CN117487721A - Lactobacillus brevis and application thereof - Google Patents
Lactobacillus brevis and application thereof Download PDFInfo
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- CN117487721A CN117487721A CN202311712284.2A CN202311712284A CN117487721A CN 117487721 A CN117487721 A CN 117487721A CN 202311712284 A CN202311712284 A CN 202311712284A CN 117487721 A CN117487721 A CN 117487721A
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- lactobacillus brevis
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- 240000001929 Lactobacillus brevis Species 0.000 title claims abstract description 26
- 235000013957 Lactobacillus brevis Nutrition 0.000 title claims abstract description 26
- 150000004676 glycans Chemical class 0.000 claims abstract description 48
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 45
- 239000005017 polysaccharide Substances 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 235000005340 Asparagus officinalis Nutrition 0.000 claims abstract description 18
- 239000000284 extract Substances 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 8
- 230000004151 fermentation Effects 0.000 claims abstract description 8
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- 241000234427 Asparagus Species 0.000 claims description 17
- 238000009835 boiling Methods 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- 235000015278 beef Nutrition 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 229940099596 manganese sulfate Drugs 0.000 claims description 5
- 239000011702 manganese sulphate Substances 0.000 claims description 5
- 235000007079 manganese sulphate Nutrition 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 5
- 229920000053 polysorbate 80 Polymers 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 2
- 239000006286 aqueous extract Substances 0.000 claims 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 abstract description 26
- 235000012000 cholesterol Nutrition 0.000 abstract description 13
- 230000001603 reducing effect Effects 0.000 abstract description 10
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 6
- 235000021395 porridge Nutrition 0.000 abstract description 2
- 244000003416 Asparagus officinalis Species 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 20
- 210000004185 liver Anatomy 0.000 description 15
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 11
- 108010023302 HDL Cholesterol Proteins 0.000 description 7
- 108010028554 LDL Cholesterol Proteins 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 229940079593 drug Drugs 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 5
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- 210000004027 cell Anatomy 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 229920002444 Exopolysaccharide Polymers 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 150000004804 polysaccharides Polymers 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 2
- 238000010876 biochemical test Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
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- 230000000813 microbial effect Effects 0.000 description 2
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- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- PCZOHLXUXFIOCF-UHFFFAOYSA-N Monacolin X Natural products C12C(OC(=O)C(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 PCZOHLXUXFIOCF-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000006694 eating habits Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- PCZOHLXUXFIOCF-BXMDZJJMSA-N lovastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 PCZOHLXUXFIOCF-BXMDZJJMSA-N 0.000 description 1
- 229960004844 lovastatin Drugs 0.000 description 1
- QLJODMDSTUBWDW-UHFFFAOYSA-N lovastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CC(C)C=C21 QLJODMDSTUBWDW-UHFFFAOYSA-N 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
Classifications
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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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- 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
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- 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
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to lactobacillus brevis and application thereof. In order to solve the problems that animal polysaccharide has long production period and high production cost, plant polysaccharide is easy to be influenced by factors such as climate environment, natural disasters and the like, and the yield and quality of the plant polysaccharide are unstable, the invention separates lactobacillus brevis (Levilactobacillus brevis) M-10 from northwest China sour porridge fermentation liquor, and inoculates the strain into a culture medium to produce extracellular polysaccharide for reducing cholesterol, wherein the formula of the culture medium is to replace distilled water in the existing MRS culture medium with asparagus old stem water extract.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to lactobacillus brevis and application thereof.
Background
The hypercholesteremia in human body can cause cardiovascular and cerebrovascular diseases, and the diseases become the biggest single cause of death of the global non-infectious diseases, and the death rate is more than 50 percent. The prevalence of cardiovascular disease has increased in recent years due to aging of the population and unhealthy eating habits. At present, the clinic treatment for high cholesterol mainly uses statin and other chemical medicines. However, statins have significant side effects that can cause damage to the liver and skeletal muscle of some patients. Thus, there is a need to find and develop natural cholesterol-lowering substances with little side effects.
Animal polysaccharides, plant polysaccharides and microbial polysaccharides have all been reported to have cholesterol lowering effects. However, animal polysaccharide is affected by individual differences of animals to cause unstable quality, and the animal polysaccharide has long production period and high production cost; the production of plant polysaccharide is easily affected by climate environment, natural disasters and other factors, and the yield and quality of the plant polysaccharide are unstable. Lactic acid bacteria are well-established and safe microorganisms, and their exopolysaccharides are high molecular polymers secreted outside the cell wall during growth, and are also safe.
Compared with animal polysaccharide and plant polysaccharide, the lactobacillus extracellular polysaccharide has large production scale, is not influenced by environment, and has high yield, low cost, short period and easy obtainment. At present, no report about the function of reducing cholesterol of extracellular polysaccharide of lactobacillus brevis exists at home and abroad.
Disclosure of Invention
The invention provides lactobacillus brevis and application thereof, and extracellular polysaccharide produced by the lactobacillus brevis is used for reducing cholesterol.
The invention adopts the following technical scheme to achieve the aim:
the invention provides a Lactobacillus brevis (Levilactobacillus brevis) M-10, the preservation list is located in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at the following address: the preservation number of the Beijing city Chaoyang area North Chen Xili No. 1 and 3 is CGMCC No.24970.
The invention also provides application of the lactobacillus brevis (Levilactobacillus brevis) M-10 strain for producing extracellular polysaccharide.
The invention also provides application of extracellular polysaccharide produced by the lactobacillus brevis (Levilactobacillus brevis) M-10 in preparing a medicament for reducing cholesterol.
The invention also provides a culture medium for producing extracellular polysaccharide by the lactobacillus brevis (Levilactobacillus brevis) M-10, which comprises the following components:
glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, tween 80 1mL/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, asparagus old stem water extract 800-1000 mL, and pH 6.2-6.6.
Further, the preparation method of the asparagus old stem water extract comprises the following steps: pulverizing 15-25 g of asparagus old stems, passing through 180-200 meshes, adding 100mL of distilled water, boiling for 2-4 hours, and filtering with filter paper to obtain filtrate, wherein the filtrate is asparagus old stem water extract.
The invention also provides a method for producing extracellular polysaccharide by lactobacillus brevis (Levilactobacillus brevis) M-10, which comprises the following steps:
inoculating Lactobacillus brevis (Levilactobacillus brevis) M-10 into the culture medium according to an inoculum size of 5% (v/v), culturing at a constant temperature of 30 ℃ for 25 hours, taking fermentation liquor, boiling in a boiling water bath for 10 minutes, cooling to room temperature, centrifuging at a rotating speed of 10000r/min for 10 minutes at 4 ℃, taking supernatant, standing at 4 ℃ overnight, centrifuging at a rotating speed of 10000r/min for 15 minutes at 4 ℃, collecting supernatant, adding 95% (v/v) ethanol with 3 times volume for precipitation for 20 hours at 4 ℃, centrifuging at a rotating speed of 10000r/min for 15 minutes, collecting precipitate, and vacuum freeze-drying for 14 hours to obtain extracellular polysaccharide.
Compared with the prior art, the invention has the following advantages:
(1) The extracellular polysaccharide is not influenced by external environment, and has the advantages of short period, low cost and stable quality.
(2) The extracellular polysaccharide has high yield, and the yield is up to 4.2g/L fermentation liquor.
(3) The extracellular polysaccharide of the invention is the first extracellular polysaccharide for reducing cholesterol from lactobacillus brevis, and the effect is higher than the cholesterol reducing effect of all polysaccharides at present and higher than the cholesterol reducing effect of the original formula.
(4) Compared with the positive medicine, the effect of the extracellular polysaccharide on reducing serum TC is 92% of that of the positive medicine; the effect of reducing serum LDL-C and liver TC is equal to that of positive medicines. Can be used for developing cholesterol-lowering drugs.
Drawings
FIG. 1 is a morphological map of the cells and a 16S rDNA sequence electrophoresis map;
FIG. 2 is the serum index in example 2;
FIG. 3 shows the contents of liver TC and TG in example 2;
FIG. 4 is the serum index in example 3;
FIG. 5 shows the content of liver TC and TG in example 3;
FIG. 6 is the serum index in example 4;
FIG. 7 shows the contents of liver TC and TG in example 4;
FIG. 8 is the serum index in example 5;
fig. 9 shows the contents of liver TC and TG in example 5.
Detailed Description
In order to further illustrate the technical scheme of the invention, the invention is further illustrated by the following examples.
EXAMPLE 1 identification of bacterial cells
1. Separation and purification
1mL of northwest jin acid porridge fermentation liquid is taken, coated on an MRS flat plate after gradient dilution, and placed in a 30 ℃ incubator for 48 hours. And (3) picking single colonies with clear falling forms, and purifying for 3 times by using a streak separation method to obtain pure colonies.
2. Authentication
(1) Morphology observation: the cell morphology (A in FIG. 1) was observed on MRS plates, and the cell morphology (B and C in FIG. 1) was observed by gram staining, which was seen as gram positive bacteria, short rods, and no branching.
(2) Physiological and biochemical test: pure colonies were inoculated into physiological and biochemical identification medium, placed in a constant temperature incubator at 30℃for cultivation for 24 to 48 hours, and experimental results were recorded (Table 1).
(3) Sequencing of 16S rDNA molecules: extracting microbial genome DNA of a tested strain by a lysozyme-SDS-proteinase-K method, amplifying a 16S rDNA sequence (D in figure 1), carrying out sequencing on the large organism, and comparing sequencing results by an NCBI database and then submitting the sequencing results to Genebank to obtain the accession number MG722900.
In summary, lactobacillus brevis (Levilactobacillus brevis) M-10 was identified.
TABLE 1 results of physiological and biochemical tests
Note that: + indicates that 90% or more of the strains are positive; -represents ≡90% strain negative; d represents 80% -90% of strains positive; d or L represents the optical activity of 90% lactic acid; DL means that 25% -75% of the total lactic acid is form L.
Example 2
Inoculating activated lactobacillus brevis M-10 into a culture medium according to an inoculum size of 5%, wherein the components of the culture medium are as follows: glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, tween 80 1mL/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, asparagus old stem water extract 800mL, and pH 6.2-6.6. The preparation method of the asparagus old stem water extract comprises the following steps: 15g of asparagus old stems are crushed, pass through 180 meshes, add 100mL of distilled water and boil for 2 hours, and filter the mixture with filter paper to obtain filtrate.
After the culture medium is inoculated, the culture is carried out for 25 hours at a constant temperature of 30 ℃ to obtain fermentation liquor, boiling is carried out for 10 minutes in boiling water bath, cooling is carried out to room temperature, centrifugation is carried out (10000 r/min,10 minutes, 4 ℃), supernatant fluid is taken, the final concentration of trichloroacetic acid in the solution is 80%, and standing is carried out at 4 ℃ for overnight. Centrifuging (10000 r/min,15min, 4deg.C), collecting supernatant, adding 95% ethanol 3 times volume, precipitating for 20 hr, centrifuging (10000 r/min,15min, 4deg.C), collecting precipitate, and vacuum lyophilizing for 14 hr to obtain extracellular polysaccharide.
The cholesterol-lowering efficacy of extracellular polysaccharide is detected by the following specific method: 40C 57BL/6N mice were selected and randomly divided into 4 groups after one week of adaptive breeding: control, model, EPS and drug groups, 10 per group. The control group was fed with normal feed, and the model group, EPS group and drug group were fed with high cholesterol feed (normal feed added with 5% lard, 1.2% cholesterol and 0.2% sodium cholate). The EPS group was filled with 0.2mL of extracellular polysaccharide solution (50 mg/kg) daily, the drug group was filled with 0.2mL of lovastatin solution (10 mg/kg) daily, and the control group and the model group were filled with an equivalent amount of physiological saline daily, weighed once a week and recorded. After 5 weeks of gastric lavage, the eyes are removed for blood taking without water inhibition for 12 hours, ether anesthesia is carried out, the whole blood is centrifuged at 4000rpm at 4 ℃, the serum is separated, the contents of Total Cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the serum are measured by an enzyme method kit, and the TC and TG contents in the liver are measured.
As shown in FIG. 2, the serum TC content of the EPS group is remarkably reduced by 13.13% (P < 0.05), the serum TG content and the HDL-C content are not remarkably changed (P > 0.05), and the serum LDL-C content is remarkably reduced (P < 0.05) compared with the model group. As shown in FIG. 3, liver TC content was significantly reduced (P < 0.05) while liver TG content was not significantly changed (P > 0.05).
Example 3:
inoculating activated lactobacillus brevis M-10 into a culture medium according to an inoculum size of 5%, wherein the components of the culture medium are as follows: glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, tween 80 1mL/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, asparagus old stem water extract 900mL, and pH 6.2-6.6. The preparation method of the asparagus old stem water extract comprises pulverizing 20g of asparagus old stem, sieving with 190 mesh sieve, adding 100mL distilled water, boiling for 3h, and filtering with filter paper to obtain filtrate.
After the culture medium is inoculated, the culture is carried out for 25 hours at a constant temperature of 30 ℃ to obtain fermentation liquor, boiling is carried out for 10 minutes in boiling water bath, cooling is carried out to room temperature, centrifugation is carried out (10000 r/min,10 minutes, 4 ℃), supernatant fluid is taken, the final concentration of trichloroacetic acid in the solution is 80%, and standing is carried out at 4 ℃ for overnight. Centrifuging (10000 r/min,15min, 4deg.C), collecting supernatant, adding 95% ethanol 3 times volume, precipitating for 20 hr, centrifuging (10000 r/min,15min, 4deg.C), collecting precipitate, and vacuum lyophilizing for 14 hr to obtain extracellular polysaccharide.
The cholesterol-lowering efficacy of extracellular polysaccharide is tested, the specific method is the same as that of example 2, the test result is shown in fig. 4, and compared with a model group, the serum TC content of the EPS group is obviously reduced by 43.2% (P < 0.01), the serum TG content and HDL-C content are not obviously changed (P > 0.05), and the serum LDL-C content is obviously reduced (P < 0.05). As shown in FIG. 5, the liver TC content was extremely reduced (P < 0.01) while the liver TG content was not significantly changed (P > 0.05).
Example 4:
inoculating activated lactobacillus brevis M-10 into a culture medium according to an inoculum size of 5%, wherein the components of the culture medium are as follows: glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, tween 80 1mL/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, asparagus old stem water extract 1000mL, and pH 6.2-6.6. The preparation method of the asparagus old stem water extract comprises pulverizing 25g asparagus old stem, sieving with 200 mesh sieve, adding 100mL distilled water, boiling for 4h, and filtering with filter paper to obtain filtrate.
Inoculating the culture medium, culturing at 30deg.C for 25 hr to obtain fermentation broth, boiling in boiling water bath for 10min, cooling to room temperature, centrifuging (10000 r/min,10min, 4deg.C), collecting supernatant, and standing at 4deg.C overnight until the final concentration of trichloroacetic acid in solution is 4%. Centrifuging (10000 r/min,15min, 4deg.C), collecting supernatant, adding 95% ethanol 3 times volume, precipitating for 20 hr, centrifuging (10000 r/min,15min, 4deg.C), collecting precipitate, and vacuum lyophilizing for 14 hr to obtain extracellular polysaccharide.
The cholesterol-lowering efficacy of extracellular polysaccharide is detected, the specific method is the same as that of example 2, the detection result is shown in figure 6, compared with a model group, the serum TC content of the EPS group is obviously reduced by 14.25 percent (P is less than 0.05), the serum TG content and HDL-C content are not obviously changed (P is more than 0.05), and the serum LDL-C content is obviously reduced (P is less than 0.05); as shown in FIG. 7, liver TC content was significantly reduced (P < 0.05) while liver TG content was not significantly changed (P > 0.05).
Example 5
An experiment was performed to compare the cholesterol lowering effect of the extracellular polysaccharide obtained in the medium of example 3 with that of the extracellular polysaccharide obtained in the original formula medium (MRS medium). The experimental results are: as shown in FIG. 8, the serum TC content of the exopolysaccharide group (new EPS group) of the present formulation was significantly reduced by 43.2% (P < 0.01) compared to the exopolysaccharide of the original formulation, while the reduction of the original formulation was 31.4% (P < 0.01), which is lower than 11.8% of the present formulation; the extracellular polysaccharide of the formula and the original formula has no obvious reduction effect on the TG content and HDL-C content of serum, and the effect of the extracellular polysaccharide and the HDL-C content is equivalent (P is more than 0.05); the serum LDL-C content of the extracellular polysaccharide group of the formula is obviously reduced by 40 percent (P is less than 0.05), and the content of the extracellular polysaccharide group of the formula is reduced by 17.1 percent (P is less than 0.01). As shown in fig. 9, the liver TC content of the extracellular polysaccharide group (new EPS group) of the present formulation was extremely significantly reduced by 36.3% (P < 0.01), whereas the original formulation was reduced by only 0.09%; the extracellular polysaccharide of the formula and the original formula has no obvious effect of reducing the content of liver TG, and the effect of the extracellular polysaccharide and the original formula is equivalent (P is more than 0.05).
While the principal features and advantages of the present invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (6)
1. The Lactobacillus brevis (Levilactobacillus brevis) M-10 is characterized in that the preservation unit of the Lactobacillus brevis (Levilactobacillus brevis) M-10 is China general microbiological culture Collection center with the preservation number of CGMCC No.24970.
2. Use of lactobacillus brevis (Levilactobacillus brevis) M-10 as claimed in claim 1 for the production of extracellular polysaccharides.
3. Use of an extracellular polysaccharide produced by lactobacillus brevis (Levilactobacillus brevis) M-10 as claimed in claim 1 for the preparation of a cholesterol-lowering medicament.
4. A medium for producing extracellular polysaccharide from lactobacillus brevis (Levilactobacillus brevis) M-10 as claimed in claim 1, wherein the medium comprises the following components:
glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, tween 80 1mL/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, asparagus old stem water extract 800-1000 mL, and pH 6.2-6.6.
5. The culture medium of claim 4, wherein the preparation method of the asparagus old stem aqueous extract comprises the following steps: pulverizing 15-25 g of asparagus old stems, passing through 180-200 meshes, adding 100mL of distilled water, boiling for 2-4 hours, and filtering with filter paper to obtain filtrate, wherein the filtrate is asparagus old stem water extract.
6. A method for producing extracellular polysaccharide by lactobacillus brevis (Levilactobacillus brevis) M-10, comprising the steps of:
inoculating Lactobacillus brevis (Levilactobacillus brevis) M-10 of claim 1 into the culture medium of claim 4 or 5 according to an inoculum size of 5% (v/v), culturing at 30 ℃ for 25 hours, taking fermentation liquor, boiling in boiling water bath for 10 minutes, cooling to room temperature, centrifuging at 4 ℃ for 10 minutes at a rotational speed of 10000r/min, taking supernatant, standing at 4 ℃ overnight, centrifuging at 4 ℃ for 15 minutes at a rotational speed of 10000r/min, collecting supernatant, adding 95% (v/v) ethanol for precipitation for 20 hours at 3 times volume, centrifuging at 4 ℃ for 15 minutes at a rotational speed of 10000r/min, collecting precipitate, and vacuum freeze-drying for 14 hours to obtain extracellular polysaccharide.
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