CN117305189B - Lactobacillus delbrueckii subspecies bulgaricus VB183 and culture device and application thereof - Google Patents
Lactobacillus delbrueckii subspecies bulgaricus VB183 and culture device and application thereof Download PDFInfo
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- CN117305189B CN117305189B CN202311608342.7A CN202311608342A CN117305189B CN 117305189 B CN117305189 B CN 117305189B CN 202311608342 A CN202311608342 A CN 202311608342A CN 117305189 B CN117305189 B CN 117305189B
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Abstract
The invention discloses Lactobacillus delbrueckii subspecies bulgaricus VB183 and a culture device and application thereof, and relates to the field of microorganisms. The preservation number of the lactobacillus delbrueckii is CGMCC No.28204. The Lactobacillus delbrueckii VB183 culture device has the advantages of high lactose degradation rate, strong beta-galactosidase activity, strong acid and alkali resistance, strong bile salt resistance and the like, has strong antibacterial capacity on common pathogenic bacteria, can be used for relieving lactose intolerance, regulating intestinal flora, preventing and treating intestinal diseases caused by pathogenic bacteria infection, has high application value, can be used for mass production of Lactobacillus delbrueckii bulgaricus VB183 fermentation products, is favorable for development of microbial agents, medicines or feed production devices, and has high economic value.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to lactobacillus delbrueckii subspecies bulgaricus VB183 and a culture device and application thereof.
Background
Lactose is a disaccharide consisting of glucose and galactose and is the major source of carbohydrates in human and other most mammalian milk. Lactose cannot be directly absorbed by the human body, and is mainly hydrolyzed into glucose and galactose by apical lactase (mainly beta-galactosidase) of villi on the mucous membrane surface of small intestine, especially jejunum, and then is absorbed by active transport of cells. The lack of lactase or reduced enzymatic activity in the small intestine of the human body can cause the inability to digest and break down lactose in breast milk or other dairy products completely, thereby causing adverse reactions such as abdominal pain, diarrhea, abdominal distension, etc.
Current lactose intolerant treatment or relief strategies include low lactose diet, lactase supplementation, supplementation with probiotics having high beta-galactosidase activity or lactose degrading phenotype, etc. Probiotics are metabolized into the body to produce various enzymes, such as lactase, lipase, protease, skin enzymes, etc., which help to treat lactose intolerance. However, strains capable of alleviating lactose intolerance are currently still under development.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides Lactobacillus delbrueckii subspecies bulgaricus VB183 and a culture device and application thereof, and also provides a fermentation product, a microbial agent, a medicine or feed, and a fermentation device and application thereof, and the novel Lactobacillus delbrueckii strain obtained by screening has the advantages of high lactose degradation rate, strong activity of beta-galactosidase, strong acid and alkali resistance, strong bile salt resistance and the like, has strong antibacterial capacity on common pathogenic bacteria, can be used for relieving lactose intolerance, regulating intestinal flora, preventing and treating intestinal diseases caused by pathogenic bacteria infection, has high application value, can be used for mass production of Lactobacillus delbrueckii subspecies bulgaricus VB183 fermentation products, is favorable for developing microbial agents, medicines or feed production devices, mass production of commercial products, and has high economic value.
For this purpose, in a first aspect of the invention, the invention proposes a Lactobacillus delbrueckii subspecies bulgaricus VB183. According to the embodiment of the invention, the preservation number of the lactobacillus delbrueckii is CGMCC No.28204.
Preservation information:
strain name: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus）VB183
Preservation date: 2023, 08 and 21 days
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Preservation number: CGMCC No.28204
The lactobacillus delbrueckii subspecies bulgaricus obtained by screening of the inventionLactobacillus delbrueckiisubsp.bulgaricus) The new strain has the advantages of high lactose degradation rate, strong beta-galactosidase activity, strong acid and alkali resistance, strong bile salt resistance and the like, has strong antibacterial capacity on common pathogenic bacteria, can be used for solving lactose intolerance, regulating intestinal flora, preventing and treating intestinal diseases caused by pathogenic bacteria infection, and has high application value.
In a second aspect of the invention, the invention provides a fermentation product. According to an embodiment of the invention, the fermentation product comprises the aforementioned lactobacillus delbrueckii.
In a third aspect of the invention, the invention provides a microbial agent. According to an embodiment of the invention, the microbial agent comprises at least one of the aforementioned lactobacillus delbrueckii, the aforementioned fermentation product. The microbial agent can be used for relieving lactose intolerance and regulating intestinal flora, and can also be used for preventing and treating intestinal diseases caused by pathogenic bacteria infection.
In a fourth aspect of the invention, the invention provides a pharmaceutical or feed product. According to an embodiment of the invention, the pharmaceutical product or feed comprises at least one of the aforementioned lactobacillus delbrueckii, the aforementioned fermentation product or the aforementioned microbial agent. The feed disclosed by the invention can be used for relieving lactose intolerance and regulating intestinal flora; the medicine of the invention can be used for preventing and/or relieving lactose intolerance and preventing and/or treating intestinal diseases caused by pathogenic bacteria infection.
In a fifth aspect of the invention, the invention proposes the use of the aforementioned lactobacillus delbrueckii, the aforementioned fermentation product or the aforementioned microbial agent for the preparation of a medicament or feed having 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.
In a sixth aspect of the invention, the invention provides a method of inhibiting the growth of a pathogenic bacterium in vitro. According to an embodiment of the invention, the method comprises: co-culturing at least one of the lactobacillus delbrueckii, the fermentation product and the microbial agent with a sample containing pathogenic bacteria. As described above, lactobacillus delbrueckii subspecies bulgaricus of the present inventionLactobacillus delbrueckiisubsp.bulgaricus) VB183 is effective in inhibiting pathogenic bacterial growth.
In a seventh aspect of the present invention, the present invention provides a culture apparatus or a production apparatus for the Lactobacillus delbrueckii, the fermentation product, the microbial agent, or the drug or feed.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a graph showing the result of a scanning electron microscope of Lactobacillus delbrueckii VB183 in example 2 of the present invention;
FIG. 2 is a graph showing the results of HPLC detection of lactose content in Lactobacillus delbrueckii VB183 fermentation broth in example 3 of the present invention;
FIG. 3 is a graph of lactose concentration standard in example 3 of the present invention, wherein the abscissa represents lactose concentration and the ordinate represents peak area of lactose standard with different concentrations measured by HPLC;
FIG. 4 is a graph showing the results of measurement of lactose degradation rate of Lactobacillus delbrueckii VB183 and commercial strain in 24 hours in example 3 of the present invention, wherein VB183 is Lactobacillus delbrueckii subsp. Bulgaricus of the present inventionLactobacillus delbrueckiisubsp.bulgaricus) VB183, NCFM is Lactobacillus Du Bangshi, BB-12 is Bifidobacterium lactis of Hansen of Danish, and LGG is Lactobacillus rhamnosus of Finnish;
FIG. 5 is a graph showing the standard concentration of o-nitrophenol (ONP) in example 4, wherein the abscissa represents the ONP standard concentration and the ordinate represents the absorbance of ONP standard at 405 and nm;
FIG. 6 is a graph showing the results of the measurement of the beta-galactosidase activities of Lactobacillus delbrueckii VB183 and commercial strains in example 4 of the present invention, wherein VB183 is Lactobacillus delbrueckii subsp. Bulgaricus of the present inventionLactobacillus delbrueckiisubsp.bulgaricus) VB183, NCFM was Lactobacillus Du Bangshi, BB-12 was Bifidobacterium lactis subspecies of Hansen animals of Danish, and LGG was Lactobacillus rhamnosus of Veri, finland.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
Herein, the term "MRS medium" is a lactic acid bacterium medium, and can be classified into two types, MRS solid medium and MRS liquid medium, depending on the level of coagulants (typically agarose).
As used herein, the term "sterile water" refers to water that is physically treated to produce bacteria-free water.
The invention provides Lactobacillus delbrueckii subspecies bulgaricus VB183, fermentation products, microbial agents, medicines or feeds and uses thereof, and the detailed description of the products is respectively provided below.
Bacterial strain
The invention provides Lactobacillus delbrueckii subspecies bulgaricus VB183.
According to the embodiment of the invention, the preservation number of the lactobacillus delbrueckii is CGMCC No.28204. The strain is a brand-new isolate strain which is preserved in China general microbiological culture Collection center (CGCC) No.28204, and the preservation address is shown in the general microbiological culture Collection center of China general microbiological culture Collection center, and is stored in the year 08 and 21 of 2023: beijing, chaoyang area, north Chenxi way No. 1, no. 3, post code: 100101.
according to the embodiment of the invention, the 16S rDNA sequencing result of the Lactobacillus delbrueckii is shown as SEQ ID NO. 1.
The lactobacillus delbrueckii subspecies bulgaricus obtained by screening of the inventionLactobacillus delbrueckiisubsp.bulgaricus) The VB183 new strain has the advantages of high lactose degradation rate, strong beta-galactosidase activity, strong acid and alkali resistance, strong bile salt resistance and the like, has strong antibacterial capacity on common pathogenic bacteria, can be used for solving lactose intolerance, regulating intestinal flora, preventing and treating intestinal diseases caused by pathogenic bacteria infection, and has high application value.
The term "Lactobacillus delbrueckii subspecies bulgaricus" is used hereinLactobacillus delbrueckiisubsp.bulgaricus) The VB183 strain "," Lactobacillus delbrueckii subspecies bulgaricus VB183 "and" Lactobacillus delbrueckii VB183 "are synonymous.
Fermentation product
The invention provides a fermentation product. According to an embodiment of the invention, the fermentation product comprises the aforementioned lactobacillus delbrueckii.
The term "fermentation product" as used herein means a solution obtained by culturing Lactobacillus delbrueckii VB183 for a period of time, which mainly contains Lactobacillus delbrueckii VB183 and its metabolites; or further treating the supernatant by means of centrifugation, filtration and the like, wherein the supernatant mainly comprises metabolites of Lactobacillus delbrueckii VB 183; or further processing the bacterial suspension by means of centrifugation, re-suspension and the like, wherein the bacterial suspension mainly comprises lactobacillus delbrueckii VB183.
According to an embodiment of the present invention, further comprising: a metabolite of the aforementioned lactobacillus delbrueckii.
It should be noted that the features and advantages described above for lactobacillus delbrueckii are equally applicable to the fermentation product and are not described here.
Microbial agent
The invention provides a microbial agent. According to an embodiment of the invention, the microbial agent comprises at least one of the aforementioned lactobacillus delbrueckii, the aforementioned fermentation product. The microbial agent can be used for relieving lactose intolerance and regulating intestinal flora, and can also be used for preventing and treating intestinal diseases caused by pathogenic bacteria infection.
It should be noted that the microbial agent of the present invention may be a microbial liquid agent, including but not limited to fermentation products, etc.; and can also be a microbial solid microbial agent, including but not limited to freeze-dried powder and the like.
In the microbial agent of the present invention, lactobacillus delbrueckii may exist in the form of living cells and/or non-living cells.
As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.
Illustratively, the living cells may be immobilized cells. As used herein, "immobilized cells" refers to living cells immobilized on a carrier, which can undergo vital activities such as growth, development, reproduction, inheritance, and metabolism in a certain spatial range.
As used herein, "non-living cells" refers to cells that do not have the ability to metabolize, reproduce, and replicate, including but not limited to stem cells. Illustratively, the microbial agent is a lyophilized powder.
In some specific embodiments, the lactobacillus delbrueckii VB183 is present as living cells, stem cells, immobilized cells, or in any other form.
In some specific embodiments, the dry cell is obtained by freeze-drying the lactobacillus delbrueckii VB183.
In some embodiments, the microbial agent may further comprise a strain acceptable to at least one of a pharmaceutical product and a feed.
In some embodiments, the microbial agent further comprises a pharmaceutically acceptable adjuvant or carrier or an acceptable adjuvant or carrier in an animal feed.
As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.
As used herein, "acceptable adjuvants or carriers in animal feed" refers to substances or compositions that are edible to animals and which can be tailored to the animal feed requirements of different countries.
Herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those of skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
As used herein, the term "pharmaceutically acceptable excipients" may include any solvent suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with lactobacillus delbrueckii VB183 of the present disclosure, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present disclosure.
It should be noted that the features and advantages described above for lactobacillus delbrueckii are equally applicable to the microbial agent, and are not described here.
Medicine or feed
The invention provides a medicine or feed. According to an embodiment of the invention, the pharmaceutical product or feed comprises at least one of the aforementioned lactobacillus delbrueckii, the aforementioned fermentation product or the aforementioned microbial agent. The feed disclosed by the invention can be used for relieving lactose intolerance and regulating intestinal flora; the medicine of the invention can be used for preventing and/or relieving lactose intolerance and preventing and/or treating intestinal diseases caused by pathogenic bacteria infection.
According to an embodiment of the invention, further comprising pharmaceutically or feed acceptable excipients or carriers.
In some specific embodiments, the aforementioned lactobacillus delbrueckii, the aforementioned fermentation product, or the aforementioned microbial agent is added or inoculated to a feed, or added to a pharmaceutical composition, whereby a feed having lactose intolerance alleviation and/or intestinal flora regulation functions is further obtained, or a drug capable of preventing and/or alleviating pathogenic bacterial infection or an intestinal disease caused by pathogenic bacterial infection is obtained.
Illustratively, the pharmaceutical product includes, but is not limited to: human medicine and veterinary medicine. The animal can be pet, livestock, or wild animal.
It should be noted that the features and advantages described above for lactobacillus delbrueckii are equally applicable to the pharmaceutical product, or the feed, and are not described here again.
Use of the same
The invention also provides the application of the lactobacillus delbrueckii, the fermentation product or the microbial agent in preparing medicines or feeds, wherein the medicines or feeds have at least one of the following applications: preventing and/or alleviating lactose intolerance; regulating intestinal flora; preventing and/or treating intestinal diseases caused by pathogenic bacteria infection.
According to an embodiment of the invention, the pathogenic bacteria are selected from at least one of staphylococcus aureus, escherichia coli, salmonella, listeria, shigella and clostridium perfringens.
In some embodiments, the medicament further comprises an excipient and/or carrier.
In some embodiments, the excipient comprises at least one selected from the group consisting of binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and slow-release agents.
In some embodiments, the carrier comprises at least one selected from the group consisting of sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, cereal hydrolytic solids.
In some specific embodiments, the dosage form of the medicament comprises at least one selected from the group consisting of oral liquid, powder, granules, capsules, tablets, and drop pills.
It should be noted that the features and advantages described above for lactobacillus delbrueckii are equally applicable to this use and are not described here again.
Method
The invention provides a method for inhibiting pathogenic bacteria growth in vitro. According to an embodiment of the invention, the method comprises: co-culturing at least one of the lactobacillus delbrueckii, the fermentation product and the microbial agent with a sample containing pathogenic bacteria. As described above, lactobacillus delbrueckii VB183 of the present invention is effective in inhibiting the growth of pathogenic bacteria.
According to an embodiment of the invention, the pathogenic bacteria are selected from at least one of staphylococcus aureus, escherichia coli, salmonella, listeria, shigella and clostridium perfringens.
It should be noted that the features and advantages described above for lactobacillus delbrueckii are equally applicable to the method and are not described here.
Device and method for controlling the same
The invention provides a culture device or a production device of the lactobacillus delbrueckii, the fermentation product, the microbial agent or the medicine or feed. The Lactobacillus delbrueckii subspecies bulgaricus VB183 culture device can be used for mass production of Lactobacillus delbrueckii subspecies bulgaricus VB183 fermentation products, is favorable for developing microbial agents, medicines or feed production devices, mass production of commercial products and has high economic value.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) Acquisition of VB183
Lactobacillus delbrueckii subspecies bulgaricus of the inventionLactobacillus delbrueckiisubsp.bulgaricus) VB183 is isolated from lactose-tolerant healthy adult feces.
The strain collecting and separating process comprises the following steps: adding 0.5 g lactose-tolerant healthy adult feces into 5 mL sterilized PBS buffer solution, mixing, diluting and coating on an MRS solid culture medium plate, culturing 48 h in an anaerobic environment at 37 ℃, respectively picking single colonies with different sizes and shapes, streaking on a fresh MRS plate, anaerobically culturing 48 h at 37 ℃, repeating the streaking purification culture for a plurality of times until the colony shapes in the plates are consistent, and identifying after microscopic examination and no bacteria.
And (3) strain preservation: culturing the obtained pure culture strain to a concentration of about 10 7 CFU/mL, 500. Mu.L of fungus solution is taken, 50% glycerol is added into 500. Mu.L to lead the glycerol concentration to reach 25%, and then ultralow temperature preservation is carried out at-80 ℃.
MRS liquid medium is: yeast extract 0.5%, glucose 2%, peptone 1.0%, beef extract 1.0%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, tween 80.1%, pH6.2, and sterilizing at 121deg.C for 15 min.
MRS solid medium is: 2% agar was added to MRS broth.
Example 2: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) Identification of VB183
The culture obtained after the separation and purification in example 1 was further confirmed to be a pure culture by streaking and smear microscopy, and then strain identification was performed by experimental means including a gram stain test, a physiological and biochemical test such as a contact enzyme, and a 16S rDNA full-sequence sequencing identification.
BLAST comparison of 16S rDNA sequencing results, final identification determination: the separated strain is a Lactobacillus delbrueckii subsp bulgaricus strain named Lactobacillus delbrueckii subsp bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.28204 in 2023 and 08 and 21.
In this example, the main species identification means and results were as follows:
the lactobacillus delbrueckii VB183 is observed by a scanning electron microscope, and the specific method is as follows: firstly, the colony with the size of rice grains is scraped in a flat plate, and a sample is fixed by adopting a glutaraldehyde and osmium acid double fixing method. The samples were then dehydrated with gradient concentrations (including five concentrations of 30%,50%,70%,80%,90% and 95%) of ethanol, each concentration being treated for 15 min and then twice with 100% ethanol for 20 min each. The dehydrated sample was dried in a Hitachi HCP-2 type critical point dryer, followed by plating. Finally, the processed sample is observed in a Hitachi SU-8010 scanning electron microscope, and a photo taken by the electron microscope is shown in fig. 1.
The scanning electron microscope and the gram staining test result show that: lactobacillus delbrueckii VB183 was gram positive, single colony white, smooth and irregular, and the cells were rod-shaped (FIG. 1).
Physiological and biochemical characterization of Lactobacillus delbrueckii VB183 was performed with specific experiments in reference to the Berger's bacteria identification manual.
The physical and chemical characteristics identification result is: the contact enzyme is negative, oxidase is negative, maltose, sucrose, raffinose, inulin, glucose, lactose, galactose, mannose, fructose, melezitose, raffinose can not hydrolyze starch and gelatin, V-P reaction is negative, and methyl red is positive.
The sequencing result of the 16S rDNA of the Lactobacillus delbrueckii VB183 is shown in SEQ ID NO. 1.
CCTTGGGCGGGCGGTGCCTATACATGCAAGTCGAGCGAGCTGAATTCAAAGATTCCTTCGGGGTGATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGTAAGCTGTCACTTTAGGATGAGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAACTGCATCGGAAACTGTCATTCTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTGTGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAAAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGTCGGTGGGATAACCTTTATAGGAGTCAGCCGCCCTAAGTCCGGGCCC（SEQ ID NO: 1）。
Example 3: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 lactose degradation capability investigation
3% lactose was added to the MRS basal liquid medium to evaluate lactose degradation ability of the test strain as an amount of lactose consumed after 24 h culture. The lactose content was quantitatively determined by High Performance Liquid Chromatography (HPLC).
The test strain is prepared by thawing frozen strain preservation tube at-80deg.C on ice, streaking 1-cycle bacteria liquid on MRS solid culture medium plate, and standing culturing in anaerobic incubator at 37deg.C for 48 h. Single colonies were transferred to MRS broth with 10 mL and cultured in anaerobic conditions at 37℃for 24℃ 24 h. The bacterial suspension was transferred to a 250 mL Erlenmeyer flask containing 50 mL lactose medium at 2% transfer, an anaerobic workstation at 37℃and allowed to stand for 24 h.
MRS liquid medium is: yeast extract 0.5%, glucose 2%, peptone 1.0%, beef extract 1.0%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, tween 80.1%, pH6.2, and sterilizing at 121deg.C for 15 min.
MRS solid medium is: 2% agar was added to MRS broth.
Lactose medium is: glucose was removed from MRS broth and 30 g/L lactose was added.
Drawing a lactose concentration standard curve, namely drawing 12.275 g lactose in a small beaker, adding 100 mL of water for dissolution, transferring to a 250 mL volumetric flask, continuously metering the volume to 250 mL by using water to prepare the lactose concentration of 49.1 mg/mL, then respectively sucking 80, 40, 20, 10 and 5 mL of the lactose concentration standard curve into different 100 mL volumetric flasks, adding water to the volume of 100 mL, and shaking uniformly to prepare 39.28, 19.64, 9.82, 4.91 and 2.46 mg/mL lactose solutions.
Lactose concentration standard curves (fig. 3) were plotted with lactose concentration on the abscissa and peak area by HPLC on the ordinate, and linear regression equations were obtained.
Determination of lactose degradation Rate the lactose degradation Rate was determined by taking Lactobacillus delbrueckii VB183 fermentation broth obtained by culturing 1 mL in step 1, centrifuging at 14000 rpm for 3 min, taking the supernatant, appropriately diluting, filtering with a 0.22 μm sterile filter membrane, quantitatively detecting lactose content by HPLC, and determining lactose degradation Rate according to the following formula based on the detected lactose content.
The HPLC detection method for lactose content is as follows: the chromatographic column is Luna 5 μm NH2 100 a (5 μm,150×4.6 mm); the flow rate is 1.2 mL/min; column temperature is 30 ℃; the sample injection volume is 10 mu L; the mobile phase is acetonitrile: water = 8:2.
the HPLC detection result of lactose content of the lactobacillus delbrueckii VB183 fermentation broth is shown in figure 2.
As shown in fig. 2, lactose retention time of lactobacillus delbrueckii VB183 was measured to be 10.3 min, and the lactose degradation rate was finally determined to be 83% based on the peak area measured by HPLC and the lactose concentration standard curve determined in step 2.
A commercial strain of Lactobacillus or Bifidobacterium having excellent lactose degrading property was selected, and the lactose degrading rate of the commercial strain was examined by the same method to further evaluate the lactose degrading property of Lactobacillus delbrueckii VB183 obtained in example 1. Wherein the commercial strain NCFM is Lactobacillus acidophilus (DuPont, U.S.), BB-12 is Bifidobacterium animalis subspecies lactis (Hansen, denmark), CECT5716 is Lactobacillus fermentum (Papanic, spanish), and LGG is Lactobacillus rhamnosus (Veriiolo, finland). The measurement results are shown in FIG. 4.
The results show that the lactose degradation rate of lactobacillus delbrueckii VB183 of example 1 is 83%, generally higher than that of commercial strains; 28% higher than the commercial lactobacillus acidophilus NCFM (lactose degradation rate 65%) with the highest lactose degradation rate.
Example 4: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 beta-galactosidase activity investigation
The most important enzyme for alleviating lactose intolerance is beta-galactosidase, which breaks down lactose into glucose and galactose. The experiment adopts the ONPG method to further determine the Lactobacillus delbrueckii subspecies bulgaricus strainLactobacillus delbrueckiisubsp.bulgaricus) Beta-galactosidase Activity of Lactobacillus delbrueckii VB183.
The principle of detecting the activity of the beta-galactosidase is as follows: beta-galactosidase catalyzes colorless o-nitrophenol beta-D-galactopyranoside (ONPG) to break off glycosidic bond, yellow o-nitrophenol (ONP) and beta-D-galactopyranoside are produced, as ONPG is decomposed into ONP, the color of solution is gradually deepened, ONP has maximum absorption peak at 420nm, so the yield of ONP can be calculated through the absorption value, and the enzyme activity of the beta-galactosidase is calculated.
The β -galactosidase enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. Mu.M ONP per minute was measured at 30 ℃.
The detection method comprises the following steps:
1. preparation of test strains
Placing the strain preservation tube frozen at-80 ℃ on ice for thawing, streaking the 1-cycle bacteria liquid on an MRS solid culture medium plate, and standing and culturing in an anaerobic incubator at 37 ℃ for 48 h. Single colonies were transferred to MRS broth with 10 mL and cultured in anaerobic conditions at 37℃for 24℃ 24 h. The bacterial suspension was transferred to a 250 mL flask containing 50 mL lactose medium at 2% transfer, placed in an anaerobic workstation at 37℃and allowed to stand for 24 h.
2. Reagent preparation
(1) PBS buffer
16.1 g disodium hydrogen phosphate, 5.5 g sodium dihydrogen phosphate, 0.75 g potassium chloride, 0.246 g magnesium sulfate and 2.7 mL 2-mercaptoethanol were dissolved in 800. 800 mL water, and 2 mol/L sodium hydroxide solution was added to adjust the pH to 6.0.+ -. 0.05. The solution was transferred to a 1L volumetric flask, fixed in volume with water, and mixed well.
(2) 10 mM substrate solution
The o-nitrobenzene-beta-D-galactopyranoside (ONPG) substrate solution is prepared by dissolving 301 mg ONPG in 80 mL PBS buffer, transferring the solution into a 100 mL volumetric flask, and fixing the volume with PBS to obtain 10 mM substrate solution.
(3) Stop solution
After dissolving 10 g sodium carbonate with water, transfer to a 100 mL volumetric flask for constant volume.
(4) Preparation of test samples
The 8 mL broth was centrifuged at 5000 rpm at 4℃for 10 min, the supernatant was discarded and washed 2 times with an equal volume of PBS buffer. The cells were resuspended in 2 mL of PBS buffer and the samples were added to the milling beads and subjected to 3 cryogenic milling in a milling apparatus using a procedure of 65 Hz for 120 s under the following disruption conditions: grinding time 60 s at 4℃and interval 60 s. The crude enzyme solution is diluted appropriately so that 0.05 to 0.25 units of beta-galactosidase are contained in each mL of final solution.
3. Drawing of O-nitrophenol (ONP) standard curve
139, mg o-nitrophenol (ONP) was accurately weighed, placed in a 25, mL small beaker, dissolved in 10 mL of 95% alcohol, and then transferred to a 1, L volumetric flask, and the volume was fixed with pure water. Pipette 2, 4, 6, 8 and 10 mL solutions to a volumetric flask of 100 mL, and mix well with 10wt% sodium carbonate solution to volume. These solutions contained 0.02, 0.04, 0.06, 0.08, 0.1. Mu.M ONP per mL, respectively.
The absorbance was measured at wavelength 405 and nm, and the linear regression equation was obtained by taking water as a control, ONP concentration as an abscissa, and absorbance of each concentration standard as an ordinate, and making a standard curve (fig. 5).
Measurement of beta-galactosidase enzyme Activity 200. Mu.L of diluted crude enzyme solution was preheated in a metal bath at 30℃for 5 min, substrate ONPG concentration solution 1 mL also preheated at 30℃for 5 min was added, mixed well, reacted at 400 rpm in the metal bath at 30℃for 10 min, then immediately followed by addition of 400. Mu.L of sodium carbonate solution to terminate the reaction, 200. Mu.L was taken in an ELISA plate, and absorbance at 420nm was measured in 30 min.
Calculating and analyzing to obtain a standard curve: y=2.5986x+0.0494, r 2 ＝0.9995
Wherein Y is the light absorption value at 420nm, and X is the concentration of ONP (mu mol).
Enzyme activity (U/mL):
wherein: OD (optical density) 420 Absorbance at 420nm for the sample to be measured; b is the intercept of the standard curve, 0.0494; v is the total volume of the reaction system, 1.6 mL; f is the dilution multiple of the measured enzyme solution; k is the slope of the standard curve, 2.5986; t is the reaction time of the catalyst,10 min; v1 is the volume of the enzyme solution, 0.2. 0.2 mL.
A commercial strain of Lactobacillus or Bifidobacterium having excellent lactobacilli activity was selected, and the commercial strain was examined for beta-galactosidase activity by the same method, and the Lactobacillus delbrueckii VB183 obtained in example 1 was further evaluated for its ability to alleviate lactose intolerance. Wherein the commercial strain NCFM is Lactobacillus acidophilus (DuPont, U.S.), BB-12 is Lactobacillus bifidus subspecies lactis (Hansen, denmark) and LGG is Lactobacillus rhamnosus (Weirio, finland). The measurement results are shown in Table 1 and FIG. 6.
TABLE 1 determination of beta-galactosidase Activity of Lactobacillus delbrueckii VB183 and control Strain
The results show that the beta-galactosidase activity of the Lactobacillus delbrueckii VB183 of example 1 is 2.619U/mL, which is higher than that of the commercial strain; is 3.6 times of Lactobacillus bifidus subspecies BB-12 of Hansen of Danish, and 10 times of Lactobacillus acidophilus NCFM of DuPont.
Example 5: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 antibacterial ability investigation
In the embodiment, the antibacterial ability of the lactobacillus delbrueckii VB183 of the present invention is evaluated by a double-plate culture method, specifically, whether the strain of the single colony has the activity of inhibiting the indicator bacteria is judged by whether the antibacterial circle and the antibacterial circle size are present around the single colony on the upper layer.
The double-layer plate culture method comprises the following specific steps: the lower layer culture medium is MRS culture medium, 2 μl of bacterial suspension of Lactobacillus delbrueckii VB183 and water are respectively inoculated on MRS solid culture medium, mature single colony is formed after anaerobic culture for 1 day at 37 ℃, and the upper layer is poured into culture medium containing indicator bacteria (staphylococcus aureus ATCC 6538, escherichia coli 8099, salmonella paratyphi B CMCC50094, listeria monocytogenes ATCC19114, shigella dysenteriae CMCC51252 and clostridium perfringens ATCC 13124) with the final concentration of the indicator bacteria of 10 to 10 mL/dish 6 CFU/mL，After solidification, placing the strain into the culture medium under the proper growth conditions of each indicator bacterium to respectively culture the strain for 16-18 h, observing and recording whether a bacteriostasis zone and the size of the bacteriostasis zone exist around the VB183 single colony, and judging whether the strain has the activity of inhibiting the indicator bacterium. The results are shown in Table 2.
Table 2 evaluation of bacteriostatic ability of Lactobacillus delbrueckii VB183 against 6 pathogenic bacteria
The results show that Lactobacillus delbrueckii VB183 of example 1 has antibacterial effect on 6 pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella, listeria, shigella, and capsular bacterium.
Example 6: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 gastric acid resistance investigation
This example examined the tolerance of Lactobacillus delbrueckii VB183 to a pH4 simulated gastric acid environment.
The treatment conditions of the tested strain are as follows: the test bacteria were treated under acidic conditions at pH4 for 2 and 4 hours, respectively, and their survival rates were measured by dilution coating. The blank is Lactobacillus delbrueckii VB183 treated with sterile water (deionized water at 121℃for 30 min) for the same time.
The calculation formula of gastric acid-resistant survival rate of the tested strain is as follows:
wherein the number of viable bacteria of the blank is represented by N0, and the number of viable bacteria of the test strain is represented by N.
The results are shown in Table 3. It can be seen that the survival rate of Lactobacillus delbrueckii VB183 of example 1 was 96.3% after 2 hours of treatment at pH 4. The survival rate is still greater than 86% when treated at pH4 for 4 hours.
The results show that the lactobacillus delbrueckii VB183 has better gastric acid resistance.
TABLE 3 Lactobacillus delbrueckii VB183 gastric acid resistance test data
Example 7: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 anti bile salt test
This example examined the tolerance of Lactobacillus delbrueckii VB183 at different bile salt concentrations.
The concentration of bile salts is 0.03%, 0.06%, 0.1%, 0.2% and 0.3%, the treatment time is 3 h, and the survival rate is detected by dilution coating. Wherein the blank is Lactobacillus delbrueckii VB183 treated with sterile water (deionized water at 121℃for 30 min) at the same time.
The calculation formula of the bile salt-resistant survival rate of the test bacteria is as follows:
wherein the number of viable bacteria of the blank is represented by N0, and the number of viable bacteria measured under different bile salt concentration treatment conditions is represented by N.
The results are shown in Table 4. It can be seen that the survival rate of Lactobacillus delbrueckii VB183 of example 1, treated for 4 hours at 0.3% bile salt concentration, was still as high as 92.1% with substantially no effect on strain viability.
The results show that the Lactobacillus delbrueckii VB183 has strong anti-bile salt capability.
TABLE 4 Lactobacillus delbrueckii VB183 bile salt test data
Example 8: lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183 intestinal juice stability investigation
This example examined the stability of Lactobacillus delbrueckii VB183 under simulated intestinal fluid conditions.
The specific scheme is as follows: the strain preservation tube frozen at-80 ℃ is placed on ice for thawing, 10 mu L to 990 mu L of sterile intestinal juice is taken, and after uniform mixing, coating counting is carried out at 0 h, 2 h and 4 h respectively. The blank sample was sterilized in 10. Mu.L to 990. Mu.L of sterile water (deionized water at 121 ℃ C. For 30 min), and the samples were uniformly mixed and then each coated and counted at 0 h.
The calculation formula of the survival rate of the simulated intestinal juice test of the test bacteria is as follows:
wherein the number of viable bacteria of the blank is represented by N0, and the number of viable bacteria measured under simulated intestinal juice conditions is represented by N.
The results are shown in Table 5. It can be seen that Lactobacillus delbrueckii VB183 of example 1 has a strong viability in intestinal simulated fluid, and the viability is still as high as 96.9% after treatment of simulated intestinal fluid with 4 h.
The results show that the Lactobacillus delbrueckii VB183 intestinal juice has strong stability and can still maintain high activity after passing through the digestive tract.
TABLE 5 Lactobacillus delbrueckii VB183 simulated intestinal juice test data
In conclusion, the lactobacillus delbrueckii VB183 has strong acid resistance, bile salt resistance and gastrointestinal fluid resistance, and can still maintain high activity after passing through the alimentary canal.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (6)
1. Lactobacillus delbrueckii subspecies bulgaricus @Lactobacillus delbrueckiisubsp.bulgaricus) VB183, characterized in that said De's milk leverThe preservation number of the bacteria is CGMCC No.28204.
2. A microbial agent comprising the Lactobacillus delbrueckii strain of claim 1.
3. A pharmaceutical product or feed comprising at least one of the lactobacillus delbrueckii of claim 1 or the microbial agent of claim 2.
4. A pharmaceutical product or feed according to claim 3, further comprising a pharmaceutically acceptable adjuvant or carrier.
5. Use of the lactobacillus delbrueckii of claim 1 or the microbial agent of claim 2 in the manufacture of a medicament or feed having 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 are selected from at least one of staphylococcus aureus, escherichia coli, salmonella, listeria, shigella and clostridium perfringens.
6. A method of inhibiting the growth of a pathogenic bacterium in vitro comprising:
co-culturing at least one of the lactobacillus delbrueckii of claim 1 and the microbial agent of claim 2 with a sample containing pathogenic bacteria;
the pathogenic bacteria are selected from at least one of staphylococcus aureus, escherichia coli, salmonella, listeria, shigella and clostridium perfringens.
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Citations (5)
| 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 |
| CN110129220A (en) * | 2019-04-30 | 2019-08-16 | 石河子大学 | A kind of lactobacillus bulgaricus BSTS6-4 and its application |
| CN111621445A (en) * | 2020-06-29 | 2020-09-04 | 张秉胜 | Production method of self-cultured lactobacillus animal beverage |
| CN112625971A (en) * | 2020-12-30 | 2021-04-09 | 杭州娃哈哈科技有限公司 | Lactobacillus delbrueckii capable of consuming a large amount of lactose and resisting acid, bile salt and oxidation and application thereof |
| CN116162562A (en) * | 2022-08-12 | 2023-05-26 | 深圳市盐田区人民医院 | Lactobacillus delbrueckii subspecies and culture method and application thereof |
-
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Patent Citations (5)
| 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 |
| CN110129220A (en) * | 2019-04-30 | 2019-08-16 | 石河子大学 | A kind of lactobacillus bulgaricus BSTS6-4 and its application |
| CN111621445A (en) * | 2020-06-29 | 2020-09-04 | 张秉胜 | Production method of self-cultured lactobacillus animal beverage |
| CN112625971A (en) * | 2020-12-30 | 2021-04-09 | 杭州娃哈哈科技有限公司 | Lactobacillus delbrueckii capable of consuming a large amount of lactose and resisting acid, bile salt and oxidation and application thereof |
| CN116162562A (en) * | 2022-08-12 | 2023-05-26 | 深圳市盐田区人民医院 | Lactobacillus delbrueckii subspecies and culture method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| "In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus";Alexander Arsov 等;《Int. J. Mol. Sci. 》;第23卷;第1-15页 * |
| "具有优良发酵特性德氏乳杆菌 保加利亚亚种的筛选及其益生特性";王磊 等;《现代食品科技》;第37卷(第5期);第43-51页 * |
| 乳酸菌的益生功能及作用机制研究进展;曹振辉 等;《食品工业科技》;第36卷(第24期);第366-370、377页 * |
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