CN118064301A - Weissella multocida and culture thereof - Google Patents
Weissella multocida and culture thereof Download PDFInfo
- Publication number
- CN118064301A CN118064301A CN202410031483.5A CN202410031483A CN118064301A CN 118064301 A CN118064301 A CN 118064301A CN 202410031483 A CN202410031483 A CN 202410031483A CN 118064301 A CN118064301 A CN 118064301A
- Authority
- CN
- China
- Prior art keywords
- weissella
- culture
- multocida
- grw
- grw3
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000202221 Weissella Species 0.000 title claims abstract description 45
- 241000272814 Anser sp. Species 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 241000192133 Weissella paramesenteroides Species 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- 239000003674 animal food additive Substances 0.000 claims description 7
- 241000191967 Staphylococcus aureus Species 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
- 230000000844 anti-bacterial effect Effects 0.000 claims description 4
- 230000003385 bacteriostatic effect Effects 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
- 239000000203 mixture Substances 0.000 claims description 3
- 238000009631 Broth culture Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 241000588914 Enterobacter Species 0.000 claims 1
- 239000004615 ingredient Substances 0.000 claims 1
- 239000003833 bile salt Substances 0.000 abstract description 10
- 241000186660 Lactobacillus Species 0.000 abstract description 8
- 229940039696 lactobacillus Drugs 0.000 abstract description 8
- 102000004142 Trypsin Human genes 0.000 abstract description 6
- 108090000631 Trypsin Proteins 0.000 abstract description 6
- 239000002253 acid Substances 0.000 abstract description 6
- 230000000968 intestinal effect Effects 0.000 abstract description 6
- 238000004321 preservation Methods 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 6
- 239000012588 trypsin Substances 0.000 abstract description 6
- 241000272517 Anseriformes Species 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 15
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 6
- 238000009395 breeding Methods 0.000 description 6
- 230000001488 breeding effect Effects 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 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 5
- 239000008272 agar Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- 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
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- 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
- 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
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 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
- 230000001580 bacterial effect Effects 0.000 description 3
- 229940093761 bile salts Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000004211 gastric acid Anatomy 0.000 description 3
- 210000001035 gastrointestinal tract 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
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000004310 lactic acid Substances 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
- 238000012216 screening Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 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
- 235000019319 peptone Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 240000001606 Adenanthera pavonina Species 0.000 description 1
- 235000011470 Adenanthera pavonina Nutrition 0.000 description 1
- 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
- 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
- 241000287828 Gallus gallus Species 0.000 description 1
- 238000003794 Gram staining Methods 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
- 241000124008 Mammalia 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
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 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
- 210000004534 cecum Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- 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 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance 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
-
- 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
Landscapes
- 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 belongs to the technical field of microorganisms, and particularly relates to Weissella multocida and a culture thereof. The preservation number of the Weissella multocida (Weissellaparamesenteroides) AK-GRw3 is as follows: CCTCC NO: M20231982. The intestinal membrane-like Weissella AK-GRw3 has excellent trypsin resistance, good acid resistance and good bile salt resistance, and when the concentration of the bile salt is 0.6%, the survival rate is as high as 210.9%. The Weissella multocida AK-GRw3 is added into goose feed, so that the growth of geese can be promoted, good intestinal flora ecology can be established, immunity can be improved, animal production and health can be influenced and improved, and a better microecological preparation reference basis is provided for expanding a goose-derived lactobacillus resource library and application.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Weissella multocida and a culture thereof.
Background
Along with the improvement of national living standard, the demands and requirements of people on the breeding industry are gradually increased, and the breeding industry of geese is rapidly developed. However, the goose breeding technology is relatively lagged relative to the mature chicken breeding mode, and the problems of scattered breeding, irregular breeding, 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. Lactic acid bacteria (lactic acidbacteria, LAB) are novel microecological preparations which are widely applied, are mature in application on a plurality of animals and have good effects, and are focused on by the characteristics of green, safety, environmental protection and the like. Meanwhile, 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, and enhance immunity and resistance of organism.
The lactobacillus strains are mostly screened and separated from natural environment or intestinal tracts of indigenous animals, and the strains separated from the environment are mostly used for fermentation and have uneven safety performance level. Because the physiological characteristics of geese are greatly different from those of domestic animals, the strain obtained by separating and screening other mammal intestinal tracts is directly applied to the production of geese, and the application effect is not ideal. But the goose-derived lactobacillus strain screening and separating resources are few at present, and the method can be truly applied to less actual production. The Weissella multocida (Weissellaparamesenteroides) belongs to one kind of lactic acid bacteria, can be used as a novel feed additive to replace partial antibiotics, but has weak tolerance to gastric acid, bile salts and the like. Therefore, a strain of Weissella multocida which is highly resistant to gastric acid and bile salts is lacking.
Disclosure of Invention
The invention aims to overcome the defect that the prior art lacks an enteric membrane Weissella with strong gastric acid and bile salt tolerance, and provides the enteric membrane Weissella and a culture thereof.
In order to solve the technical problems, the invention adopts the following technical scheme.
The first aspect of the present invention provides an enteroid Weissella (Weissellaparamesenteroides) AK-GRw3, characterized in that the preservation number is: CCTCC NO: M20231982.
In a second aspect, the invention provides a culture of the Weissella enterica AK-GRw, which is a fermentation broth.
In some embodiments of the invention, the Weissella multocida AK-GRw is inoculated into a broth culture medium containing MRS, shake culture is carried out for 20-26 hours, and thalli are removed, thus obtaining the culture of the Weissella multocida AK-GRw.
In a third aspect, the invention provides the use of said enteroid Weissella multocida AK-GRw3 or said culture in the preparation of a bacteriostatic formulation.
In some embodiments of the invention, the bacteriostatic species comprises staphylococcus aureus.
In a fourth aspect, the invention provides the use of said enteroid Weissella multocida AK-GRw3 or said culture in the manufacture of a medicament for the treatment of a disease caused by infection with Staphylococcus aureus and/or Escherichia coli.
In a fifth aspect, the invention provides the use of said enteroid Weissella multocida AK-GRw3 or said culture in the preparation of a feed additive for promoting goose growth.
In a sixth aspect, the invention provides a bacteriostatic agent, the active ingredient of which is the Weissella enterica AK-GRw3 or the culture.
In a seventh aspect, the present invention provides a medicament comprising the Weissella enterica AK-GRw or the culture as an active ingredient.
According to an eighth aspect of the present invention, there is provided a feed additive comprising the enteroid Weissella AK-GRw3 or the culture as an active ingredient.
Compared with the prior art, the invention has the following beneficial effects: the invention screens the goose-source lactobacillus intestinal membrane Weissella AK-GRw3 with the probiotic effect from the intestinal tracts of healthy geese. The goose-source lactobacillus strain with the characteristics of inhibiting the growth and the colonization of pathogenic bacteria, having strong adhesion colonization capacity and the like is screened from a large number of strains, and the method provides a better reference basis for a microecological preparation for expanding a goose-source lactobacillus resource library and application.
The survival rate of the Weissella multocida AK-GRw3 provided by the invention can reach 99.58% when the pH=2, and the survival rate can reach 210.9% when the concentration of bile salt is 0.6%. The trypsin-resistant performance is excellent, and the survival rate reaches 272.62% when the trypsin concentration is 0.8%.
Preservation of biological materials
The intestinal membrane-like Weissella Weissellaparamesenteroides AK-GRw is preserved in China center for common microorganism bacterial strain preservation management (CCTCC) at the year of 2023 and the month of 10 and 23, the preservation number is CCTCC NO: M20231982, and the preservation address is eight paths of Lopa nationality in Wuchang district of Wuhan, hubei province.
Drawings
FIG. 1 is a diagram showing characteristics of an agar culture of Weissella multocida AK-GRw MRS.
FIG. 2 is a graph showing the results of culture of Weissella multocida AK-GRw MRS broth.
FIG. 3 is a graph showing the results of gram staining of Weissella multocida AK-GRw 3.
FIG. 4 is a graph showing the results of the test for the formation of indole, nitrate reduction, VP and methyl red by Weissella multocida AK-GRw.
FIG. 5 is a graph showing the results of measurement of sucrose, glucose, maltose and lactose by Weissella multocida AK-GRw.
FIG. 6 is a diagram showing the results of 16S rRNA sequencing of Weissella multocida AK-GRw.
FIG. 7 is a graph showing the result of the inhibition of E.coli by Weissella multocida AK-GRw 3.
FIG. 8 is a graph showing the results of the inhibition of Weissella multocida AK-GRw against Staphylococcus aureus.
FIG. 9 is a 26h growth curve of Weissella multocida AK-GRw 3.
FIG. 10 is a graph showing the 26hpH variation of Weissella multocida AK-GRw 3.
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-GRw. 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-GRw3 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 4-5, which show that AK-GRw 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 was detected by agarose gel electrophoresis and sent to Shanghai Biotechnology Co., ltd for sequencing, the sequence of which is shown as SEQ ID NO.1, and the sequencing result is shown in FIG. 6. Sequencing results were compared in a nucleic acid database on NCBI website and found to be 16S rRNA genes of AK-GRw strain and Weissella enterica FL3 strain, genBank accession numbers: KC 118737.1, homology of 99.93%, proves that the bacteria to be tested are Weissella enterica-like bacteria. The results are shown in Table 1.
TABLE 1 results of comparative analysis in nucleic acid databases
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-GRw3 cell-free fermentation supernatant.
(4) The method comprises the steps of absorbing a certain concentration of the indicator fungus suspension on a flat plate in one step, uniformly coating by using a sterile glass rake, slightly placing sterile oxford cups into a culture dish by using tweezers, and uniformly placing 3 or 4 oxford cups into a horizontally placed flat plate.
(5) AK-GRw cell fermentation supernatant was added to the well of oxford cup and spread for 12h in a refrigerator at 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 at 37℃with shaking at 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-GRw 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-GRw bacteria is rapidly increased at 6h, and the acid yield is stabilized at 18h, so that the pH value in the fermentation liquor is close to 4.
The lactobacillus AK-GRw strain with excellent performance is separated from the cecum of the goose, and is identified as the Weissella with intestinal membrane. Experiments show that the AK-GRw3 strain obtained by separation has good acid resistance, general cholate resistance, excellent trypsin resistance and poor antibacterial performance.
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. An enterobacter jejuni (Weissellaparamesenteroides) AK-GRw3, characterized by a deposit number of: CCTCC NO: M20231982.
2. A culture of weissella multocida AK-GRw3 according to claim 1, characterized in that the culture is a fermentation broth.
3. A method of preparing a culture according to claim 2, comprising the steps of: inoculating the Weissella multocida AK-GRw to a broth culture medium containing MRS, shake culturing for 20-26h, and removing thalli to obtain a culture of the Weissella multocida AK-GRw 3.
4. Use of a weissella multocida AK-GRw3 according to claim 1 or a culture according to claim 2 for the preparation of a bacteriostatic formulation.
5. The use according to claim 4, wherein the bacteriostatic species comprises staphylococcus aureus.
6. Use of an enteroid weissella AK-GRw as claimed in claim 1 or a culture as claimed in claim 2 for the preparation of a medicament for the treatment of diseases caused by infection with staphylococcus aureus and/or escherichia coli.
7. Use of an enteroid weissella AK-GRw as defined in claim 1 or a culture as defined in claim 2 for the preparation of a feed additive, wherein the feed additive is used to promote goose growth.
8. An antibacterial preparation, characterized in that the effective component is the Weissella enterica AK-GRw as claimed in claim 1 or the culture as claimed in claim 2.
9. A medicament characterized in that the active ingredient is the enteroid weissella AK-GRw as defined in claim 1 or the culture as defined in claim 2.
10. A feed additive comprising the culture of Weissella multocida AK-GRw as claimed in claim 1 or claim 2 as effective ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410031483.5A CN118064301A (en) | 2024-01-09 | 2024-01-09 | Weissella multocida and culture thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410031483.5A CN118064301A (en) | 2024-01-09 | 2024-01-09 | Weissella multocida and culture thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118064301A true CN118064301A (en) | 2024-05-24 |
Family
ID=91101031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410031483.5A Pending CN118064301A (en) | 2024-01-09 | 2024-01-09 | Weissella multocida and culture thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118064301A (en) |
-
2024
- 2024-01-09 CN CN202410031483.5A patent/CN118064301A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112111433B (en) | Lactobacillus plantarum LZU-J-QA85 with acid-resistant and bile salt-resistant activities and application thereof | |
| CN110029074B (en) | Bacillus subtilis and application thereof in prevention and treatment of fish and shrimp culture diseases | |
| CN106282044B (en) | A kind of preparation method of Hyphomicrobium and pyrroloquinoline quinone | |
| CN108179122B (en) | High-adhesion probiotic enterococcus faecium and application thereof | |
| CN110028560B (en) | Bacteriocin produced by bacillus coagulans and application thereof | |
| CN112063566B (en) | Enterococcus faecium and application thereof | |
| CN111733117B (en) | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof | |
| CN114806944B (en) | Lactobacillus plantarum LP11, fermentation broth thereof, and preparation method and application thereof | |
| CN113717887B (en) | Goose-source lactobacillus plantarum and application thereof | |
| CN116421632A (en) | Application of pediococcus acidilactici in preparing food, health-care product or medicine with lipid-lowering effect | |
| CN114410514B (en) | Bacillus stereiensis and application thereof | |
| CN114231464B (en) | Bacillus coagulans and application thereof | |
| CN113061550B (en) | Lactobacillus new strain Z6 and application thereof in food | |
| CN112322539B (en) | Enterococcus faecium R-NTR-1 from ocean and screening method and application thereof | |
| CN105132320B (en) | The culture medium of high density solid state fermentation culture lactobacillus and method | |
| CN118064301A (en) | Weissella multocida and culture thereof | |
| CN103865832A (en) | Lactobacillus plantarum BS10 and application thereof in regulating pig blood fat | |
| CN109161501B (en) | Feeding bacillus licheniformis and application thereof | |
| CN117987302A (en) | Pediococcus pentosaceus and bacteriostat prepared from same | |
| CN110747137A (en) | Method for separating, identifying and screening saccharomyces cerevisiae in dairy cow excrement | |
| CN114395514B (en) | Lactobacillus acidophilus, microbial inoculum and application thereof | |
| CN117106676B (en) | Bacillus subtilis and application thereof in feed production | |
| CN117223790B (en) | Biological fermentation feed and liquid-solid double-phase fermentation method thereof | |
| CN117965340B (en) | Bacillus subtilis for preventing and treating nocardia and regulating blood sugar and blood fat and application thereof | |
| CN116121085B (en) | Cold-resistant yeast suitable for low-temperature aquaculture and biocontrol application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |