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WO2025112581A1 - 一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用 - Google Patents

一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用 Download PDF

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Publication number
WO2025112581A1
WO2025112581A1 PCT/CN2024/107503 CN2024107503W WO2025112581A1 WO 2025112581 A1 WO2025112581 A1 WO 2025112581A1 CN 2024107503 W CN2024107503 W CN 2024107503W WO 2025112581 A1 WO2025112581 A1 WO 2025112581A1
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lactobacillus delbrueckii
present
bulgaricus
food
medicine
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French (fr)
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梁天鑫
周洁雯
张光旭
赵新
刘宇
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Hangzhou Vicrobx Biotech Co Ltd
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Hangzhou Vicrobx Biotech Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of microorganisms, and in particular to a Lactobacillus delbrueckii subspecies bulgaricus VB183 and a culture device and application thereof.
  • Lactose is a disaccharide composed of glucose and galactose. It is the main source of carbohydrates in the milk of humans and most other mammals. Lactose cannot be directly absorbed by the human body. It is mainly hydrolyzed into glucose and galactose by lactase (mainly ⁇ -galactosidase) at the tip of the villi on the surface of the small intestine, especially the jejunum mucosa, and then absorbed through active transport of cells.
  • lactase mainly ⁇ -galactosidase
  • lactose intolerance Current strategies for the treatment or relief of lactose intolerance include low-lactose diet, lactase supplementation, and probiotics with high ⁇ -galactosidase activity or lactose-degrading phenotypes.
  • Probiotics can metabolize into the body to produce a variety of enzymes, such as lactase, lipase, protease, peptidase, etc., which help treat lactose intolerance.
  • strains that can alleviate lactose intolerance have yet to be developed.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.
  • the present invention provides a kind of Lactobacillus delbrueckii subspecies bulgaricus VB183 and its culture device and application
  • the present invention also provides fermentation product, microbial agent, food, medicine, health product or feed and its fermentation device and application
  • the new strain of Lactobacillus delbrueckii screened by the present invention has the advantages of high lactose degradation rate, strong ⁇ -galactosidase activity, strong acid and alkali resistance and bile salt resistance, strong antibacterial ability to common pathogens, can be used to alleviate lactose intolerance, regulate intestinal flora, prevent and treat intestinal diseases caused by pathogen infection, high application value, Lactobacillus delbrueckii subspecies bulgaricus VB183 culture device of the present invention can mass produce Lactobacillus delbrueckii subspecies bulgaricus VB183 fermentation product, is
  • the present invention proposes a Lactobacillus delbrueckii subspecies bulgaricus VB183.
  • the preservation number of the Lactobacillus delbrueckii is CGMCC No.28204.
  • the new strain of Lactobacillus delbrueckii subsp. bulgaricus screened by the present invention has the advantages of high lactose degradation rate, strong ⁇ -galactosidase activity, strong acid and alkali resistance and bile salt resistance, etc., has strong antibacterial ability against common pathogens, can be used to resolve lactose intolerance, regulate intestinal flora, prevent and treat intestinal diseases caused by pathogenic bacteria infection, and has high application value.
  • the present invention provides a fermentation product.
  • the fermentation product includes the aforementioned Lactobacillus delbrueckii.
  • the present invention provides a microbial agent.
  • the microbial agent includes at least one of the aforementioned Lactobacillus delbrueckii and the aforementioned fermentation product.
  • the microbial agent of the present invention can be used to alleviate lactose intolerance, regulate intestinal flora, and can also be used to prevent and treat intestinal diseases caused by pathogenic bacteria infection.
  • the present invention provides a food, medicine, health product or feed.
  • the food, medicine, health product or feed comprises at least one of the aforementioned Lactobacillus delbrueckii, 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 infection.
  • the present invention proposes the use of the aforementioned Lactobacillus delbrueckii, 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.
  • the present invention proposes the use of the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product or the aforementioned microbial agent in the preparation of 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.
  • the present invention proposes a method for inhibiting the growth of pathogens in vitro.
  • the method comprises: co-culturing at least one of the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product and the aforementioned microbial agent with a sample containing pathogens.
  • the Lactobacillus delbrueckii subsp. bulgaricus VB183 of the present invention can effectively inhibit the growth of pathogens.
  • the present invention provides a culture device or a production device for the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product, the aforementioned microbial agent, or the aforementioned food, medicine, health product, or feed.
  • Fig. 1 is a scanning electron microscopy result diagram of Lactobacillus delbrueckii VB183 in Example 2 of the present invention
  • Example 2 is a graph showing the HPLC test results of lactose content in the fermentation broth of Lactobacillus delbrueckii VB183 in Example 3 of the present invention
  • 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;
  • Example 4 is a graph showing the results of the 24-hour lactose degradation rate determination of Lactobacillus delbrueckii VB183 and commercial strains in Example 3 of the present invention, wherein VB183 is Lactobacillus delbrueckii subsp. bulgaricus VB183 of the present invention, NCFM is Lactobacillus acidophilus of DuPont, BB-12 is Bifidobacterium animalis subsp. lactis of Chr. Hansen, Denmark, and LGG is Lactobacillus rhamnosus of Vilio of Finland;
  • 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 ONP standard and the ordinate is the absorbance of ONP standard of different concentrations at 405nm;
  • Example 6 is a graph showing the results of ⁇ -galactosidase activity assay of Lactobacillus delbrueckii VB183 and commercial strains in Example 4 of the present invention, wherein VB183 is Lactobacillus delbrueckii subsp. bulgaricus VB183 of the present invention, NCFM is Lactobacillus acidophilus of DuPont, BB-12 is Bifidobacterium animalis subsp. lactis of Chr. Hansen, Denmark, and LGG is Lactobacillus rhamnosus of Villio, Finland.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
  • 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).
  • sterile water refers to water that has been treated by physical methods to be free of bacteria. of water.
  • the present invention provides Lactobacillus delbrueckii subspecies bulgaricus VB183, fermentation products, microbial agents, food, medicine, health products or feed and uses thereof, which will be described in detail below.
  • the invention provides a Lactobacillus delbrueckii subspecies bulgaricus VB183.
  • the Lactobacillus delbrueckii has a preservation number of CGMCC No. 28204.
  • the strain is a brand-new isolate, which has been deposited in the "General Microbiological Center of China Microbiological Culture Collection Administration Committee" on August 21, 2023, with a preservation number of CGCC No. 28204, and a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.
  • the 16S rDNA sequencing result of the Lactobacillus delbrueckii is shown as SEQ ID NO: 1.
  • the new strain of Lactobacillus delbrueckii subsp. bulgaricus VB183 screened by the present invention has the advantages of high lactose degradation rate, strong ⁇ -galactosidase activity, strong acid and alkali resistance and bile salt resistance, etc., has strong antibacterial ability against common pathogens, can be used to resolve lactose intolerance, regulate intestinal flora, prevent and treat intestinal diseases caused by pathogenic bacteria infection, and has high application value.
  • the present invention provides a fermentation product.
  • the fermentation product includes the aforementioned Lactobacillus delbrueckii.
  • the "fermentation product" of the present invention refers to a solution obtained after culturing Lactobacillus delbrueckii VB183 for a period of time, which solution mainly contains Lactobacillus delbrueckii VB183 and its metabolites; or a supernatant further treated by centrifugation, filtration, etc., which supernatant mainly contains the metabolites of Lactobacillus delbrueckii VB183; or a bacterial suspension further treated by centrifugation, resuspension, etc., which bacterial suspension mainly contains Lactobacillus delbrueckii VB183.
  • the present invention further comprises: a metabolite of the aforementioned Lactobacillus delbrueckii.
  • Lactobacillus delbrueckii are also applicable to the fermentation product and will not be described in detail here.
  • the present invention provides a microbial agent.
  • the microbial agent includes at least one of the aforementioned Lactobacillus delbrueckii and the aforementioned fermentation product.
  • the microbial agent of the present invention can be used to alleviate lactose intolerance, regulate intestinal flora, and can also be used to prevent and treat intestinal diseases caused by pathogenic bacteria infection.
  • the microbial agent of the present invention may be a liquid microbial agent, including but not limited to fermentation products, etc.; it may also be a solid microbial agent, including but not limited to freeze-dried powder, etc.
  • Lactobacillus delbrueckii may exist in the form of living cells and/or non-living cells.
  • living cells refer to cells that have the ability to metabolize, reproduce or replicate.
  • the living cells may be immobilized cells.
  • 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.
  • non-living cells refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria.
  • the microbial agent is a freeze-dried powder.
  • the Lactobacillus delbrueckii VB183 exists in the form of living cells, dried bacteria, immobilized cells or any other forms.
  • the dry bacteria are obtained by freeze-drying the Lactobacillus delbrueckii VB183.
  • the microbial agent may also contain at least one strain acceptable to food, medicine, health care product and feed.
  • 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.
  • accepted in food refers to substances or compositions that can be consumed by humans, which may be adjusted according to the food requirements of different countries.
  • accepted 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.
  • 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 mammals treated therewith.
  • pharmaceutically acceptable refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, particularly humans.
  • 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.
  • pharmaceutically acceptable carrier includes any solvent, drug 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 treatment or pharmaceutical compositions is covered.
  • the term "pharmaceutically acceptable excipient” may include any solvent suitable for a specific target dosage form.
  • any conventional excipients incompatible with the Lactobacillus delbrueckii VB183 disclosed in the present invention such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.
  • Lactobacillus delbrueckii are also applicable to this microbial agent and will not be repeated here.
  • the present invention provides a food, medicine, health product or feed.
  • the food, medicine, health product or feed comprises at least one of the aforementioned Lactobacillus delbrueckii, 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 infection.
  • auxiliary materials or carriers acceptable in pharmacy, food, health care products or feed are provided.
  • the aforementioned Lactobacillus delbrueckii, 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 food, health products 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.
  • 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.
  • the medicine includes but is not limited to: medicine for human use and medicine for veterinary use.
  • the medicine for veterinary use can be for pets, livestock, and wild animals.
  • the health care products include but are not limited to: health care products for humans and health care products for animals.
  • Lactobacillus delbrueckii are also applicable to the food, medicine, health care product or feed, and will not be repeated here.
  • the present invention proposes the use of the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product or the aforementioned microbial agent in preparing food or health products, wherein the food or health products are used to alleviate lactose intolerance and/or regulate intestinal flora.
  • the present invention also proposes the use of the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product or the aforementioned microbial agent in the preparation of 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.
  • the pathogenic bacteria is selected from at least one of Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsula perfringens.
  • the medicament further contains an excipient and/or a carrier.
  • 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.
  • 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 hydrolyzed cereal solids.
  • the dosage form of the drug includes at least one selected from oral solution, powder, granule, capsule, tablet, and pill.
  • the present invention provides a method for inhibiting the growth of pathogens in vitro.
  • the method comprises: co-culturing at least one of the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product and the aforementioned microbial agent with a sample containing pathogens.
  • the Lactobacillus delbrueckii VB183 of the present invention can effectively inhibit the growth of pathogens.
  • the pathogenic bacteria is at least one selected from Staphylococcus aureus, Escherichia coli, Salmonella, Listeria, Shigella and Capsula perfringens.
  • Lactobacillus delbrueckii are also applicable to this method and will not be described in detail here.
  • the present invention provides a culture device or production device for the aforementioned Lactobacillus delbrueckii, the aforementioned fermentation product, the aforementioned microbial agent, or the aforementioned food, medicine, health product, or feed.
  • the Lactobacillus delbrueckii subspecies bulgaricus VB183 culture device of the present invention can mass-produce Lactobacillus delbrueckii subspecies bulgaricus VB183 fermentation product, which is beneficial to the development of microbial agent, food, medicine, health product, or feed production device, mass production of commercial products, and high economic value.
  • Example 1 Obtaining Lactobacillus delbrueckii subsp. bulgaricus VB183
  • Lactobacillus delbrueckii subsp. bulgaricus VB183 of the present invention is isolated from the feces of healthy lactose-tolerant adults.
  • strain collection and isolation Take 0.5g of lactose-tolerant healthy adult feces and add it to 5mL sterilized PBS buffer. After mixing, dilute and spread it on MRS solid culture medium plate, and culture it in anaerobic environment at 37°C for 48h. Pick out single colonies of different sizes and shapes and streak them on fresh MRS plates, and culture them anaerobically at 37°C for 48h. Repeat the streak purification culture several times until the colonies in the plate have consistent morphology and are identified after no foreign bacteria are found under microscopic examination.
  • the obtained pure culture strain was cultured to a concentration of about 10 7 CFU/mL, 500 ⁇ L of the bacterial solution was added with 500 ⁇ L of 50% glycerol to make the glycerol concentration reach 25%, and then ultra-low temperature preservation was performed at -80°C.
  • MRS liquid culture medium is: 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.
  • MRS solid culture medium is: 2% agar is added to MRS liquid culture medium.
  • Example 1 After the culture isolated and purified in Example 1 was further confirmed to be a pure culture by streaking and smear microscopy, the bacterial species was identified by experimental means including Gram staining test, contact enzyme and other physiological and biochemical tests and 16S rDNA full sequence sequencing identification.
  • the 16S rDNA sequencing results were compared by BLAST, and finally identified as a strain of Lactobacillus delbrueckii subsp. bulgaricus, named Lactobacillus delbrueckii subsp. bulgaricus VB183, which was deposited in the "General Microbiology Center of China Microbiological Culture Collection Administration" on August 21, 2023, with the deposit number CGMCC No. 28204.
  • Lactobacillus delbrueckii VB183 was observed by scanning electron microscopy.
  • the specific method is as follows: First, a colony the size of a rice grain was scraped from the plate, and the sample was fixed by the double fixation method of glutaraldehyde and osmium acid. The sample was then dehydrated with an ethanol solution of gradient concentrations (including five concentrations of 30%, 50%, 70%, 80%, 90% and 95%), each concentration was treated for 15 minutes, and then treated twice with 100% ethanol, each time for 20 minutes. The dehydrated sample was dried in a Hitachi HCP-2 critical point dryer and then coated. Finally, the treated sample was observed in a Hitachi SU-8010 scanning electron microscope, and the electron microscope photos are shown in Figure 1.
  • Lactobacillus delbrueckii VB183 was Gram-positive, with single colonies being white, smooth and irregular in shape, and the cells being rod-shaped ( Figure 1 ).
  • Lactobacillus delbrueckii VB183 The physiological and biochemical characteristics of Lactobacillus delbrueckii VB183 were analyzed, and the specific experiments were carried out with reference to the Bergey's Bacterial Identification Manual.
  • catalase negative can utilize maltose, sucrose, raffinose, inulin, glucose, lactose, galactose, mannose, fructose, turanose, and raffinose, cannot hydrolyze starch and gelatin, V-P reaction negative, methyl red positive.
  • the 16S rDNA sequencing result of Lactobacillus delbrueckii VB183 is shown in SEQ ID NO:1.
  • 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 detected by high performance liquid chromatography (HPLC).
  • MRS liquid culture medium is: 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.
  • MRS solid culture medium is: 2% agar is added to MRS liquid culture medium.
  • Lactose culture medium glucose is removed from MRS liquid culture medium and 30g/L lactose is added.
  • a lactose concentration standard curve was drawn with lactose concentration as the abscissa and the peak area measured by HPLC as the ordinate ( FIG. 3 ), and a linear regression equation was obtained.
  • the lactose retention time of Lactobacillus delbrueckii VB183 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 83%.
  • lactobacillus or bifidobacterium commercial strains with excellent lactose degradation performance were selected, and the lactose degradation rate of the commercial strains was detected by the same method to further evaluate the lactose degradation performance of Lactobacillus delbrueckii VB183 obtained in Example 1.
  • the commercial strain NCFM is Lactobacillus acidophilus (DuPont, USA)
  • BB-12 is Bifidobacterium animalis subsp. lactis (Chr. Hansen, Denmark)
  • CECT5716 is Lactobacillus fermentum (Bai Shi, Spain)
  • LGG Lactobacillus rhamnosus (Virio, Finland). The measurement results are shown in Figure 4.
  • lactose degradation rate of Lactobacillus delbrueckii VB183 in Example 1 was 83%, which was generally higher than commercial strains; and 28% higher than the commercially available Lactobacillus acidophilus NCFM with the highest lactose degradation rate (lactose degradation rate 65%).
  • the most important enzyme for relieving lactose intolerance is ⁇ -galactosidase, which can break down lactose into glucose and galactose.
  • the ONPG method was used to further determine the ⁇ -galactosidase activity of Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii VB183.
  • ⁇ -galactosidase activity detection is as follows: ⁇ -galactosidase catalyzes the cleavage of the glycosidic bond of colorless o-nitrophenol ⁇ -D-galactopyranoside (ONPG) to produce yellow o-nitrophenol (ONP) and ⁇ -D-pyranose. As ONPG decomposes into ONP, the color of the solution will gradually deepen. ONP has a maximum absorption peak at 420nm, so the yield of ONP can be calculated by the absorbance value, thereby calculating the enzyme activity of ⁇ -galactosidase.
  • the ⁇ -galactosidase activity unit (U) is defined as the amount of enzyme required to produce 1 ⁇ M ONP per minute at 30°C.
  • the detection method is as follows:
  • the substrate solution of o-nitrophenyl- ⁇ -D-galactopyranoside 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.
  • o-nitrophenol o-nitrophenol
  • Each mL of these solutions contains 0.02, 0.04, 0.06, 0.08, and 0.1 ⁇ M ONP, respectively.
  • the absorbance was measured at a wavelength of 405 nm, with water as the control, the concentration of ONP as the abscissa, and the absorbance of the standard substances at various concentrations as the ordinate, to make a standard curve ( Figure 5) and obtain a linear regression equation.
  • Y is the light absorption value at 420 nm
  • X is the concentration of ONP ( ⁇ mol).
  • OD 420 is the absorbance of the sample to be tested at 420nm; b is the intercept of the standard curve, 0.0494; V is the total volume of the reaction system, 1.6mL; f is the dilution factor of the enzyme solution; K is the slope of the standard curve, 2.5986; t is the reaction time, 10min; V1 is the volume of the enzyme solution, 0.2mL.
  • lactobacillus or bifidobacterium commercial strains with excellent lactosidase activity were selected, and the ⁇ -galactosidase activity of the commercial strains was detected by the same method to further evaluate the ability of Lactobacillus delbrueckii VB183 obtained in Example 1 to alleviate lactose intolerance.
  • the commercial strain NCFM is Lactobacillus acidophilus (DuPont, USA)
  • BB-12 is Bifidobacterium animalis subsp. lactis (Chr. Hansen, Denmark)
  • LGG Lactobacillus rhamnosus (Virio, Finland).
  • the measurement results are shown in Table 1 and Figure 6.
  • the double-layer plate culture method is used to evaluate the antibacterial ability of the Lactobacillus delbrueckii VB183 of the present invention. Specifically, whether there is an inhibition zone around the upper single colony and the size of the inhibition zone are used to determine whether the strain of the single colony has the activity of inhibiting indicator bacteria.
  • the specific method of the double-layer plate culture method is as follows: the lower culture medium is MRS culture medium, 2 ⁇ l of the bacterial suspension of Lactobacillus delbrueckii VB183 and water are taken, and inoculated on the MRS solid culture medium. After anaerobic culture at 37°C for 1 day, a mature single colony is 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/dish, and the final concentration of indicator bacteria is 10 6 CFU/mL. After solidification, it is placed under the growth conditions suitable for each indicator bacteria and cultured for 16-18 hours respectively. Observe and record whether there is an inhibition zone around the VB183 single colony and the size of the inhibition zone to determine whether it has the activity of inhibiting the indicator bacteria. The results of the investigation are shown in Table 2.
  • indicator bacteria Staphylococcus aureus ATCC 6538, Escherichia coli 8099, Salmonella paratyphi B CMCC50094, Listeria monocytogenes ATCC19114, Shigella dysenteriae
  • Lactobacillus delbrueckii VB183 of Example 1 is effective against Escherichia coli, Staphylococcus aureus, Salmonella, It has antibacterial effects on six pathogenic bacteria, including Listeria, Shigella, and Clostridium perfringens.
  • test bacteria were treated under pH 4 acidic conditions for 2 and 4 hours, and their survival rates were detected by dilution and coating.
  • the blank control was Lactobacillus delbrueckii VB183 treated with sterile water (deionized water sterilized at 121°C for 30 minutes) for the same period of time.
  • the number of viable bacteria in the blank control is represented by N0
  • the number of viable bacteria in the test strain is represented by N.
  • Example 7 Anti-bile salt test of Lactobacillus delbrueckii subsp. bulgaricus VB183
  • 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 Lactobacillus delbrueckii VB183 treated with sterile water (deionized water 121°C sterilized for 30 minutes) for the same time.
  • the number of live bacteria in the blank control is represented by N0
  • the number of live bacteria measured under different bile salt concentrations is represented by N.
  • Example 8 Study on the intestinal fluid stability of Lactobacillus delbrueckii subsp. bulgaricus VB183
  • the specific scheme is as follows: thaw the strain storage tube frozen at -80°C on ice, take 10 ⁇ L to 990 ⁇ L sterile intestinal fluid, mix well and apply the count at 0h, 2h and 4h.
  • the blank control sample is 10 ⁇ L to 990 ⁇ L sterile water (deionized water sterilized at 121°C for 30min), mix well and apply the count at 0h.
  • the survival rate calculation formula of the test bacteria in the simulated intestinal fluid test is as follows:
  • the number of live bacteria in the blank control is represented by N0
  • the number of live bacteria measured under simulated intestinal fluid conditions is represented by N.
  • Lactobacillus delbrueckii VB183 of the present invention has strong intestinal fluid stability and can maintain high activity after passing through the digestive tract.
  • the Lactobacillus delbrueckii VB183 of the present invention has strong acid and bile resistance and gastrointestinal fluid resistance. It can maintain high activity after passing through the digestive tract.

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Abstract

提供一种德氏乳杆菌保加利亚亚种VB183及其培养装置和应用，涉及微生物领域。所述德氏乳杆菌的保藏号为CGMCC No.28204。本发明的德氏乳杆菌具有乳糖降解率高、β-半乳糖苷酶活力强、耐酸碱和耐胆盐能力强等优点，对常见病原菌有强抑菌能力，可用于缓解乳糖不耐受、调节肠道菌群、预防和治疗病原菌感染引起的肠道疾病，应用价值高，提供的德氏乳杆菌保加利亚亚种VB183培养装置可以批量生产德氏乳杆菌保加利亚亚种VB183发酵产物，利于开发微生物菌剂、食品、药品、保健品或饲料生产装置，批量生产商业化产品，经济价值高。

Description

一种德氏乳杆菌保加利亚亚种VB183及其培养装置和应用 技术领域
本发明涉及微生物领域，尤其是涉及一种德氏乳杆菌保加利亚亚种VB183及其培养装置和应用。
背景技术
乳糖是由葡萄糖和半乳糖组成的双糖，是人类以及其他大多数哺乳动物乳汁中碳水化合物的主要来源。乳糖无法直接被人体吸收，主要是由小肠尤其是空肠黏膜表面绒毛的顶端乳糖酶(主要为β-半乳糖苷酶)水解成葡萄糖和半乳糖，然后通过细胞的主动运输从而被吸收。人体在小肠中缺少乳糖酶或乳糖酶酶活降低的情况下，会导致不能完全消化分解母乳或其他乳制品中的乳糖，从而产生腹痛、腹泻、腹胀等不良反应。
目前乳糖不耐受的治疗或缓解策略包括低乳糖饮食、补充乳糖酶、补充具有高β-半乳糖糖苷酶活性或有降解乳糖表型的益生菌等。益生菌进入体内可代谢产生多种酶，如乳糖酶、脂肪酶、蛋白酶、肤酶等等，有助于治疗乳糖不耐受症。然而，目前能够缓解乳糖不耐受的菌种仍有待开发。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提供了一种德氏乳杆菌保加利亚亚种VB183及其培养装置和应用，本发明还提供了发酵产物、微生物菌剂、食品、药品、保健品或饲料及其发酵装置和应用，本发明筛选得到的德氏乳杆菌新菌株具有乳糖降解率高、β-半乳糖苷酶活力强、耐酸碱和耐胆盐能力强等优点，对常见病原菌有强抑菌能力，可用于缓解乳糖不耐受、调节肠道菌群、预防和治疗病原菌感染引起的肠道疾病，应用价值高，本发明的德氏乳杆菌保加利亚亚种VB183培养装置可以批量生产德氏乳杆菌保加利亚亚种VB183发酵产物，利于开发微生物菌剂、食品、药品、保健品或饲料生产装置，批量生产商业化产品，经济价值高。
为此，在本发明的第一方面，本发明提出了一种德氏乳杆菌保加利亚亚种VB183。根据本发明的实施例，所述德氏乳杆菌的保藏号为CGMCC No.28204。
保藏信息：
菌株名称：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183
保藏日期：2023年08月21日
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心
保藏编号：CGMCC No.28204
本发明筛选得到的德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)新菌株具有乳糖降解率高、β-半乳糖苷酶活力强、耐酸碱和耐胆盐能力强等优点，对常见病原菌有强抑菌能力，可用于解乳糖不耐受、调节肠道菌群、预防和治疗病原菌感染引起的肠道疾病，应用价值高。
在本发明的第二方面，本发明提出了一种发酵产物。根据本发明的实施例，所述发酵产物包括前述德氏乳杆菌。
在本发明的第三方面，本发明提出了一种微生物菌剂。根据本发明的实施例，所述微生物菌剂包括前述德氏乳杆菌、前述发酵产物中的至少之一。本发明的微生物菌剂可用于缓解乳糖不耐受、调节肠道菌群，还可以用于预防和治疗病原菌感染引起的肠道疾病。
在本发明的第四方面，本发明提出了一种食品、药品、保健品或饲料。根据本发明的实施例，所述食品、药品、保健品或饲料包括前述德氏乳杆菌、前述发酵产物或前述微生物菌剂中的至少一种。本发明的食品、保健品或饲料可用于缓解乳糖不耐受、调节肠道菌群；本发明的药品可用于预防和/或缓解乳糖不耐受，预防和/或治疗病原菌感染引起的肠道疾病。
在本发明的第五方面，本发明提出了前述德氏乳杆菌、前述发酵产物或前述微生物菌剂在制备食品或保健品中的用途，所述食品或保健品用于缓解乳糖不耐受和/或调节肠道菌群。
在本发明的第六方面，本发明提出了前述德氏乳杆菌、前述发酵产物或前述微生物菌剂在制备药物或饲料中的用途，所述药物或饲料具有下列至少一种用途：预防和/或缓解乳糖不耐受；调节肠道菌群；预防和/或治疗病原菌感染引起的肠道疾病。
在本发明的第七方面，本发明提出了一种体外抑制病原菌生长的方法。根据本发明的实施例，所述方法包括：将前述德氏乳杆菌、前述发酵产物和前述微生物菌剂中的至少一种与含有病原菌的样本共培养。如前所述，本发明德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183可以有效抑制病原菌生长。
在本发明的第八方面，本发明提出了前述德氏乳杆菌、前述发酵产物、前述微生物菌剂或前述食品、药品、保健品或饲料的培养装置或生产装置。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本发明实施例2中德氏乳杆菌VB183的扫描电镜结果图；
图2为本发明实施例3中德氏乳杆菌VB183发酵菌液的乳糖含量HPLC检测结果图；
图3为本发明实施例3中乳糖浓度标准曲线图，其中，横坐标为乳糖浓度，纵坐标为HPLC测得的不同浓度乳糖标准品的峰面积；
图4为本发明实施例3中德氏乳杆菌VB183与商品菌株的24小时乳糖降解率测定结果图，其中，VB183为本发明德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183，NCFM为美国杜邦嗜酸乳杆菌，BB-12为丹麦科汉森动物双歧杆菌乳亚种，LGG为芬兰维利奥鼠李糖乳杆菌；
图5为本发明实施例4中邻硝基酚(ONP)浓度标准曲线图，其中，横坐标为ONP标准品浓度，纵坐标为405nm条件下不同浓度ONP标准品吸光度；
图6为本发明实施例4中德氏乳杆菌VB183与商品菌株的β-半乳糖苷酶活力测定结果图，其中，VB183为本发明德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183，NCFM为美国杜邦嗜酸乳杆菌，BB-12为丹麦科汉森动物双歧杆菌乳亚种，LGG为芬兰维利奥鼠李糖乳杆菌。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
在本文中，术语“MRS培养基”为乳酸细菌培养基，依据凝固剂(通常是琼脂糖)的含量高低，可分为MRS固体培养基和MRS液体培养基两类。
在本文中，术语“无菌水”为通过物理方法对含有菌团的水进行处理而得到不含细菌 的水。
本发明提出了德氏乳杆菌保加利亚亚种VB183、发酵产物、微生物菌剂、食品、药品、保健品或饲料及其用途，下面将分别对其进行详细描述。
菌种
本发明提出了一种德氏乳杆菌保加利亚亚种VB183。
根据本发明的实施例，所述德氏乳杆菌的保藏号为CGMCC No.28204。该菌种是一株全新的分离株，已于2023年08月21日保藏在“中国微生物菌种保藏管理委员会普通微生物中心”，保藏号为CGCC No.28204，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101。
根据本发明的实施例，所述德氏乳杆菌的16S rDNA测序结果如SEQ ID NO:1所示。
本发明筛选得到的德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183新菌株具有乳糖降解率高、β-半乳糖苷酶活力强、耐酸碱和耐胆盐能力强等优点，对常见病原菌有强抑菌能力，可用于解乳糖不耐受、调节肠道菌群、预防和治疗病原菌感染引起的肠道疾病，应用价值高。
在本文中，术语“德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183菌株”、“德氏乳杆菌保加利亚亚种VB183”和“德氏乳杆菌VB183”同义。
发酵产物
本发明提出了一种发酵产物。根据本发明的实施例，所述发酵产物包括前述德氏乳杆菌。
需要说明的是，本发明的“发酵产物”是指将德氏乳杆菌VB183培养一段时间后得到的溶液，该溶液主要包含德氏乳杆菌VB183及其代谢产物；或进一步经离心、过滤等手段处理后的上清液，该上清液主要包含德氏乳杆菌VB183的代谢产物；或进一步经离心、重悬等手段处理后的菌悬液，该菌悬液主要包含德氏乳杆菌VB183。
根据本发明的实施例，进一步包括：前述德氏乳杆菌的代谢产物。
需要说明的是，前面针对德氏乳杆菌所描述的特征和优点，同样适用于该发酵产物，在此不再赘述。
微生物菌剂
本发明提出了一种微生物菌剂。根据本发明的实施例，所述微生物菌剂包括前述德氏乳杆菌、前述发酵产物中的至少之一。本发明的微生物菌剂可用于缓解乳糖不耐受、调节肠道菌群，还可以用于预防和治疗病原菌感染引起的肠道疾病。
需要说明的是，本发明的微生物菌剂可为微生物液体菌剂，包括但不限于发酵产物等；也可为微生物固体菌剂，包括但不限于冻干粉等。
需要说明的是，本发明的微生物菌剂中，德氏乳杆菌可以以活细胞和/或非活细胞的形式存在。
在本文中，“活细胞”是指具有新陈代谢、繁殖或复制能力的细胞。
示例性地，所述活细胞可为固定化的细胞。本文中，“固定化的细胞”是指将活细胞固定在载体上，可在一定的空间范围进行生长、发育、繁殖、遗传和新陈代谢等生命活动。
在本文中，“非活细胞”是指不具有新陈代谢、繁殖和复制能力的细胞，包括但不限于干菌体。示例性地，所述微生物菌剂为冻干粉。
在一些具体的实施方式中，所述德氏乳杆菌VB183是以活细胞、干菌体、固定化的细胞或者以其他任何形式存在的。
在一些具体的实施方式中，所述干菌体是所述德氏乳杆菌VB183经冷冻干燥处理获得的。
在一些具体的实施方式中，所述微生物菌剂中还可以含有食品、药品、保健品和饲料中的至少之一可接受的菌株。
在一些具体的实施方式中，所述微生物菌剂进一步包括药学上可接受的辅料或载体、食品或保健品中可接受的辅料或载体或者动物饲料中可接受的辅料或载体。
在本文中，“食品中可接受的”是指可被人类食用的物质或组合物，其可根据不同国家的食品要求进行调整。
在本文中，“保健品中可接受的”是指可被人类食用的物质或组合物，其可根据不同国家的保健品要求进行调整。
在本文中，“药学上可接受的”是指物质或组合物必须与包含制剂的其它成分和/或用其治疗的哺乳动物化学上和/或毒理学上相容。优选地，本发明所述的“药学上可接受的”是指联邦监管机构或国家政府批准的或美国药典或其他一般认可药典上列举的在动物中、特别是人体中使用的。
在本文中，“动物饲料中可接受的辅料或载体”是指可被动物食用的物质或组合物，其可根据不同国家的动物饲料要求进行调整。
在本文中，术语“药学上可接受的载体”包括任何溶剂，药物稳定剂，或其组合物，这些载体都是所属技术领域技术人员已知的。除了任意常规载体与活性成分不相容的情况外，涵盖其在治疗或药物组合物中的用途。
在本文中，术语“药学上可接受的辅料”均可包括任何溶剂，适合于特有的目标剂型。除了任何常规的辅料与本发明公开的德氏乳杆菌VB183不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本公开所考虑的范围。
需要说明的是，前面针对德氏乳杆菌所描述的特征和优点，同样适用于该微生物菌剂，在此不再赘述。
食品、药品、保健品或饲料
本发明提出了一种食品、药品、保健品或饲料。根据本发明的实施例，所述食品、药品、保健品或饲料包括前述德氏乳杆菌、前述发酵产物或前述微生物菌剂中的至少一种。本发明的食品、保健品或饲料可用于缓解乳糖不耐受、调节肠道菌群；本发明的药品可用于预防和/或缓解乳糖不耐受，预防和/或治疗病原菌感染引起的肠道疾病。
根据本发明的实施例，进一步包括药学、食品、保健品或饲料上可接受的辅料或载体。
在一些具体的实施方式中，将前述的德氏乳杆菌、前述发酵产物或前述微生物菌剂添加或接种到食品、保健品或饲料中，或添加至药物组合物中，由此，进一步得到具有缓解乳糖不耐受和/或调节肠道菌群功能的食品、保健品或饲料，或得到能预防和/或缓解病原菌感染或病原菌感染引起的肠道疾病的药品。
示例性地，所述食品包括不限于：益生菌片、发酵乳制品(比如益生菌酸奶)、益生菌固体饮料、益生菌乳粉、益生菌奶酪、益生菌豆制品、益生菌糖果、益生菌发酵蔬菜等。
示例性地，所述药品包括不限于：人用药、兽用药。所述兽用可以为宠物用、家畜用、野生动物用。
示例性地，所述保健品包括不限于：人用保健品、兽用保健品。
需要说明的是，前面针对德氏乳杆菌所描述的特征和优点，同样适用于该食品、药品、保健品或饲料，在此不再赘述。
用途
本发明提出了前述德氏乳杆菌、前述发酵产物或前述微生物菌剂在制备食品或保健品中的用途，所述食品或保健品用于缓解乳糖不耐受和/或调节肠道菌群。
本发明还提出了前述德氏乳杆菌、前述发酵产物或前述微生物菌剂在制备药物或饲料中的用途，所述药物或饲料具有下列至少一种用途：预防和/或缓解乳糖不耐受；调节肠道菌群；预防和/或治疗病原菌感染引起的肠道疾病。
根据本发明的实施例，所述病原菌选自金黄色葡萄球菌、大肠杆菌、沙门氏菌、李斯特菌、志贺氏菌和产气荚膜菌中的至少之一。
在一些具体的实施方式中，所述药物进一步含有赋形剂和/或载体。
在一些具体的实施方式中，所述赋形剂包括选自粘合剂、崩解剂、润滑剂、助流剂、稳定剂、填充剂、稀释剂、缓释剂中的至少之一。
在一些具体的实施方式中，所述载体包括选自糖类、纤维素及其衍生物、磷酸钙类、硬脂酸碱土金属盐、植物油类、非离子表面活性剂、阳离子表面活性剂、负离子表面活性剂、脂肪醇类、谷物水解固形物中的至少之一。
在一些具体的实施方式中，所述药物的剂型包括选自口服液、散剂、颗粒剂、胶囊剂、片剂、滴丸剂中的至少之一。
需要说明的是，前面针对德氏乳杆菌所描述的特征和优点，同样适用于该用途，在此不再赘述。
方法
本发明提出了一种体外抑制病原菌生长的方法。根据本发明的实施例，所述方法包括：将前述德氏乳杆菌、前述发酵产物和前述微生物菌剂中的至少一种与含有病原菌的样本共培养。如前所述，本发明德氏乳杆菌VB183可以有效抑制病原菌生长。
根据本发明的实施例，所述病原菌选自金黄色葡萄球菌、大肠杆菌、沙门氏菌、李斯特菌、志贺氏菌和产气荚膜菌中的至少之一。
需要说明的是，前面针对德氏乳杆菌所描述的特征和优点，同样适用于该方法，在此不再赘述。
装置
本发明提出了前述德氏乳杆菌、前述发酵产物、前述微生物菌剂或前述食品、药品、保健品或饲料的培养装置或生产装置。本发明的德氏乳杆菌保加利亚亚种VB183培养装置可以批量生产德氏乳杆菌保加利亚亚种VB183发酵产物，利于开发微生物菌剂、食品、药品、保健品或饲料生产装置，批量生产商业化产品，经济价值高。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183的获得
本发明德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183是从乳糖耐受的健康成人粪便中分离获得的。
菌种采集及分离过程：取0.5g乳糖耐受的健康成人粪便加入到5mL灭菌的PBS缓冲液，混匀后稀释涂布于MRS固体培养基平板上，于37℃厌氧环境中培养48h，分别挑取大小形态不一的单菌落在新鲜的MRS平板上划线，在37℃厌氧培养48h，重复进行多次划线纯化培养，直至平皿中的菌落形态一致，经镜检无杂菌后进行鉴定。
菌种保藏：对获得的纯培养菌株进行培养，至浓度约为107CFU/mL，取500μL菌液添加50％甘油500μL，使其甘油浓度达到25％，然后进行-80℃超低温保藏。
MRS液体培养基为：酵母提取物0.5％、葡萄糖2％、蛋白胨1.0％、牛肉膏1.0％、磷酸氢二钾0.2％、柠檬酸氢二铵0.2％、乙酸钠0.5％、硫酸镁0.02％、硫酸锰0.005％、吐温80 0.1％，pH6.2，121℃灭菌15min。
MRS固体培养基为：MRS液体培养基中加入2％琼脂。
实施例2：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183的鉴定
将实施例1中分离纯化后的培养物通过划线、涂片镜检，进一步确定其为纯培养物后，采用包括革兰氏染色试验、接触酶等生理生化试验及16S rDNA全序列测序鉴定在内的实验手段进行菌种鉴定。
将16S rDNA测序结果进行BLAST比较，最终鉴定确定：分离得到的菌株为一株德氏乳杆菌保加利亚亚种菌株，命名为德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183，已于2023年08月21日保藏在“中国微生物菌种保藏管理委员会普通微生物中心”，保藏号为CGMCC No.28204。
该实施例中，主要菌种鉴定手段及结果如下：
将德氏乳杆菌VB183进行扫描电镜观察，具体方法如下：首先在平板中刮取米粒大小的菌落，采用戊二醛和锇酸双固定法固定样品。随后用梯度浓度(包括30％，50％，70％，80％，90％和95％五种浓度)的乙醇溶液对样品进行脱水处理，每种浓度处理15min，再用100％的乙醇处理两次，每次20min。脱水后的样品在Hitachi HCP-2型临界点干燥仪中进行干燥，随后进行镀膜。最后将处理好的样品在Hitachi SU-8010型扫描电镜中进行观察，电镜拍摄照片见图1。
扫描电镜及革兰氏染色试验结果显示：德氏乳杆菌VB183为革兰氏阳性，单菌落白色，光滑不规则状，细胞呈杆状(图1)。
对德氏乳杆菌VB183进行生理生化特征分析，具体实验参照伯杰氏细菌鉴定手册进行。
理化特征鉴定结果为：接触酶阴性，氧化酶阴性，可利用麦芽糖、蔗糖、棉子糖、菊糖、葡萄糖、乳糖、半乳糖、甘露糖、果糖、松二糖、棉子糖，不能水解淀粉和明胶，V-P反应阴性，甲基红阳性。
德氏乳杆菌VB183的16S rDNA测序结果如SEQ ID NO:1所示。
实施例3：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183 乳糖降解能力考察
在MRS基础液体培养基中添加3％乳糖，以培养24h后消耗的乳糖量作为试验菌株乳糖降解能力的评估标准。乳糖含量用高效液相色谱(HPLC)定量检测。
1.试验菌株准备
将冻存在﹣80℃的菌株保藏管置于冰上解冻，取1环菌液于MRS固体培养基平板进行划线，于37℃厌氧培养箱中静置培养48h。将单菌落转接至装有10mL的MRS液体培养基中，37℃厌氧环境静置培养24h。按照2％转接量，将菌悬液转接至装有50mL的乳糖培养基的250mL三角瓶中，37℃厌氧工作站，静置培养24h。
MRS液体培养基为：酵母提取物0.5％、葡萄糖2％、蛋白胨1.0％、牛肉膏1.0％、磷酸氢二钾0.2％、柠檬酸氢二铵0.2％、乙酸钠0.5％、硫酸镁0.02％、硫酸锰0.005％、吐温80 0.1％，pH6.2，121℃灭菌15min。
MRS固体培养基为：MRS液体培养基中加入2％琼脂。
乳糖培养基为：MRS液体培养基中去除葡萄糖，加入30g/L乳糖。
2.乳糖浓度标准曲线的绘制
取12.275g乳糖于小烧杯中，加入100mL水溶解后转移至250mL容量瓶，继续用水定容至250mL，配制成49.1mg/mL的浓度，随后分别吸取80、40、20、10、5mL溶液至不同100mL容量瓶中，加入水定容至100mL，摇匀后制成39.28、19.64、9.82、4.91、2.46mg/mL的乳糖溶液。
以乳糖浓度为横坐标，HPLC测得的峰面积为纵坐标绘制乳糖浓度标准曲线(图3)，得出线性回归方程。
3.乳糖降解率的测定
取1mL经步骤1培养得到的德氏乳杆菌VB183发酵菌液，14000rpm离心3min，取上清液进行适当稀释，0.22μm无菌滤膜过滤后，通过HPLC定量检测乳糖含量，基于测得的乳糖含量，按如下公式确定乳糖降解率。
乳糖含量HPLC检测方法如下：色谱柱为Luna 5μm NH2(5μm，150×4.6mm)；流速为1.2mL/min；柱温30℃；进样体积为10μL；流动相为乙腈：水＝8：2。
德氏乳杆菌VB183发酵菌液乳糖含量HPLC检测结果见图2。
如图2所示，测得德氏乳杆菌VB183的乳糖保留时间为10.3min，基于HPLC测得的峰面积和步骤2确定的乳糖浓度标准曲线，最终确定其乳糖降解率为83％。
选择部分市售乳糖降解性能优良的乳杆菌或双歧杆菌商品菌株，用同样的方法检测商品菌株的乳糖降解率，进一步评估实施例1获得的德氏乳杆菌VB183的乳糖降解性能。其中，商品菌株NCFM为嗜酸乳杆菌(美国杜邦)，BB-12为动物双歧杆菌乳亚种(丹麦科汉森)，CECT5716为发酵乳杆菌(西班牙百施)，LGG为鼠李糖乳杆菌(芬兰维利奥)。测定结果见图4。
结果显示，实施例1的德氏乳杆菌VB183的乳糖降解率为83％，普遍高于商品菌株；比市售乳糖降解率最高的嗜酸乳杆菌NCFM(乳糖降解率65％)高28％。
实施例4：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183β-半乳糖苷酶活力考察
缓解乳糖不耐受最主要的酶是β-半乳糖苷酶，可将乳糖分解为葡萄糖和半乳糖。本实验采用ONPG法，进一步测定德氏乳杆菌保加利亚亚种菌株(Lactobacillus delbrueckiisubsp.bulgaricus)德氏乳杆菌VB183的β-半乳糖苷酶活力。
β-半乳糖苷酶活力检测原理如下：β-半乳糖苷酶催化无色的邻硝基苯酚β-D-半乳吡喃糖苷(ONPG)发生糖苷键的断裂，产生黄色的邻硝基酚(ONP)和β-D-吡喃半乳糖，随着ONPG分解为ONP，溶液颜色会逐渐加深，ONP在420nm具有最大吸收峰，因此可以通过吸光值来计算ONP的产量，从而计算出β-半乳糖苷酶的酶活。
β-半乳糖苷酶酶活力单位(U)定义为：在30℃下，每分钟产生1μM ONP所需的酶量。
检测方法如下：
1.试验菌株准备
将冻存在-80℃的菌株保藏管置于冰上解冻，取1环菌液于MRS固体培养基平板进行划线，于37℃厌氧培养箱中静置培养48h。将单菌落转接至装有10mL的MRS液体培养基中，37℃厌氧环境静置培养24h。按照2％转接量，将菌悬液转接至装有50mL乳糖培养基的250mL三角瓶中，置于37℃厌氧工作站，静置培养24h。
2.试剂配制
(1)PBS缓冲液
在800mL水中溶解16.1g磷酸氢二钠、5.5g磷酸二氢钠、0.75g氯化钾、0.246g硫酸镁和2.7mL 2-巯基乙醇，加入2mol/L的氢氧化钠溶液，调pH至6.0±0.05。将溶液转移到1L容量瓶，用水定容，混匀。
(2)10mM底物溶液
邻硝基苯-β-D-吡喃半乳糖苷(ONPG)底物溶液为用80mL PBS缓冲液溶解301mg的ONPG，将溶液转入100mL容量瓶，用PBS定容，作为10mM底物溶液。
(3)终止溶液
用水溶解10g碳酸钠后，转入100mL容量瓶定容。
(4)测试样制备
取8mL发酵液于5000rpm，4℃离心10min，弃上清，用等体积PBS缓冲液洗涤2次。用2mL PBS缓冲液重悬菌体，将样品加入研磨珠，在研磨仪中用65Hz，120s的程序进行3次低温研磨，破碎条件为：4℃，研磨时间60s，间隔时间60s。适当稀释粗酶液，使得每mL的最终溶液中含有0.05～0.25单位的β-半乳糖苷酶。
3.邻-硝基苯酚(ONP)标准曲线的绘制
准确称取139mg邻-硝基苯酚(ONP)，放入25mL小烧杯中，加入10mL 95％酒精溶解，随后转入1L容量瓶，用纯水定容。用移液管分别吸取2、4、6、8和10mL的溶液至100mL的容量瓶，用10wt％的碳酸钠溶液定容，混匀。这些溶液中每mL分别含有0.02、0.04、0.06、0.08、0.1μM的ONP。
在波长405nm条件下测吸光度，以水为对照，ONP的浓度为横坐标，各浓度标准物的吸光度为纵坐标，做标准曲线(图5)，得到线性回归方程。
4.β-半乳糖苷酶酶活力的测定
取200μL稀释后的粗酶液于30℃金属浴中预热5min，加入同样在30℃预热5min的底物ONPG浓度溶液1mL，混匀，在30℃金属浴转速400rpm，反应10min，随后立即加入400μL碳酸钠溶液来终止反应，吸取200μL于酶标板中，于30min内测定420nm处的吸光值。
5.计算
经计算分析得到标准曲线：Y＝2.5986X+0.0494，R2＝0.9995
其中，Y为420nm处光吸收值，X为ONP的浓度(μmol)。
酶活力(U/mL)：
式中：OD420为待测样本在420nm处的吸光值；b为标准曲线的截距，0.0494；V为反应体系总体积，1.6mL；f为测定酶液的稀释倍数；K为标准曲线的斜率，2.5986；t为反应时间，10min；V1为酶液的体积，0.2mL。
选择部分市售乳糖苷酶活力优良的乳杆菌或双歧杆菌商品菌株，用同样的方法检测商品菌株的β-半乳糖苷酶活力，进一步评估实施例1获得的德氏乳杆菌VB183缓解乳糖不耐受的能力。其中，商品菌株NCFM为嗜酸乳杆菌(美国杜邦)，BB-12为动物双歧杆菌乳亚种(丹麦科汉森)，LGG为鼠李糖乳杆菌(芬兰维利奥)。测定结果见表1、图6。
表1德氏乳杆菌VB183与对照菌株的β-半乳糖苷酶活力测定结果
上述结果显示，实施例1的德氏乳杆菌VB183的β-半乳糖苷酶活力为2.619U/mL，均高于商品菌株；是丹麦科汉森的动物双歧杆菌乳亚种BB-12的3.6倍，是美国杜邦的嗜酸乳杆菌NCFM的10倍。
实施例5：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183抑菌能力考察
在本实施例中，采用双层板培养法来评估本发明德氏乳杆菌VB183的抑菌能力，具体地，通过上层单菌落周围是否有抑菌圈及抑菌圈大小，判断该单菌落的菌株是否具有抑制指示菌的活性。
双层板培养法具体方法如下：下层培养基选用MRS培养基，取德氏乳杆菌VB183的菌悬液和水各2μl，点种于MRS固体培养基上，37℃厌氧培养1天后形成成熟单菌落，上层倒入含指示菌(金黄色葡萄球菌ATCC 6538、大肠杆菌8099、乙型副伤寒沙门氏菌CMCC50094、单增李斯特氏菌ATCC19114、痢疾志贺氏菌CMCC51252、产气荚膜梭菌ATCC 13124)的培养基，7～10mL/皿，指示菌终浓度为106CFU/mL，待凝固后，置于各指示菌适宜的生长条件下分别培养16～18h，观察并记录VB183单菌落周围是否有抑菌圈及抑菌圈大小，判断是否具有抑制该指示菌活性。考察结果见表2。
表2德氏乳杆菌VB183对6种致病菌的抑菌能力评估
上述结果显示，实施例1的德氏乳杆菌VB183对大肠杆菌、金黄色葡萄球菌、沙门氏菌、 李斯特氏菌、志贺氏菌、产气荚膜菌等6种致病菌均有抑菌作用。
实施例6：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183耐胃酸能力考察
本实施例考察了德氏乳杆菌VB183对pH 4模拟胃酸环境的耐受性。
受试菌株处理条件为：pH 4酸性条件分别处理受试菌2、4小时，稀释涂布检测其存活率。空白对照为同等时间下用无菌水(去离子水121℃灭菌30min)处理的德氏乳杆菌VB183。
受试菌株耐胃酸存活率计算公式如下：
其中，空白对照的活菌数用N0表示，受试菌株的活菌数用N表示。
结果如表3所示。可以看出，实施例1的德氏乳杆菌VB183在pH4条件下处理2小时后，存活率为96.3％。在pH为4条件下处理4小时，存活率仍大于86％。
以上结果表明，本发明的德氏乳杆菌VB183具有较好的耐胃酸能力。
表3德氏乳杆菌VB183耐胃酸试验数据
实施例7：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183抗胆盐试验
本实施例考察了德氏乳杆菌VB183在不同胆盐浓度下的耐受性。
胆盐浓度分别为0.03％、0.06％、0.1％、0.2％、0.3％，处理时间为3h，稀释涂布检测其存活率。其中空白对照为同等时间下用无菌水(去离子水121℃灭菌30min)处理的德氏乳杆菌VB183。
受试菌耐胆盐存活率计算公式如下：
其中，空白对照的活菌数用N0表示，不同胆盐浓度处理条件下测得的活菌数用N表示。
结果如表4所示。可以看出，实施例1的德氏乳杆菌VB183在0.3％胆盐浓度下处理4 小时，其存活率仍高达92.1％，菌株活率基本无影响。
以上结果表明，本发明的德氏乳杆菌VB183具有很强的抗胆盐能力。
表4德氏乳杆菌VB183胆盐试验数据
实施例8：德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.bulgaricus)VB183肠液稳定性考察
本实施例考察了德氏乳杆菌VB183在模拟肠液条件下的稳定性。
具体方案如下：将冻存在-80℃的菌株保藏管置于冰上解冻，取10μL至990μL无菌肠液中，混匀后分别于0h、2h、4h进行涂布计数。其中空白对照样品为10μL至990μL无菌水(去离子水121℃灭菌30min)中，混匀后分别于0h涂布计数。
受试菌模拟肠液试验存活率计算公式如下：
其中，空白对照的活菌数用N0表示，模拟肠液条件下测得的活菌数用N表示。
结果如表5所示。可以看出，实施例1的德氏乳杆菌VB183在肠模拟液中的存活能力强，模拟肠液处理4h后，存活率仍高达96.9％。
以上结果表明，本发明的德氏乳杆菌VB183肠液稳定性强，可在通过消化道后依然维持高活性。
表5德氏乳杆菌VB183模拟肠液试验数据
综上所述，本发明德氏乳杆菌VB183具有较强的耐酸耐胆盐以及抵抗胃肠液的能力， 可在通过消化道后依然维持高活性。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (12)

  1. 一种德氏乳杆菌保加利亚亚种VB183，其特征在于，所述德氏乳杆菌的保藏号为CGMCC No.28204。
  2. 一种发酵产物，其特征在于，包括权利要求1所述德氏乳杆菌。
  3. 根据权利要求2所述的发酵产物，其特征在于，进一步包括：
    权利要求1所述德氏乳杆菌的代谢产物。
  4. 一种微生物菌剂，其特征在于，包括权利要求1所述德氏乳杆菌、权利要求2或3所述发酵产物中的至少之一。
  5. 一种食品、药品、保健品或饲料，其特征在于，包括权利要求1所述德氏乳杆菌、权利要求2或3所述发酵产物或权利要求4所述微生物菌剂中的至少一种。
  6. 根据权利要求5所述的食品、药品、保健品或饲料，其特征在于，进一步包括药学、食品、保健品上可接受的辅料或载体。
  7. 权利要求1所述德氏乳杆菌、权利要求2或3所述发酵产物或权利要求4所述微生物菌剂在制备食品或保健品中的用途，所述食品或保健品用于缓解乳糖不耐受和/或调节肠道菌群。
  8. 权利要求1所述德氏乳杆菌、权利要求2或3所述发酵产物或权利要求4所述微生物菌剂在制备药物或饲料中的用途，所述药物或饲料具有下列至少一种用途：
    预防和/或缓解乳糖不耐受；
    调节肠道菌群；
    预防和/或治疗病原菌感染引起的肠道疾病。
  9. 根据权利要求8所述的用途，其特征在于，所述病原菌选自金黄色葡萄球菌、大肠杆菌、沙门氏菌、李斯特菌、志贺氏菌和产气荚膜菌中的至少之一。
  10. 一种体外抑制病原菌生长的方法，其特征在于，包括：
    将权利要求1所述德氏乳杆菌、权利要求2或3所述发酵产物和权利要求4所述微生物菌剂中的至少一种与含有病原菌的样本共培养。
  11. 根据权利要求10所述的方法，其特征在于，所述病原菌选自金黄色葡萄球菌、大肠杆菌、沙门氏菌、李斯特菌、志贺氏菌和产气荚膜菌中的至少之一。
  12. 权利要求1所述德氏乳杆菌、权利要求2或3所述发酵产物、权利要求4所述微生物菌剂或权利要求5所述食品、药品、保健品或饲料的培养装置或生产装置。
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CN119286742B (zh) * 2024-12-13 2025-03-14 内蒙古农业大学 一种德氏乳杆菌乳酸亚种yn-01及由其制备的产品和应用
CN120496623B (zh) * 2025-07-08 2025-09-19 杭州微致生物科技有限公司 用于确定微生物发酵工艺的参数组合的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5416020A (en) * 1992-09-29 1995-05-16 Bio-Technical Resources Lactobacillus delbrueckii ssp. bulgaricus strain and fermentation process for producing L-(+)-lactic acid
WO2011050426A2 (en) * 2009-10-27 2011-05-05 "Selur Vk Holding" Eood New strains of lactic acid bacteria and their combinations producing probiotic preparations
CN110129220A (zh) * 2019-04-30 2019-08-16 石河子大学 一种保加利亚乳杆菌bsts6-4及其应用
CN112625971A (zh) * 2020-12-30 2021-04-09 杭州娃哈哈科技有限公司 一株可大量消耗乳糖且兼具耐酸、耐胆盐和抗氧化性的德氏乳杆菌及其应用
US20220304323A1 (en) * 2019-08-30 2022-09-29 Chr. Hansen A/S Production of lactase enzymes using altered regulation strains
CN117305189A (zh) * 2023-11-29 2023-12-29 杭州微致生物科技有限公司 一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015074054A1 (en) * 2013-11-18 2015-05-21 The Trustees Of Columbia University In The City Of New York Improving microbial fitness in the mammalian gut
CN111621445A (zh) * 2020-06-29 2020-09-04 张秉胜 一种自培养乳酸菌动物饮料生产方法
CN116162562B (zh) * 2022-08-12 2024-12-13 深圳市盐田区人民医院 一种德氏乳杆菌sunkii亚种及其培养方法与应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5416020A (en) * 1992-09-29 1995-05-16 Bio-Technical Resources Lactobacillus delbrueckii ssp. bulgaricus strain and fermentation process for producing L-(+)-lactic acid
WO2011050426A2 (en) * 2009-10-27 2011-05-05 "Selur Vk Holding" Eood New strains of lactic acid bacteria and their combinations producing probiotic preparations
CN110129220A (zh) * 2019-04-30 2019-08-16 石河子大学 一种保加利亚乳杆菌bsts6-4及其应用
US20220304323A1 (en) * 2019-08-30 2022-09-29 Chr. Hansen A/S Production of lactase enzymes using altered regulation strains
CN112625971A (zh) * 2020-12-30 2021-04-09 杭州娃哈哈科技有限公司 一株可大量消耗乳糖且兼具耐酸、耐胆盐和抗氧化性的德氏乳杆菌及其应用
CN117305189A (zh) * 2023-11-29 2023-12-29 杭州微致生物科技有限公司 一种德氏乳杆菌保加利亚亚种vb183及其培养装置和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOKAR A, KARBASSI A: "Determination of Proper Conditions for the Production of Crude Beta-galactosidase Using Lactobacillus delbrueckii ssp. bulgaricus", JOURNAL OF AGRICULTURAL SCIENCE AND TECHNOLOGY, vol. 11, 1 January 2009 (2009-01-01), pages 301 - 308, XP093319731 *
MOEZ RHIMI, NUSHIN AGHAJARI, BASSEM JAOUADI , MICHEL JUY , SAMIRA BOUDEBBOUZE, EMMANUELLE MAGUIN, RICHARD HASER , SAMIR BEJAR: "Exploring the acidotolerance of beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus: an attractive enzyme for lactose bioconversion", RESEARCH IN MICROBIOLOGY, vol. 160, 26 September 2009 (2009-09-26), pages 775 - 784, XP055814483 *
WEN ZHANG; CHUAN WANG; CHENG-YU HUANG; QIAN YU; HENG-CHUAN LIU; CHAO-WU ZHANG; XIAO-FANG PEI; XIN XU; GUO-QING WANG: "Analysis of beta-galactosidase production and their genes of two strains of Lactobacillus bulgaricus", BIOTECHNOLOGY LETTERS, SPRINGER NETHERLANDS, DORDRECHT, vol. 34, no. 6, 15 February 2012 (2012-02-15), Dordrecht , pages 1067 - 1071, XP035047254, ISSN: 1573-6776, DOI: 10.1007/s10529-012-0870-2 *

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