CN117987302A - Pediococcus pentosaceus and bacteriostat prepared from same - Google Patents
Pediococcus pentosaceus and bacteriostat prepared from same Download PDFInfo
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- CN117987302A CN117987302A CN202410031480.1A CN202410031480A CN117987302A CN 117987302 A CN117987302 A CN 117987302A CN 202410031480 A CN202410031480 A CN 202410031480A CN 117987302 A CN117987302 A CN 117987302A
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- pediococcus pentosaceus
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- 241000191996 Pediococcus pentosaceus Species 0.000 title claims abstract description 62
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 11
- 241000588724 Escherichia coli Species 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 239000000022 bacteriostatic agent Substances 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims description 12
- 230000003385 bacteriostatic effect Effects 0.000 claims description 11
- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 238000009472 formulation Methods 0.000 claims description 8
- 239000004480 active ingredient Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000003674 animal food additive Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000009631 Broth culture Methods 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 206010061126 Escherichia infection Diseases 0.000 claims 1
- 206010041925 Staphylococcal infections Diseases 0.000 claims 1
- 208000020612 escherichia coli infection Diseases 0.000 claims 1
- 241000186660 Lactobacillus Species 0.000 abstract description 10
- 229940039696 lactobacillus Drugs 0.000 abstract description 10
- 241001465754 Metazoa Species 0.000 abstract description 7
- 241000272814 Anser sp. Species 0.000 abstract description 6
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- 239000002253 acid Substances 0.000 abstract description 6
- 239000012588 trypsin Substances 0.000 abstract description 6
- 230000005764 inhibitory process Effects 0.000 abstract description 4
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- 230000036039 immunity Effects 0.000 abstract description 3
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- 244000005700 microbiome Species 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 15
- 239000003833 bile salt Substances 0.000 description 10
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 6
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- 239000008272 agar Substances 0.000 description 5
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 4
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
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- 238000012986 modification Methods 0.000 description 4
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- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 210000004211 gastric acid Anatomy 0.000 description 3
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 3
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 3
- 210000004877 mucosa Anatomy 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
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- 230000001580 bacterial effect Effects 0.000 description 2
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- 239000011248 coating agent Substances 0.000 description 2
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- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241000446292 Pediococcus pentosaceus SL4 Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
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- 210000005000 reproductive tract Anatomy 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
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- 239000008223 sterile water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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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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- 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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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus pentosaceus and a bacteriostatic agent prepared from the pediococcus pentosaceus. The Pediococcus pentosaceus (Pediococcuspentosaceus) AK-GRw1 has the preservation number of: CCTCCNO: M20231980. The pediococcus pentosaceus AK-GRw1 has excellent trypsin resistance and acid resistance, has good inhibition performance on escherichia coli and staphylococcus aureus, can promote the growth of geese, establish good intestinal flora ecology, improve immunity, influence and improve animal production and health when being added into goose feed, and provides a better reference basis for microecologics for expanding goose-source lactobacillus resource libraries and application.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus pentosaceus and a bacteriostatic agent prepared from the pediococcus pentosaceus.
Background
In the livestock breeding industry, the microecological preparation can be used as an environment-friendly feed additive to improve the microecological environment of the intestinal tracts of livestock, promote the proliferation of beneficial bacteria in the intestinal tracts of the livestock, prevent or inhibit the proliferation of harmful bacteria, adjust the balance of intestinal flora, obviously reduce the ammonia odor of animal feces, reduce mosquitoes, flies and insects, improve and optimize the ecological environment of livestock breeding, reduce environmental pollution, improve the anti-stress capability and immunity of animals, improve the conversion rate of animal feeds, reduce the production cost and increase the economic benefit. With the gradual rise of requirements and demands of the breeding industry, the breeding industry of geese also develops rapidly. However, compared with other mature poultry cultivation modes, the goose cultivation technology is relatively lagged, and the problems of scattered cultivation, non-standardization, low efficiency, frequent occurrence of bacterial diseases and the like appear. Nowadays, antibiotic-free cultivation becomes an inevitable trend of the development of the animal husbandry in the future, and people increasingly pursue safe and green foods. Accordingly, efforts are underway to find feed additives that can replace antibiotics.
The lactobacillus can decompose toxic and harmful substances generated in the culture environment, inhibit the growth of harmful bacteria in the water body, and play a role in purifying the water quality; has stronger protease, lipase and amylase activities, and promotes the absorption and utilization efficiency of feed; can stimulate the development of immune organs, enhance the immunity and the resistance of organisms, can improve the intestinal functions of organisms through substances such as bacteriocins and the like generated by the organisms, and is one of common strains widely applied to the treatment and prevention of digestive tract and genital tract diseases of people and animals.
However, most lactobacillus strains of existing lactobacillus preparations in the market are screened and separated from natural environment or intestinal tracts of original animals, and the strains separated from the environment are used for fermentation, so that the antibacterial effect, gastric acid bile salt and other tolerance capacities are not strong, the safety performance is also uneven, and the application of the lactobacillus preparations is limited. Therefore, the lactobacillus with stable bacteriostasis and strong gastric acid and bile salt tolerance is found to have wide prospect.
Disclosure of Invention
The invention aims to overcome the defects of unstable bacteriostasis capability of lactobacillus and weak tolerance capability to gastric acid bile salt in the prior art and provide pediococcus pentosaceus and a bacteriostat prepared from the pediococcus pentosaceus.
In order to solve the technical problems, the invention adopts the following technical scheme.
In a first aspect, the invention provides a pediococcus pentosaceus (Pediococcus pentosaceus) AK-GRw1 with the preservation number: CCTCC NO: M20231980.
The second aspect of the invention provides an application of Pediococcus pentosaceus AK-GRw1 in preparing a bacteriostatic preparation.
In some embodiments of the invention, the bacteriostatic formulation comprises a culture of Pediococcus pentosaceus AK-GRw 1.
In some embodiments of the invention, the culture is a fermentation broth of Pediococcus pentosaceus AK-GRw 1.
In some embodiments of the present invention, the preparation method of the fermentation broth of Pediococcus pentosaceus AK-GRw1 comprises: inoculating the Pediococcus pentosaceus AK-GRw to a broth culture medium containing MRS, shake culturing for 20-26h, and removing thalli to obtain fermentation broth of Pediococcus pentosaceus AK-GRw 1.
In some embodiments of the invention, the bacteriostatic species of the bacteriostatic formulation include staphylococcus aureus and/or escherichia coli.
In a third aspect, the invention provides the use of Pediococcus pentosaceus AK-GRw1 in the manufacture of a medicament for the treatment of diseases caused by infection with Staphylococcus aureus and/or Escherichia coli.
In a fourth aspect, the present invention provides a bacteriostatic agent comprising as an active ingredient a culture of Pediococcus pentosaceus AK-GRw1 or Pediococcus pentosaceus AK-GRw.
In a fifth aspect, the present invention provides a medicament comprising as an active ingredient the Pediococcus pentosaceus AK-GRw1 or a culture of the Pediococcus pentosaceus AK-GRw 1.
In a sixth aspect, the present invention provides a feed additive, wherein the active ingredient is the Pediococcus pentosaceus AK-GRw1 or the Pediococcus pentosaceus AK-GRw1 culture.
Compared with the prior art, the invention has the following beneficial effects: the invention screens the pediococcus pentosaceus AK-GRw1 which is a goose source lactobacillus and has a probiotic effect from the intestinal tracts of healthy geese. The Pediococcus pentosaceus AK-GRw1 with the characteristics of inhibiting the growth and the colonization of pathogenic bacteria, having strong adhesion colonization capacity and the like is screened out from a large number of strains, and a better microecological preparation reference basis is provided for expanding a goose-source lactobacillus resource library and application.
The Pediococcus pentosaceus AK-GRw1 provided by the invention has the survival rate of 90.63% when the pH value is=2; the survival rate reaches 47.79% at a bile salt concentration of 0.2%, and even at a bile salt concentration of 0.6%, the survival rate can reach 28.60%. The trypsin-resistant performance is excellent, and the survival rate reaches 120.72% when the trypsin concentration is 0.8%. Pediococcus pentosaceus AK-GRw1 has excellent bacteriostasis to colibacillus and staphylococcus aureus, and has bacteriostasis area of 26mm to staphylococcus aureus and 14mm to colibacillus.
Preservation of biological materials
Pediococcus pentosaceus PediococcuspentosaceusAK-GRw1 is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of M20231980 and at the preservation address of eight paths of Lojia mountain in Wuchang area of Wuhan, hubei province at 10 and 23 days.
Drawings
FIG. 1 is a graph showing the characteristics of Pediococcus pentosaceus AK-GRw MRS agar culture.
FIG. 2 is a graph showing the results of culture of Pediococcus pentosaceus AK-GRw MRS broth.
FIG. 3 is a graph showing the results of gram staining of Pediococcus pentosaceus AK-GRw 1.
FIG. 4 is a graph showing the results of the Pediococcus pentosaceus AK-GRw1 indole formation assay, nitrate reduction assay, VP assay, and methyl red assay.
FIG. 5 is a graph showing the results of determination of sucrose, glucose, maltose and lactose by Pediococcus pentosaceus AK-GRw.
FIG. 6 is a graph showing the results of 16S rRNA sequencing of Pediococcus pentosaceus AK-GRw 1.
FIG. 7 is a graph showing the result of inhibition of E.coli by Pediococcus pentosaceus AK-GRw 1.
FIG. 8 is a graph showing the result of a circle of Pediococcus pentosaceus AK-GRw1 against Staphylococcus aureus.
FIG. 9 is a 26h growth plot of Pediococcus pentosaceus AK-GRw 1.
FIG. 10 is a graph of 26hpH variation of Pediococcus pentosaceus AK-GRw 1.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
EXAMPLE 1 isolation culture and purification of seed
Scraping the mucosa of the intestinal segment of the healthy goose by utilizing the back of a sterile scalpel in an ultra-clean workbench, putting the mucosa into a sterile test tube filled with normal saline, shaking the mucosa uniformly, immediately carrying out gradient dilution by using a raw Lianshui, taking a diluent 100 with a dilution gradient of 10 -4,10-5,10-6, and dripping the diluent 100 into an agar culture medium of MRS-CaCO 3 for coating a flat plate. Oxygen in the tank is consumed by a glowing jar method, anaerobic culture is carried out overnight, and plates with good growth vigor and not crowded colonies are selected for further screening. Colony selection criteria were: the colony has good growth vigor, white color, raised round surface, regular moist edge, and large calcium dissolving ring. The conditioned colonies were streaked with the loop and cultured in agar medium of MRS-CaCO 3, and the cycle was repeated at least 5 times. Therefore, a plurality of pure strain can be primarily screened, and the strain is named AK-GRw1. Colonies on agar medium of MRS-CaCO 3 are shown in FIG. 1, and growth after overnight culture in MRS liquid medium is shown as turbidity of white viscous precipitant liquid at bottom as shown in FIG. 2.
2. Smear dyeing inspection
The single purified colony was picked for smear, gram stain and microscopic oil observation, and AK-GRw1 bacteria were found to be single or multiple arranged, spherical blue-violet gram positive bacteria, and the results are shown in FIG. 3.
3. Biochemical test
Glucose, lactose, maltose, sucrose fermentation test, methyl red test, VP test, nitrate reduction test and indole formation test were performed separately: the bacteria to be detected are inoculated into peptone water and glucose peptone water respectively by an inoculating loop or an inoculating needle, and sugar fermentation tubes (glucose, lactose, maltose and sucrose) are cultivated for 24-48h at 37 ℃.
The results are shown in figures 2-3, which show that AK-GRw1 can decompose glucose, maltose, sucrose and lactose; the methyl red test and the indole formation test are positive, the color is changed into red, the VP test and the nitrate reduction test are negative, and no color change exists.
4. And (3) PCR identification: PCR amplification was performed using the universal primers 27F and 1525R of the bacterial 16S rRNA gene.
(1) Nucleic acid extraction: the individual colonies of the bacteria were picked with an inoculating loop and inoculated into MRS broth, cultured with shaking at 37℃and 120rpm for 24 hours. Then, the nucleic acid was extracted using Ezup column type bacterial genomic DNA extraction kit from Shanghai Biotechnology Co., ltd.
(2) PCR amplification and sequencing analysis of 16 SrRNA: the PCR reaction system was 50. Mu.L: PCRMASTER MIX. Mu.L, 2. Mu.L of upper primer, 2. Mu.L of lower primer, 5. Mu.L of DNA template and 16. Mu.L of sterile water. PCR reaction conditions: 95 ℃ for 5min;95℃30s,55℃30s,72℃1min,30 cycles; and at 72℃for 10min. The PCR product is detected by agarose gel electrophoresis and then sent to Shanghai Biotechnology, inc. for sequencing, the sequence of the PCR product is shown as SEQ ID NO:1, the sequencing result is shown as figure 6, the sequencing result is compared and analyzed in a nucleic acid database on NCBI website, the 16S rRNA genes of AK-GRw bacteria and Pediococcus pentosaceus SL4 strain are found, genBank accession number: CP006854.1, homology of 100.00%, confirms that the test bacteria are Pediococcus pentosaceus. The results are shown in Table 1.
TABLE 1 results of comparative analysis in NCBI nucleic acid database
5. Acid and bile salt resistance and digestive enzyme resistance test
Adding 100 mu L of AK-GRw bacteria liquid into 10mLMRS broth, and shake culturing at 37deg.C and 120rpm for 24 hr; centrifuging at 8000r/min for 10min to collect thalli; the cells were suspended in MRS broth having pH values of 2 and 3, bile salt concentrations of 0.2% and 0.6%, and trypsin concentrations of 0.8% and 1.2%, respectively, and incubated at 37℃for 0 and 4 hours, and absorbance was sampled and measured, respectively, to calculate survival rate. The calculation formula is as follows: survival = X2/X1X 100%. Wherein: x1 is the number of viable bacteria in MRS broth with different pH values, bile salts and trypsin concentrations when cultured for 0 hour, and X2 is the number of viable bacteria in cfu/mL when cultured for 4 hours. See in particular table 2.
TABLE 2 acid and bile salt resistance and digestive enzyme resistance test results
6. Oxford cup plate method bacteriostasis test
(1) Preparing a nutrient agar plate and a MAIKAI agar plate, and placing the plates into a refrigerator at 4 ℃ for later use;
(2) Culturing the indicator bacteria in a proper liquid culture medium and under proper culture conditions overnight;
(3) Inoculating AK-GRw strain into a 10mL MRS broth test tube, culturing at 37deg.C under shaking at 120rpm for 24h, centrifuging at 5000rpm for 15min, removing thallus, and collecting supernatant to obtain AK-GRw1 cell-free fermentation supernatant.
(4) Sucking a certain concentration of the indicator fungus suspension on a flat plate in one step, uniformly coating with a sterile glass rake, slightly placing sterile oxford cups into a culture dish with forceps, and uniformly placing 3 or 4 oxford cups in the horizontally placed flat plate;
(5) AK-GRw1 cell-free fermentation supernatant is added into the hole of the oxford cup, and the mixture is diffused for 12 hours in a refrigerator at the temperature of 4 ℃;
(6) After culturing at 37 ℃ for 24 hours, the diameter of the inhibition zone is measured by a vernier caliper.
1 Goose-derived pathogenic escherichia coli and 1 staphylococcus aureus which are separately stored in the laboratory are respectively selected as indicator bacteria, and antibacterial tests are respectively carried out. The results show that the specific inhibition zone size separated in the invention is shown in Table 3, and the phenomenon is shown in figures 7-8.
TABLE 3 antibacterial test results of AK-GRw strain on pathogenic bacteria
7. Analysis of growth Properties and lactic acid production Capacity
AK-GRw strain was inoculated into MRS broth, cultured with shaking at 37℃and 120rpm, sampled 1 time every 2 hours, and OD and pH values at 600nm were determined with respect to the sterile MRS broth, and corresponding curves were drawn with respect to the culture time on the abscissa and the OD and pH values on the ordinate, respectively. See fig. 9 and 10. From the graph, AK-GRw1 bacteria starts to increase rapidly when entering the logarithmic phase at 6h, and the concentration change is small when entering the platform phase at about 20 h. The acid yield of AK-GRw1 bacteria is rapidly increased at 6h, and the acid yield is stabilized at 18h, so that the pH value in the fermentation liquor is about 4.
The lactobacillus AK-GRw1 strain with excellent performance is separated from the cecum of the goose, and is identified as Pediococcus pentosaceus. Experiments show that the AK-GRw1 strain obtained by separation has good acid resistance, general bile salt resistance and excellent trypsin resistance, and the AK-GRw strain has good antibacterial performance on escherichia coli and staphylococcus aureus, can reach 26mm for the antibacterial zone of staphylococcus aureus and can also reach 14mm for the antibacterial zone of escherichia coli.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. Pediococcus pentosaceus (Pediococcuspentosaceus) AK-GRw1 is characterized in that the preservation number is CCTCC NO: M20231980.
2. Use of pediococcus pentosaceus AK-GRw1 as defined in claim 1 for the preparation of a bacteriostatic formulation.
3. Use of pediococcus pentosaceus AK-GRw1 according to claim 2 for the preparation of a bacteriostatic formulation containing a culture of pediococcus pentosaceus AK-GRw 1.
4. Use of pediococcus pentosaceus AK-GRw1 according to claim 3 for the preparation of a bacteriostatic formulation, characterized in that the culture is a fermentation broth of pediococcus pentosaceus AK-GRw 1.
5. The use of pediococcus pentosaceus AK-GRw1 as claimed in claim 4 for the preparation of a bacteriostatic formulation, characterized in that the preparation method of the fermentation broth of pediococcus pentosaceus AK-GRw1 comprises:
Inoculating the Pediococcus pentosaceus AK-GRw to a broth culture medium containing MRS, shake culturing for 20-26h, and removing thalli to obtain a Pediococcus pentosaceus AK-GRw1 culture.
6. Use of pediococcus pentosaceus AK-GRw1 according to claim 2 for the preparation of a bacteriostatic formulation, characterized in that the bacteriostatic species of the bacteriostatic formulation comprise staphylococcus aureus and/or escherichia coli.
7. Use of pediococcus pentosaceus AK-GRw1 as claimed in claim 1 for the manufacture of a medicament for the treatment of diseases caused by staphylococcus aureus and/or escherichia coli infections.
8. A bacteriostatic agent comprising the pediococcus pentosaceus AK-GRw1 or a culture of pediococcus pentosaceus AK-GRw1 as an active ingredient.
9. A medicament, characterized in that the active ingredient is Pediococcus pentosaceus AK-GRw1 or a culture of Pediococcus pentosaceus AK-GRw1 according to claim 1.
10. A feed additive comprising Pediococcus pentosaceus AK-GRw1 or a culture of Pediococcus pentosaceus AK-GRw1 as an active ingredient.
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