteri has the advantages of high lactose degradation rate, strong β-galactosidase activity, strong acid and alkali resistance, and strong cholate resistance etc., has strong antibacterial 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. The application value is high. The lactobacillus reuteri VB319 culture apparatus can be used for batch producing fermentation products of the lactobacillus reuteri VB319, thereby facilitating the development of microbial agents, foods, medicines, health care products or feed production apparatuses. Commercial products can be produced in batches, and the economic value is high.

Description

Lactobacillus reuteri VB319 and its culture device and application Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to Lactobacillus reuteri VB319 and a culture device and application thereof. Background Art

[0002] Lactose is a disaccharide composed of glucose and galactose and is the main source of carbohydrates in the milk of humans and most other mammals. Lactose cannot be directly absorbed by the human body and is mainly hydrolyzed into glucose and galactose by lactase (mainly β-galactosidase) at the tip of the villi on the mucosal surface of the small intestine, especially the jejunum, and then absorbed through active transport by cells. When the human body lacks lactase in the small intestine or the lactase activity is reduced, it will not be able to completely digest and decompose the lactose in breast milk or other dairy products, resulting in adverse reactions such as abdominal pain, diarrhea, and bloating. Current treatment or relief strategies for lactose intolerance include a low-lactose diet, lactase supplementation, and supplementation with probiotics with high β-galactosidase activity or a lactose-degrading phenotype.

[0003] Lactobacillus reuteri is a beneficial bacteria that has been shown to regulate intestinal flora balance and enhance immunity. However, its ability to alleviate lactose intolerance remains unclear. Currently, strains of bacteria that can alleviate lactose intolerance are still under development.

[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 Lactobacillus reuteri VB319 strain and a culture device and application thereof. The present invention also provides a fermentation product, a microbial agent, a food, a medicine, a health product, or a feed, and a fermentation device and application thereof. The new strain of Lactobacillus reuteri screened by the present invention has advantages such as a high lactose degradation rate, strong β-galactosidase activity, strong acid, alkali, and bile salt tolerance. It has strong antibacterial ability against common pathogens and can be used to alleviate lactose intolerance, regulate intestinal flora, and prevent and treat intestinal diseases caused by pathogenic bacteria infection. It has high application value. The Lactobacillus reuteri VB319 culture device of the present invention can mass-produce Lactobacillus reuteri VB319 fermentation products, facilitating the development of microbial agent, food, medicine, health product, or feed production equipment, and mass production of commercial products, thus having high economic value.

[0006] To this end, in a first aspect of the present invention, the present invention provides a Lactobacillus reuteri VB319. According to an embodiment of the present invention, the Lactobacillus reuteri has a deposit number of CGMCC No. 28158.

[0007] Collection information:

[0008] Strain name: Limosilactobacillus reuteri VB319

[0009] Deposit date: August 14, 2023

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

[0011] Deposit number: CGMCC No.28158

[0012] The new strain VB319 of Lactobacillus reuteri obtained by screening in the present invention has the advantages of high lactose degradation rate, strong β-galactosidase activity, strong acid and alkali resistance and bile salt resistance, etc., has strong antibacterial ability against common pathogens, 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 Lactobacillus reuteri.

[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 Lactobacillus reuteri 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 Lactobacillus reuteri, 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 Lactobacillus reuteri, 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 proposes the use of the aforementioned Lactobacillus reuteri, 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: preventing and / or alleviating lactose intolerance; regulating intestinal flora; preventing and / or treating intestinal diseases caused by pathogenic bacteria infection.

[0018] In its seventh aspect, the present invention provides an in vitro method for inhibiting pathogen growth. According to an embodiment of the present invention, the method comprises co-culturing a sample containing pathogens with at least one of the aforementioned Lactobacillus reuteri, the aforementioned fermentation product, and the aforementioned microbial agent. As previously described, the present invention's Lactobacillus reuteri VB319 can effectively inhibit pathogen growth.

[0019] In an eighth aspect of the present invention, the present invention provides a culture device or a production device for the aforementioned Lactobacillus reuteri, the aforementioned fermentation product, the aforementioned microbial agent, or the aforementioned food, medicine, health product, or feed.

[0020] 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

[0021] 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:

[0022] FIG1 is a scanning electron microscopy image of Lactobacillus reuteri VB319 in Example 2 of the present invention;

[0023] FIG2 is a graph showing the HPLC detection results of lactose content in the fermentation broth of Lactobacillus reuteri VB319 in Example 3 of the present invention;

[0024] FIG3 is a standard curve of lactose concentration in Example 3 of the present invention, wherein the abscissa is lactose concentration and the ordinate is the peak area of ​​lactose standards of different concentrations measured by HPLC;

[0025] Figure 4 is a graph showing the 24-hour lactose degradation rate measurement results of Lactobacillus reuteri VB319 and commercial strains in Example 3 of the present invention, wherein VB319 is Lactobacillus reuteri VB319 of the present invention, NCFM is Lactobacillus acidophilus of DuPont, BB-12 is Bifidobacterium animalis subsp. lactis of Chr. Hansen, Denmark, CECT5716 is Lactobacillus fermentum of Spain, and LGG is Lactobacillus rhamnosus of Finland;

[0026] Figure 5 is a standard curve of o-nitrophenol (ONP) concentration in Example 4 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;

[0027] Figure 6 is a graph showing the results of β-galactosidase activity assay of Lactobacillus reuteri VB319 and commercial strains in Example 4 of the present invention, wherein VB319 is Lactobacillus reuteri VB319 of the present invention, NCFM is Lactobacillus acidophilus of DuPont, 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

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In this article, the term "MRS medium" refers to a lactic acid bacteria culture medium, which can be divided into two categories: MRS solid culture medium and MRS liquid culture medium according to the content of the coagulant (usually agarose).

[0032] As used herein, the term "sterile water" refers to water that is free of bacteria by treating water containing bacterial masses through physical methods.

[0033] The present invention provides Lactobacillus reuteri, fermentation products, microbial agents, foods, medicines, health products or feeds and uses thereof, which will be described in detail below.

[0034] strains

[0035] The invention provides Lactobacillus reuteri VB319.

[0036] According to an embodiment of the present invention, the Lactobacillus reuteri is deposited with CGMCC No. 28158. This strain is a new isolate and was deposited on August 14, 2023, at the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGCC No. 28158. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0037] According to an embodiment of the present invention, the 16S rDNA sequencing result of the Lactobacillus reuteri is shown as SEQ ID NO: 1.

[0038] The new strain of Lactobacillus reuteri VB319 screened by the present invention has the advantages of high lactose degradation rate, 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.

[0039] Herein, the terms "Limosilactobacillus reuteri VB319" and "Lactobacillus reuteri VB319" are synonymous.

[0040] fermentation products

[0041] The present invention provides a fermentation product. According to an embodiment of the present invention, the fermentation product includes the aforementioned Lactobacillus reuteri.

[0042] It should be noted that the "fermentation product" of the present invention refers to the solution obtained after culturing Lactobacillus reuteri VB319 for a period of time, which solution mainly contains Lactobacillus reuteri VB319 and its metabolites; or the supernatant after further treatment by means of centrifugation, filtration, etc., which supernatant mainly contains the metabolites of Lactobacillus reuteri VB319; or the bacterial suspension after further treatment by means of centrifugation, resuspension, etc., which bacterial suspension mainly contains Lactobacillus reuteri VB319.

[0043] According to an embodiment of the present invention, the present invention further comprises: a metabolite of the aforementioned Lactobacillus reuteri.

[0044] It should be noted that the characteristics and advantages described above for Lactobacillus reuteri are also applicable to this fermentation product and will not be described in detail here.

[0045] microbial agents

[0046] 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 Lactobacillus reuteri 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.

[0047] 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.

[0048] It should be noted that, in the microbial agent of the present invention, Lactobacillus reuteri may exist in the form of living cells and / or non-living cells.

[0049] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce, or replicate.

[0050] For example, the living cells may 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.

[0051] 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.

[0052] In some specific embodiments, the Lactobacillus reuteri VB319 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.

[0053] In some specific embodiments, the dried bacteria are obtained by freeze-drying the Lactobacillus reuteri VB319.

[0054] In some specific embodiments, the microbial agent may further contain at least one strain acceptable to food, medicine, health care product and feed.

[0055] In some specific embodiments, the 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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 reuteri VB319 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.

[0062] It should be noted that the characteristics and advantages described above for Lactobacillus reuteri are also applicable to this microbial agent and will not be repeated here.

[0063] Food, medicine, health products or feed

[0064] The present invention provides a food, medicine, health product, or feed. According to an embodiment of the present invention, the food, medicine, health product, or feed comprises at least one of the aforementioned Lactobacillus reuteri, 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.

[0065] 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.

[0066] In some specific embodiments, the aforementioned Lactobacillus reuteri, 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 drug that can prevent and / or alleviate pathogenic bacteria infection or intestinal diseases caused by pathogenic bacteria infection.

[0067] Exemplarily, the food includes but is 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.

[0068] For example, the medicines include but are not limited to: medicines for human use and medicines for veterinary use. The medicines for veterinary use may be for pets, livestock, or wild animals.

[0069] Illustratively, the health care products include but are not limited to: health care products for humans and health care products for animals.

[0070] It should be noted that the characteristics and advantages described above for Lactobacillus reuteri are also applicable to the food, medicine, health product or feed, and will not be repeated here.

[0071] use

[0072] The present invention provides the use of the aforementioned Lactobacillus reuteri, 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.

[0073] The present invention also proposes the use of the aforementioned Lactobacillus reuteri, 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: preventing and / or alleviating lactose intolerance; regulating intestinal flora; preventing and / or treating intestinal diseases caused by pathogenic bacteria infection.

[0074] According to an embodiment of the present invention, the pathogenic bacteria is selected from at least one of Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsule perfringens.

[0075] In some specific embodiments, the drug further contains an excipient and / or a carrier.

[0076] In some specific embodiments, 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.

[0077] In some specific embodiments, 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.

[0078] In some specific embodiments, the dosage form of the drug includes at least one selected from oral solution, powder, granule, capsule, tablet, and pill.

[0079] It should be noted that the characteristics and advantages described above for Lactobacillus reuteri are also applicable to this use and will not be repeated here.

[0080] method

[0081] The present invention provides a method for inhibiting pathogen growth in vitro. According to an embodiment of the present invention, the method comprises co-culturing a sample containing pathogens with at least one of the aforementioned Lactobacillus reuteri, the aforementioned fermentation product, and the aforementioned microbial agent. As previously mentioned, Lactobacillus reuteri VB319 can effectively inhibit the growth of pathogens.

[0082] According to an embodiment of the present invention, the pathogenic bacteria is selected from at least one of Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsule perfringens.

[0083] It should be noted that the characteristics and advantages described above for Lactobacillus reuteri are also applicable to this method and will not be repeated here.

[0084] Device

[0085] The present invention provides a culture device or production device for the aforementioned Lactobacillus reuteri, the aforementioned fermentation product, the aforementioned microbial agent, or the aforementioned food, medicine, health product, or feed. The Lactobacillus reuteri VB319 culture device of the present invention can mass-produce Lactobacillus reuteri VB319 fermentation product, facilitating the development of production devices for microbial agents, food, medicine, health product, or feed, and enabling mass production of commercial products with high economic value.

[0086] 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.

[0087] Example 1: Acquisition of Limosilactobacillus reuteri VB319

[0088] The Lactobacillus reuteri VB319 of the present invention is isolated from feces of healthy adults with lactose tolerance.

[0089] Bacterial strain collection and isolation process: Take 0.5g of lactose-tolerant healthy adult feces and add it to 5mL of sterile PBS buffer. After mixing, dilute and spread it on MRS solid culture medium plates. Incubate it in an anaerobic environment at 37℃ for 48h. Pick single colonies of different sizes and morphologies and streak them on fresh MRS plates. Incubate it anaerobically at 37℃ for 48h. Repeat the streak purification culture several times until the colonies in the plate have consistent morphology. Identify them after no other bacteria are found under microscopic examination.

[0090] Strain preservation: The obtained pure culture strains are cultured to a concentration of about 10 9 CFU / mL, take 500 μL of bacterial solution and add 500 μL of 40% glycerol to make the glycerol concentration reach 20%, and then store at -80℃ ultra-low temperature.

[0091] MRS liquid culture medium consists of: 0.5% yeast extract, 2% glucose, 1.0% peptone, 1.0% beef extract, 0.2% dipotassium hydrogen phosphate, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.02% magnesium sulfate, 0.005% manganese sulfate, 0.1% Tween 80, pH 6.2, sterilized at 121°C for 15 min.

[0092] MRS solid culture medium is: 2% agar is added to MRS liquid culture medium.

[0093] Example 2: Identification of Limosilactobacillus reuteri VB319

[0094] 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.

[0095] The 16S rDNA sequencing results were compared by BLAST, and it was finally identified that the isolated strain was a Lactobacillus reuteri, named Limosilactobacillus reuteri VB319, which was deposited in the "General Microbiology Center of China Culture Collection Administration" on August 14, 2023, with the deposit number CGMCC No. 28158.

[0096] In this embodiment, the main bacterial species identification methods and results are as follows:

[0097] Lactobacillus reuteri VB319 was observed under a scanning electron microscope using the following method: First, a rice-sized colony was scraped from a plate and fixed using a double fixation method with glutaraldehyde and osmium. The sample was then dehydrated using a gradient of ethanol solutions (30%, 50%, 70%, 80%, 90%, and 95%) for 15 minutes at each concentration, followed by two 20-minute treatments with 100% ethanol. The dehydrated sample was then dried in a Hitachi HCP-2 critical point dryer and subsequently plated. Finally, the treated sample was observed using a Hitachi SU-8010 scanning electron microscope. Microscopic images are shown in Figure 1.

[0098] Scanning electron microscopy and Gram staining test results showed that Lactobacillus reuteri VB319 was Gram-positive, with single colonies being milky white, smooth and round, and the cells being rod-shaped ( Figure 1 ).

[0099] The physiological and biochemical characteristics of Lactobacillus reuteri VB319 were analyzed, and the specific experiments were carried out according to Bergey's Bacterial Identification Manual.

[0100] The results of physical and chemical characteristics identification were: catalase negative, oxidase negative, able to utilize maltose, sucrose, raffinose, glucose, D-ribose, lactose, galactose, mannose, fructose, xylose, trehalose, mesidic biose, turanose, unable to hydrolyze starch and gelatin, VP reaction negative, methyl red negative.

[0101] The 16S rDNA sequencing result of Lactobacillus reuteri VB319 is shown in SEQ ID NO: 1.

[0102] Example 3: Investigation of Lactose Degradation Ability of Limosilactobacillus reuteri VB319

[0103] 3% lactose was added to the MRS basal liquid medium, and the amount of lactose consumed after 24 hours of culture was used as the evaluation standard for the lactose degradation ability of the test strain. The lactose content was quantitatively determined by high performance liquid chromatography (HPLC).

[0104] 1. Preparation of Test Strains

[0105] Thaw strain collection tubes frozen at -80°C on ice. Take one loopful of the suspension and streak it onto an MRS solid medium plate. Incubate in an anaerobic incubator at 37°C for 48 hours. Transfer a single colony to 10 mL of MRS liquid medium and incubate at 37°C for 24 hours. Transfer the suspension to 50 mL of lactose medium in a 250 mL Erlenmeyer flask at a 2% transfer volume and incubate in an anaerobic workstation at 37°C for 24 hours.

[0106] MRS liquid culture medium consists of: 0.5% yeast extract, 2% glucose, 1.0% peptone, 1.0% beef extract, 0.2% dipotassium hydrogen phosphate, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.02% magnesium sulfate, 0.005% manganese sulfate, 0.1% Tween 80, pH 6.2, sterilized at 121°C for 15 min.

[0107] MRS solid culture medium is: 2% agar is added to MRS liquid culture medium.

[0108] Lactose culture medium is: glucose is removed from MRS liquid culture medium and 30g / L lactose is added.

[0109] 2. Drawing of lactose concentration standard curve

[0110] Take 12.275g of lactose in a small beaker, add 100mL of water to dissolve it, and then transfer it to a 250mL volumetric flask. Continue to dilute to 250mL with water to prepare a concentration of 49.1mg / mL. Then, draw 80, 40, 20, 10, and 5mL of the solution into different 100mL volumetric flasks, add water to dilute to 100mL, and shake well to prepare 39.28, 19.64, 9.82, 4.91, and 2.46mg / mL lactose solutions.

[0111] A lactose concentration standard curve was drawn with lactose concentration as the abscissa and the peak area measured by HPLC as the ordinate ( FIG3 ), and a linear regression equation was obtained.

[0112] 3. Determination of lactose degradation rate

[0113] Take 1 mL of the fermentation broth of Lactobacillus reuteri VB319 obtained by culturing in step 1, centrifuge at 14000 rpm for 3 min, take the supernatant, dilute it appropriately, filter it with a 0.22 μm sterile filter membrane, and quantitatively detect the lactose content by HPLC. Based on the measured lactose content, determine the lactose degradation rate according to the following formula.

[0114] The HPLC method for lactose content is as follows: the chromatographic column is Luna 5μm NH2 (5 μm, 150×4.6 mm); flow rate: 1.2 mL / min; column temperature: 30°C; injection volume: 10 μL; mobile phase: acetonitrile: water = 8:2.

[0115] The HPLC test results of lactose content in VB319 fermentation broth are shown in Figure 2.

[0116] As shown in FIG2 , the lactose retention time of Lactobacillus reuteri VB319 was measured to be 10.3 min. Based on the peak area measured by HPLC and the lactose concentration standard curve determined in step 2, its lactose degradation rate was finally determined to be 100%.

[0117] Several commercially available lactose-degrading strains of Lactobacillus or Bifidobacterium were selected, and their lactose degradation rates were measured using the same method to further evaluate the lactose degradation performance of Lactobacillus reuteri VB319 obtained in Example 1. The commercial strains NCFM were Lactobacillus acidophilus (DuPont, USA), BB-12 were Bifidobacterium animalis subsp. lactis (Chr. Hansen, Denmark), CECT5716 were Lactobacillus fermentum (Biesch, Spain), and LGG were Lactobacillus rhamnosus (Virio, Finland). The results are shown in Table 1 and Figure 4.

[0118] Table 1 Lactose degradation rate test results of Lactobacillus reuteri VB319 and commercial strains

[0119] The above results show that the lactose degradation rate of VB319 in Example 1 is 100%, which is generally higher than that of commercial strains; compared with Lactobacillus acidophilus NCFM (lactose degradation rate 65.2%), the lactose degradation rate is increased by 34.8%.

[0120] Example 4: Investigation of β-galactosidase activity of Lactobacillus reuteri VB319

[0121] 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 Lactobacillus reuteri VB319.

[0122] 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.

[0123] The β-galactosidase activity unit (U) was defined as the amount of enzyme required to produce 1 μM ONP per minute at 30°C.

[0124] The detection method is as follows:

[0125] 1. Preparation of Test Strains

[0126] Thaw strain collection tubes stored at -80°C on ice. Take one loopful of the suspension and streak it onto an MRS solid medium plate. Incubate in an anaerobic incubator at 37°C for 48 hours. Transfer a single colony to 10 mL of MRS liquid medium and incubate at 37°C for 24 hours. Using a 2% transfer volume, transfer the suspension to a 250 mL Erlenmeyer flask containing 50 mL of lactose medium. Place the suspension in an anaerobic workstation at 37°C for 24 hours.

[0127] 2. Reagent Preparation

[0128] (1) PBS buffer

[0129] 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.

[0130] (2) 10 mM substrate solution

[0131] 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.

[0132] (3) Stop solution

[0133] Dissolve 10 g of sodium carbonate in water and transfer to a 100 mL volumetric flask to volume.

[0134] (4) Test sample preparation

[0135] 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.

[0136] 3. Plotting of the standard curve of o-nitrophenol (ONP)

[0137] 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.

[0138] 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 5) to obtain a linear regression equation.

[0139] 4. Determination of β-galactosidase activity

[0140] 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.

[0141] 5. Calculation

[0142] The standard curve obtained by calculation and analysis is: Y=2.5986X-0.0006, R 2 =0.9995

[0143] Wherein, Y is the light absorption value at 420 nm, and X is the concentration of ONP (μmol).

[0144] Enzyme activity (U / mL):

[0145] Where: OD 420is 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.

[0146] 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 Lactobacillus reuteri VB319 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 2 and Figure 6.

[0147] Table 2 β-galactosidase activity assay results of Lactobacillus reuteri VB319 and control strains

[0148] The above results show that the β-galactosidase activity of Lactobacillus reuteri VB319 in Example 1 is 0.940 U / mL, which is higher than that of commercial strains; compared with the animal Bifidobacterium lactis subspecies BB-12 of Chr. Hansen of Denmark, it is increased by 29%, compared with the Lactobacillus acidophilus NCFM of DuPont of the United States, it is increased by 259%, and compared with the Lactobacillus fermentum CECT5716 of B.S. of Spain, it is increased by 172%.

[0149] Example 5: Investigation of the antibacterial ability of Lactobacillus reuteri VB319

[0150] In this example, a double-layer plate culture method was used to evaluate the antibacterial ability of Lactobacillus reuteri VB319 of the present invention. Specifically, whether the strain of the single colony in the upper layer had the activity of inhibiting indicator bacteria was determined by whether there was an inhibition zone around the single colony in the upper layer and the size of the inhibition zone.

[0151] The specific method of the double-layer plate culture method is as follows: the lower culture medium is MRS solid medium, 2 μl of the bacterial suspension of Lactobacillus reuteri VB319 and water are taken and spotted on the MRS solid medium. After anaerobic culture at 37°C for 1 day, mature single colonies are formed. The 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) is poured into the upper layer, 7-10 mL / plate, and the final concentration of indicator bacteria is 10 6After 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 VB319 colonies were observed and recorded to determine whether the colonies had activity against the indicator bacterium. The results are shown in Table 3.

[0152] Table 3 Evaluation of the antibacterial ability of Lactobacillus reuteri VB319 against 6 pathogenic bacteria

[0153] The above results show that Lactobacillus reuteri VB319 of Example 1 has an antibacterial effect on six pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Listeria, Shigella, and Capsula perfringens.

[0154] Example 6: Investigation of gastric acid resistance of Lactobacillus reuteri VB319 This example investigated the tolerance of Lactobacillus reuteri VB319 to a pH 4 simulated gastric acid environment.

[0155] The test strains were treated under acidic conditions of pH 4 for 2 hours, and their survival rates were measured by dilution and plating. The blank control was Lactobacillus reuteri VB319 treated with sterile water (deionized water sterilized at 121°C for 30 minutes) for the same period of time.

[0156] The calculation formula of gastric acid resistance survival rate of the test strain is as follows:

[0157] 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.

[0158] The results are shown in Table 4. It can be seen that the survival rate of Lactobacillus reuteri VB319 of Example 1 was 76.4% after being treated at pH 4 for 2 hours.

[0159] The above results show that the Lactobacillus reuteri VB319 of the present invention has good gastric acid resistance.

[0160] Table 4 Gastric acid resistance test data of Lactobacillus reuteri VB319

[0161] Example 7: Bile salt resistance test of Lactobacillus reuteri VB319

[0162] This example investigates the tolerance of Lactobacillus reuteri VB319 at different bile salt concentrations.

[0163] 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 and coating. The blank control was Lactobacillus reuteri VB319 treated with sterile water (deionized water sterilized at 121℃ for 30 minutes) for the same time.

[0164] The formula for calculating the bile salt-resistant survival rate of the test bacteria is as follows:

[0165] Among them, 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.

[0166] The results are shown in Table 5. It can be seen that the survival rate of Lactobacillus reuteri VB319 in Example 1 was still as high as 98.1% after being treated at a bile salt concentration of 0.3% for 4 hours, and the strain viability was basically unaffected.

[0167] The above results show that the Lactobacillus reuteri VB319 of the present invention has a strong bile salt resistance.

[0168] Table 5 Lactobacillus reuteri VB319 bile salt test data

[0169] Example 8: Study on the intestinal fluid stability of Lactobacillus reuteri VB319

[0170] This example investigates the stability of Lactobacillus reuteri VB319 under simulated intestinal fluid conditions.

[0171] 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.

[0172] The calculation formula for the survival rate of the test bacteria in the simulated intestinal fluid test is as follows:

[0173] Among them, 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.

[0174] The results are shown in Table 6. It can be seen that the Lactobacillus reuteri VB319 from Example 1 showed strong survival in simulated intestinal fluid, with a survival rate of 87.70% even after 4 hours of treatment. These results demonstrate that the Lactobacillus reuteri VB319 of the present invention has strong intestinal fluid stability and maintains high activity even after passing through the digestive tract.

[0175] Table 6 Lactobacillus reuteri VB319 simulated intestinal fluid test data

[0176] In summary, the Lactobacillus reuteri VB319 of the present invention has strong acid and bile salt resistance and gastrointestinal fluid resistance, and can maintain high activity after passing through the digestive tract.

[0177] 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 reuteri VB319, characterized in that The deposit number of the Lactobacillus reuteri is CGMCC No.28158.

2. A fermentation product, characterized in that The invention comprises the Lactobacillus reuteri described in claim 1.

3. The fermentation product according to claim 2, characterized in that Further including: The metabolite of Lactobacillus reuteri according to claim 1.

4. A microbial agent, characterized in that: The method comprises at least one of the Lactobacillus reuteri described in claim 1 and the fermentation product described in claim 2 or 3.

5. A food, medicine, health product or feed, characterized in that: The method comprises at least one of the Lactobacillus reuteri described in claim 1, the fermentation product described in claim 2 or 3, or the microbial agent described in 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 Lactobacillus reuteri of claim 1, the fermentation product of claim 2 or 3, or the microbial agent of 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 Lactobacillus reuteri of claim 1, the fermentation product of claim 2 or 3, or the microbial agent of 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 Lactobacillus reuteri of claim 1, the fermentation product of claim 2 or 3, and the microbial agent of 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.

12. A culture device or production device for the Lactobacillus reuteri of claim 1, the fermentation product of claim 2 or 3, the microbial agent of claim 4, or the food, medicine, health product or feed of claim 5.

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