CN106967630B - Lactobacillus fermentum and application thereof - Google Patents
Lactobacillus fermentum and application thereof Download PDFInfo
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- CN106967630B CN106967630B CN201611091698.8A CN201611091698A CN106967630B CN 106967630 B CN106967630 B CN 106967630B CN 201611091698 A CN201611091698 A CN 201611091698A CN 106967630 B CN106967630 B CN 106967630B
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- 241000186840 Lactobacillus fermentum Species 0.000 title claims abstract description 33
- 229940012969 lactobacillus fermentum Drugs 0.000 title claims abstract description 33
- 241000588724 Escherichia coli Species 0.000 claims abstract description 10
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 10
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 2
- 230000000844 anti-bacterial effect Effects 0.000 claims 1
- 239000003833 bile salt Substances 0.000 abstract description 15
- 239000002253 acid Substances 0.000 abstract description 10
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 8
- 238000004321 preservation Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 16
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 238000009630 liquid culture Methods 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000006041 probiotic Substances 0.000 description 4
- 235000018291 probiotics Nutrition 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 229940099352 cholate Drugs 0.000 description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound 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 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 230000000529 probiotic effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 2
- 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 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- 235000007079 manganese sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 239000001393 triammonium citrate Substances 0.000 description 2
- 235000011046 triammonium citrate Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000004584 Tamarindus indica Species 0.000 description 1
- 235000004298 Tamarindus indica Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
Classifications
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- 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
-
- 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
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- 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
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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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- Virology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses lactobacillus fermentum and application thereof. The preservation number is as follows: GDMCC No. 60112. The Lactobacillus fermentum DJ8A has strong acid resistance and bile salt resistance, and has obvious bacteriostatic effect on escherichia coli and staphylococcus aureus, so that the Lactobacillus fermentum DJ8A can be used for preparing antibacterial drugs and has wide market prospect.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to lactobacillus fermentum and application thereof.
Background
The feed added with antibiotics can resist diseases, promote the growth of animals and accelerate the development of intensive animal husbandry. However, with the continuous enhancement of the concept of green, safety and environmental protection and the continuous emphasis on the health degree, the antibiotic additive brings great economic benefits to people and simultaneously has gradually prominent negative effects, and the most typical characteristics are drug resistance, endogenous infection, secondary infection and drug residue caused by antibiotics, which bring serious threats to the breeding industry, the feed industry and animals, thereby endangering the health of people through the food chain. Therefore, there is an increasing call to limit or even ban the use of antibiotics in the feed and aquaculture industries, and research and development of new antibiotic substitutes is slow.
In recent years, probiotics have been the primary choice as antibiotic substitutes because they are safe, non-toxic, non-residue, do not induce drug resistance, and have significant effects on animal growth. The lactobacillus is a main strain in the probiotics, is an animal digestive tract inhabiting microorganism, has better functions in preventing and treating gastrointestinal tract infection, regulating immunologic function and regulating the balance of microbial flora of intestinal tracts, and is a promising probiotic production strain.
At present, the development and utilization of lactic acid bacteria products become a new trend for the development of the feed industry in China, but some problems to be solved urgently exist. Such as poor probiotic effect, unstable product quality, insufficient viable bacteria amount, poor stress resistance and the like. Therefore, the screening of lactobacillus products with good probiotic performance, strong tolerance, high propagation speed and stable quality has great significance for improving the market competitiveness of the products and promoting the development of animal husbandry.
Disclosure of Invention
The invention aims to provide the Lactobacillus fermentum DJ8A which has strong acid resistance and bile salt resistance and obvious bacteriostatic effect on escherichia coli and staphylococcus aureus.
Lactobacillus fermentum DJ8A, deposited at the guangdong province collection of microorganisms (GDMCC) at 11/15 days 2016, address: five stories of the experimental building of the microbial institute, one hundred provinces, in the city of Guangzhou, China, the postcode: 510070, accession number: GDMCC No. 60112.
The second purpose of the invention is to provide the application of Lactobacillus fermentum DJ8A in preparing antibacterial drugs.
The antibacterial drug is a drug for resisting staphylococcus aureus or escherichia coli.
The Lactobacillus fermentum DJ8A has strong acid resistance and bile salt resistance, and has obvious bacteriostatic effect on escherichia coli and staphylococcus aureus, so that the Lactobacillus fermentum DJ8A can be used for preparing antibacterial drugs and has wide market prospect.
Lactobacillus fermentum DJ8A, deposited at the guangdong province collection of microorganisms (GDMCC) at 11/15 days 2016, address: five stories of the experimental building of the microbial institute, one hundred provinces, in the city of Guangzhou, China, the postcode: 510070, accession number: GDMCC No. 60112.
description of the drawings:
FIG. 1 shows the morphological characteristics of the cultured colonies of Lactobacillus fermentum DJ8A according to the invention on MRS medium.
FIG. 2 is a microscopic morphological feature of Lactobacillus fermentum DJ8A of the present invention.
FIG. 3 shows the bacteriostatic ability of fermentation supernatant of Lactobacillus fermentum DJ8A of the invention against Escherichia coli, 1: fermentation supernatant of Lactobacillus fermentum (DJ 8A); 2: fermentation supernatant of strain DJ 9B; secondly, the step of: pH 4.45MRS blank medium; ③: pH 6.60MRS blank medium.
Fig. 4 is the bacteriostatic ability of the fermentation supernatant of Lactobacillus fermentum DJ8A against staphylococcus aureus, 1: fermentation supernatant of Lactobacillus fermentum (DJ 8A); 2: fermentation supernatant of strain DJ 9B; secondly, the step of: pH 4.45MRS blank medium; ③: pH 6.60MRS blank medium.
The specific implementation mode is as follows:
The following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications made to the invention in light of the spirit thereof are intended to be included within the scope of the present invention as claimed.
Example 1: separation and purification of bacterial strains
The method comprises the steps of purchasing fermented flavor food (pickled Chinese cabbage, tamarind and preserved Meicai) sold in the Guangzhou Jielie Zhongcai market place, adding proper normal saline for aseptic grinding, diluting grinding liquid to a proper concentration by using the normal saline, coating 3 bacterial liquids with proper dilution on CaCO 3 -MRS (peptone 10g, beef extract 10g, yeast powder 4g, dipotassium hydrogen phosphate 2g, triammonium citrate 2g, sodium acetate 5g, glucose 20g, calcium carbonate 20g, Tween-801.08 g, magnesium sulfate 0.2g, manganese sulfate 0.05g, tertiary water to a constant volume of 1L, adjusting pH to 7.2, sterilizing and preparing a plate for later use), carrying out anaerobic culture at 37 ℃ for 48H, selecting bacteria with larger calcium-dissolving rings on MRS (formula as above, without calcium carbonate) plates for streaking and separating, repeatedly purifying, carrying out gram staining, microscopic examination and H 2 O 2 contact enzyme tests on purified bacterial colonies, selecting gram-positive bacterial strains without spores and contact enzyme-negative bacterial strains, placing the bacterial strains in a 10% reserved glycerin tube, and carrying out 80 ℃ common separation on the bacterial strains without spores and the gram-positive bacterial strains.
Adjusting MRS liquid culture medium (10 g of peptone, 10g of beef extract, 4g of yeast powder, 2g of dipotassium phosphate, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 801.08 g of tween-801.08, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, and three-stage water to a constant volume of 1L, adjusting the pH to 7.2, and sterilizing for later use) to pH 4.0, 3.0 and 2.5, activating and culturing lactic acid bacteria obtained by primary screening until logarithmic growth phase, respectively inoculating the lactic acid bacteria into MRS liquid culture medium with pH 4.0, 3.0 and 2.5 according to the inoculation amount of 2% of volume ratio, carrying out anaerobic culture at 37 ℃ for 24h, measuring the OD 600nm value of each group of bacteria liquid by taking the uninoculated culture medium as a control, and selecting the lactic acid bacteria with better acid resistance for subsequent experiments.
The experiment shows that 48 lactic acid bacteria can grow in MRS liquid culture medium with pH 4.0, and 10 lactic acid bacteria can grow in environment with pH 3.0, wherein strains DJ8A and DJ10C grow well in environment with pH 3.0.
The strains obtained by the above tests and purification are all preserved in Guangdong province microorganism strain preservation center.
Example 2: acid and bile salt resistance test of strain
Selecting a strain capable of growing at pH 3.0, re-culturing to a logarithmic growth phase, inoculating the strain into an MRS liquid culture medium containing 0.1%, 0.2% and 0.3% of cholate according to an inoculation amount of 2% in volume ratio, carrying out anaerobic culture at 37 ℃ for 24h, measuring the OD 600nm value of each group of bacteria liquid by taking an uninoculated culture medium with corresponding cholate content as a control, and selecting lactobacillus with better cholate resistance for subsequent experiments.
In the experiment, 10 strains which can grow under the environment of pH 3.0 and are mentioned in example 1 can grow in 0.1% of the bovine bile salt, but can not grow in 0.2% and 0.3% of the bovine bile salt, and the strain DJ8A can well grow in 0.1% of the bovine bile salt.
Example 3 species identification
(1) Morphological identification: spreading and separating the screened strains on an MRS plate, carrying out anaerobic culture at 37 ℃ for 24h, and observing the colony characteristics; taking a single colony smear, gram staining, and observing the individual morphology under a microscope.
After the strain DJ8A is cultured on an MRS plate at 37 ℃ for 48 hours, the bacterial colony is round, flat and white, the surface is smooth and moist, the edge is neat, and the diameter is 3-4mm (figure 1); the microscopic morphology of strain DJ8A was characterized by short rods, single, paired or long strands, and by the absence of sporulation, gram-positive (FIG. 2).
(2) Molecular identification of 16S rRNA:
Extracting genome DNA of a strain DJ8A to be identified by adopting a CTAB method, and carrying out PCR amplification by adopting a bacterial 16S rRNA universal primer. The PCR product was sent to McJK, Inc., for sequencing. And submitting the sequencing result to GenBank for Blast comparison analysis.
The 16S rRNA gene sequence of the strain DJ8A is sequenced (the sequence is shown as SEQ ID NO. 1), analyzed and compared, and the homology of the sequence and the reported 16S rRNA gene sequence of Lactobacillus fermentum CECT562(T) reaches 99.65 percent, and the strain is identified as Lactobacillus fermentum DJ8A which is preserved in Guangdong province microbial culture collection center (GDMCC) at 2016, 11 and 15 days, and the address: five stories of the experimental building of the microbial institute, one hundred provinces, in the city of Guangzhou, China, the postcode: 510070, accession number: GDMCC No. 60112.
Example 4 Lactobacillus fermentum DJ8A acid production ability and bacteriostatic ability examination
(1) Acid-producing ability: activating and culturing Lactobacillus fermentum DJ8A for 48h, selecting single colony, inoculating to MRS liquid culture medium with pH 6.60 and pH 3.85, performing anaerobic culture at 37 deg.C for 15h, centrifuging at 4 deg.C and 10000 rpm for 10min, collecting supernatant, measuring pH, and performing equal treatment with non-inoculated MRS liquid culture medium as blank control.
After 15h of cultivation, Lactobacillus fermentum DJ8A lowered the pH of MRS liquid medium at pH 6.60 and pH 3.85 to 4.49 and 3.34, respectively.
(2) Taking fermentation supernatant of Lactobacillus fermentum DJ8A, taking escherichia coli and staphylococcus aureus as indicator bacteria, taking MRS culture medium adjusted to corresponding pH as a control, taking NA as culture medium, and carrying out bacteriostasis experiment by using an Oxford cup punching method, transferring escherichia coli and staphylococcus aureus again according to 1% inoculation amount, carrying out shaking culture at 28 ℃, 180rpm for 17h, wherein the thallus concentration is about 10 7 cfu/ml, taking 2ml of cultured escherichia coli or 2ml of cultured staphylococcus aureus and 200ml of NA culture medium which is melted and kept at about 45 ℃ to be uniformly mixed, placing 4 sterilized Oxford cups on a sterile plate, sucking 20ml of cover plate by using a sterile pipette, taking out the Oxford cups after solidification, respectively adding 100uL of Lactobacillus supernatant sample or MRS control culture medium adjusted to corresponding pH, carrying out culture at 37 ℃ for 24h, and observing the result.
The experiment result shows that the fermentation supernatant (pH 4.49) of the strain DJ8A has obvious bacteriostatic effect on escherichia coli and staphylococcus aureus (Table 1, figure 3 and figure 4).
TABLE 1 bacteriostatic ability of fermented supernatants of Lactobacillus fermentum DJ8A
Example 5 Lactobacillus fermentum DJ8A acid and bile salt tolerance study
Preparing an MRS liquid culture medium with pH of 2.0 and no bile salt and an MRS liquid culture medium with pH of 6.6 and containing 0.3% of bile salt, transferring Lactobacillus fermentum DJ8A bacterial liquid which is anaerobically cultured for 15 hours at 37 ℃ into the MRS liquid culture medium with pH of 2.0 and the MRS liquid culture medium containing 0.3% of bile salt according to the inoculation amount of 1% of the volume ratio, anaerobically culturing for 3 hours and 6 hours at 37 ℃, coating plates by proper dilution times, repeating three parallel experiments for each group, and selecting the plates with the colony number ranging from 30 to 300 for colony counting after culturing for 48 hours at 37 ℃.
The results show that Lactobacillus fermentum DJ8A can grow under the environment of pH 3.0 or 0.1% of bile salt, the viable count is still above 10 7 cfu/mL after 3 hours of treatment under the condition of pH 2.0 or 0.3% of bile salt, the viable count is still above 10 6 cfu/mL after 6 hours of treatment, and the strain DJ8A has strong acid resistance and bile salt resistance (table 2).
TABLE 2 acid and bile salt resistance of Lactobacillus fermentum DJ8A
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> lactobacillus fermentum and application thereof
<160>1
<210>1
<211>861
<212>DNA
<213> Lactobacillus fermentum DJ8A
<400> 1
TGCAGTCGAA CGCGTTGGCC CAATTGATTG ATGGTGCTTG CACCTGATTG ATTTTGGTCG 60
CCAACGAGTG GCGGACGGGT GAGTAACACG TAGGTAACCT GCCCAGAAGC GGGGGACAAC 120
ATTTGGAAAC AGATGCTAAT ACCGCATAAC AACGTTGTTC GCATGAACAA CGCTTAAAAG 180
ATGGCTTCTC GCTATCACTT CTGGATGGAC CTGCGGTGCA TTAGCTTGTT GGTGGGGTAA 240
TGGCCTACCA AGGCGATGAT GCATAGCCGA GTTGAGAGAC TGATCGGCCA CAATGGGACT 300
GAGACACGGC CCATACTCCT ACGGGAGGCA GCAGTAGGGA ATCTTCCACA ATGGGCGCAA 360
GCCTGATGGA GCAACACCGC GTGAGTGAAG AAGGGTTTCG GCTCGTAAAG CTCTGTTGTT 420
AAAGAAGAAC ACGTATGAGA GTAACTGTTC ATACGTTGAC GGTATTTAAC CAGAAAGTCA 480
CGGCTAACTA CGTGCCAGCA GCCGCGGTAA TACGTAGGTG GCAAGCGTTA TCCGGATTTA 540
TTGGGCGTAA AGAGAGTGCA GGCGGTTTTC TAAGTCTGAT GTGAAAGCCT TCGGCTTAAC 600
CGGAGAAGTG CATCGGAAAC TGGATAACTT GAGTGCAGAA GAGGGTAGTG GAACTCCATG 660
TGTAGCGGTG GAATGCGTAG ATATATGGAA GAACACCAGT GGCGAAGGCG GCTACCTGGT 720
CTGCAACTGA CGCTGAGACT CGAAAGCATG GGTAGCGAAC AGGATTAGAT ACCCTGGTAG 780
TCCATGCCGT AAACGATGAG TGCTAGGTGT TGGAGGGTTT CCGCCCTTCA GTGCCGGAGC 840
TAACGCATTA AGCACTCCGC C 861
Claims (2)
1. Lactobacillus fermentum DJ8A with accession number GDMCC No. 60112.
2. Use of a Lactobacillus fermentum (DJ 8A) according to claim 1 for the preparation of an antibacterial medicament; the antibacterial drug is a drug for resisting staphylococcus aureus or escherichia coli.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN101067119A (en) * | 2006-11-29 | 2007-11-07 | 康哲医药研究(深圳)有限公司 | Fermenting lactobacillus CMS II002 and its application |
| CN105132328A (en) * | 2015-09-14 | 2015-12-09 | 西南大学 | Lactobacillus fermentum strain suo capable of preventing gastric ulcers and application of lactobacillus fermentum strain suo |
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