CN115044505B - Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods - Google Patents
Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods Download PDFInfo
- Publication number
- CN115044505B CN115044505B CN202210651979.3A CN202210651979A CN115044505B CN 115044505 B CN115044505 B CN 115044505B CN 202210651979 A CN202210651979 A CN 202210651979A CN 115044505 B CN115044505 B CN 115044505B
- Authority
- CN
- China
- Prior art keywords
- bacillus
- antibacterial
- fermentation
- bailii
- lipopeptide
- 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.)
- Active
Links
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 77
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 75
- 235000013305 food Nutrition 0.000 title abstract description 26
- 239000002537 cosmetic Substances 0.000 title abstract description 15
- 108010028921 Lipopeptides Proteins 0.000 claims abstract description 52
- 241000186427 Cutibacterium acnes Species 0.000 claims abstract description 24
- 229940055019 propionibacterium acne Drugs 0.000 claims abstract description 24
- 244000005700 microbiome Species 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 40
- 230000004151 fermentation Effects 0.000 claims description 40
- 241000894006 Bacteria Species 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 241000222122 Candida albicans Species 0.000 claims description 14
- 241000233866 Fungi Species 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 13
- 229940095731 candida albicans Drugs 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 230000000813 microbial effect Effects 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 241000235395 Mucor Species 0.000 claims description 10
- 241000191967 Staphylococcus aureus Species 0.000 claims description 10
- 241000194031 Enterococcus faecium Species 0.000 claims description 9
- 241000186781 Listeria Species 0.000 claims description 9
- 241000589776 Pseudomonas putida Species 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 241000228197 Aspergillus flavus Species 0.000 claims description 8
- 240000006439 Aspergillus oryzae Species 0.000 claims description 8
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims description 8
- 241000203233 Aspergillus versicolor Species 0.000 claims description 8
- 241000235402 Rhizomucor Species 0.000 claims description 8
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 241001331771 Aspergillus costaricaensis Species 0.000 claims description 6
- 241001443859 Cladosporium subcinereum Species 0.000 claims description 5
- 230000000845 anti-microbial effect Effects 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 229940124350 antibacterial drug Drugs 0.000 claims description 3
- 238000003745 diagnosis Methods 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
- 239000012528 membrane Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 241000222290 Cladosporium Species 0.000 claims 1
- 238000002604 ultrasonography Methods 0.000 claims 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 abstract description 36
- 230000001580 bacterial effect Effects 0.000 abstract description 18
- 244000052616 bacterial pathogen Species 0.000 abstract description 10
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 abstract description 7
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 abstract description 7
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 6
- 230000003042 antagnostic effect Effects 0.000 abstract description 4
- 238000010227 cup method (microbiological evaluation) Methods 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 description 18
- 239000007788 liquid Substances 0.000 description 18
- 239000007787 solid Substances 0.000 description 18
- 239000000047 product Substances 0.000 description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 241000831652 Salinivibrio sharmensis Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 238000005903 acid hydrolysis reaction Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 239000002207 metabolite Substances 0.000 description 5
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000003755 preservative agent Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000008485 antagonism Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 240000008415 Lactuca sativa Species 0.000 description 3
- 235000003228 Lactuca sativa Nutrition 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 208000017520 skin disease Diseases 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000007711 solidification Methods 0.000 description 3
- 230000008023 solidification Effects 0.000 description 3
- 230000004763 spore germination Effects 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 208000002874 Acne Vulgaris Diseases 0.000 description 2
- 206010007134 Candida infections Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 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
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010000496 acne Diseases 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 101150017091 bacD gene Proteins 0.000 description 2
- -1 bacteriostaproteins Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 201000003984 candidiasis Diseases 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009920 food preservation Methods 0.000 description 2
- 239000003517 fume Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ZOCSXAVNDGMNBV-UHFFFAOYSA-N 5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile Chemical compound NC1=C(S(=O)C(F)(F)F)C(C#N)=NN1C1=C(Cl)C=C(C(F)(F)F)C=C1Cl ZOCSXAVNDGMNBV-UHFFFAOYSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- AFWTZXXDGQBIKW-UHFFFAOYSA-N C14 surfactin Natural products CCCCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 AFWTZXXDGQBIKW-UHFFFAOYSA-N 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000005899 Fipronil Substances 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 241000813090 Rhizoctonia solani Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000005282 brightening Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 108010002015 fengycin Proteins 0.000 description 1
- CUOJDWBMJMRDHN-VIHUIGFUSA-N fengycin Chemical compound C([C@@H]1C(=O)N[C@H](C(=O)OC2=CC=C(C=C2)C[C@@H](C(N[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCC(N)=O)C(=O)N1)[C@@H](C)O)=O)NC(=O)[C@@H](CCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)C[C@H](O)CCCCCCCCCCCCC)[C@@H](C)CC)C1=CC=C(O)C=C1 CUOJDWBMJMRDHN-VIHUIGFUSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 229940013764 fipronil Drugs 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 235000021574 pickled cabbage Nutrition 0.000 description 1
- 229930001119 polyketide Natural products 0.000 description 1
- 125000000830 polyketide group Chemical group 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000007692 rcm medium Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- NJGWOFRZMQRKHT-UHFFFAOYSA-N surfactin Natural products CC(C)CCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-UHFFFAOYSA-N 0.000 description 1
- NJGWOFRZMQRKHT-WGVNQGGSSA-N surfactin C Chemical compound CC(C)CCCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-WGVNQGGSSA-N 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000007218 ym medium Substances 0.000 description 1
Classifications
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C12R2001/07—Bacillus
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Nutrition Science (AREA)
- Dermatology (AREA)
- Virology (AREA)
- Birds (AREA)
- Animal Husbandry (AREA)
- Physiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an antibacterial lipopeptide produced by bacillus bailii and application thereof in cosmetics and foods, belonging to the technical field of bioengineering. The invention separates bacterial strains from the surface microorganisms of pickled Chinese cabbage, and screens the bacterial strains by adopting an oxford cup method to obtain a microorganism capable of antagonizing propionibacterium acnes with high efficiency. The strain is identified as Bacillus velezensis, belongs to Bacillus bailii and is deposited in the microorganism strain collection of Guangdong province with the deposit number of GDMCCNo:62393, the preservation date is 2022, 4 and 18. The antibacterial substances of the strain are extracted to obtain the bacillus bailii antibacterial lipopeptide, and the antibacterial lipopeptide has broad-spectrum antibacterial effect on common food-related strains and skin pathogenic bacteria, and can be applied to the aspects of foods, cosmetics, skin care products and the like.
Description
Technical Field
The invention relates to an antibacterial lipopeptide produced by bacillus bailii and application thereof in cosmetics and foods, belonging to the technical field of bioengineering.
Background
Skin micro-ecology refers to an ecosystem composed of various microorganisms together with tissues on the skin surface and the like. Skin disorders such as characteristic dermatitis, psoriasis, acne and the like occur due to skin microecological and in-vivo metabolic imbalance, and an important cause of the skin disorders is microbial infection, for example, acne may be caused by massive abnormal propagation of propionibacterium acnes, atopic dermatitis may be caused by staphylococcus aureus, candida albicans may cause skin candidiasis, and the skin candidiasis is well developed at skin folds (fossa, inguinal, under breast, perianal and nail folds, between fingers), skin flushing, wetness, brightening, sometimes covered with a layer of white or broken matter, and small blisters around lesions. Therefore, the antibacterial substance capable of inhibiting skin pathogenic bacteria in a broad spectrum has potential application value in the aspects of treatment of skin diseases and protection of skin micro-ecology.
Food spoilage causes significant losses to the food industry. Food spoilage is statistically responsible for about 10% -20% of the food lost each year worldwide. The mass propagation of microorganisms is one of the decisive factors for the spoilage of foods. As one of methods of inhibiting spoilage, preservatives are added, and the preservatives currently mainly used are chemical preservatives. However, chemical preservatives have certain toxicity to human bodies, and as people continuously raise the attention to food safety, it is becoming more and more important to find more possible natural, green and safe preservatives which can effectively inhibit the growth of food spoilage bacteria.
The species taxonomic status of bacillus beleiensis was determined later, was isolated earlier in 1999, and was reported and named for the first time until 2005. However, in recent years, guan Beilai bacillus has been reported more and more at home and abroad, and researches mainly focus on promoting growth of animals and plants, antagonizing pathogenic bacteria, inducing system resistance, identifying antibacterial substances and gene cluster antagonizing action mechanisms and the like, and the bacteria have important roles in biological control, drug research and development, food fermentation, industrial application and the like. In particular, in bacteriostasis, bacillus beleiensis achieves the inhibition effect on pathogenic bacteria by synthesizing secondary metabolites, wherein the secondary metabolites mainly comprise bacteriocins, bacteriostaproteins, lipopeptides, polyketides and the like. Among them, antibacterial lipopeptides play an important role. Lipopeptides produced by bacillus belicus include surfactants, iturin, fipronil, bacitracin, and the like. Liu Xuejiao and the like find that bacillus belicus 3A3-15 can generate antibacterial substances such as surfactant and the like, can cause the hypha of fusarium oxysporum to expand, bend, twist, spiral, internode shortening, hypha breaking and the like, has obvious teratogenic effect, and has the inhibition rate of spore germination as high as 93.2 percent. The chogdhury et al study showed that bacillus beleiensis FZB42 strain is capable of producing Surfactin, fengycin and bacillus D during interaction with lettuce (lettuce) root system, and that its metabolites lipopeptides and not only directly inhibit rhizoctonia solani, but also mediate the defensive reaction of lettuce. Lipopeptides (75 μg/mL) produced by bacillus beleiensis Y6 can inhibit fungal spore germination by about 60%, wherein iturin exhibits a strong inhibition on fungal spore germination, ubiquitin exhibits a weak antifungal activity, and surfactant has no obvious fungal inhibition.
Although many researches on the inhibition effect of antibacterial lipopeptid produced by bacillus bailii on pathogenic bacteria are carried out at present, most researches are still mainly focused on aspects of agriculture, biological control, aquaculture and the like. Antagonism of bacillus bailii-produced antimicrobial lipopeptides against skin pathogenic bacteria and food spoilage bacteria to protect skin micro-ecology and food preservation has not been seen. Lipopeptides have unique advantages of low toxicity, good biodegradability, useful physicochemical properties, biological activity and the like, which make them widely applicable in various aspects of cosmetics, medical care, biomedicine, agriculture and animal husbandry, food industry and the like. Bacillus belicus and antibacterial lipopeptid thereof have potential application value and huge market demands in the aspects of cosmetics, skin care products, foods and the like.
Disclosure of Invention
The invention provides bacillus beijerinckii (Bacillus velezensis) ST7-6 which is preserved in the microorganism strain collection center of Guangdong province, and the preservation number is GDMCC No:62393, 4 th year 2022 and 18 th year, taxonomic designation Bacillus velezensis.
The bacillus beleiensis (Bacillus velezensis) ST7-6 provided by the invention is derived from pickled Chinese cabbage, and the bacillus beleiensis (Bacillus velezensis) ST7-6 presents milky small dots on a solid LB culture medium and has rough and irregular edges. The bacteria are screened from the surface microorganisms of pickled Chinese cabbage, colony PCR is carried out on the bacteria through bacterial universal primers 27F and 1492R, after the products are sent to Songan company in Shanghai for sequencing, BLAST is used for comparing the sequences with the previously released 16S rRNA sequences of the bacteria in NCBI database, the homology is 99%, the nucleotide sequence is shown as SEQ ID NO.1, and the bacteria are identified as bacillus belicus.
The invention provides a microbial agent, which contains the bacillus beijerinus (Bacillus velezensis) ST7-6 or fermentation liquid, or extract or metabolite thereof.
In one embodiment of the present invention, the bacillus belicus (Bacillus velezensis) ST7-6 is added to the microbial agent in an amount of: the bacterial concentration is at least 10 8 cfu/ml。
The invention also provides a product which contains one or more of the bacillus bailii (Bacillus velezensis) ST7-6, the fermentation liquid of bacillus bailii (Bacillus velezensis) ST7-6 and the metabolite of bacillus bailii (Bacillus velezensis) ST7-6.
In one embodiment of the invention, the product is a food, a pharmaceutical, a feed additive, a skin care product or a cosmetic.
In one embodiment of the invention, the Bacillus bailii (Bacillus velezensis) ST7-6 has a bacterial concentration in the product of at least 10 8 cfu/ml。
The invention also provides an antibacterial drug which contains the bacillus bailii (Bacillus velezensis) ST7-6 or any microbial agent.
The invention also provides application of the bacillus belicus (Bacillus velezensis) ST7-6 or fermentation liquor or metabolite thereof or the microbial agent in inhibiting bacteria or fungi, wherein the application is not aimed at diagnosis or treatment of diseases.
In one embodiment of the invention, the bacteria include, but are not limited to, propionibacterium acnes, staphylococcus aureus, listeria, enterococcus faecium, pseudomonas putida; the fungi include, but are not limited to, aspergillus oryzae, aspergillus flavus, aspergillus versicolor, rhizomucor minutissima, mucor umbrella, aspergillus costaricensis, cladosporium subcinereum, and Candida albicans.
The invention also provides application of the bacillus belicus (Bacillus velezensis) ST7-6 or fermentation liquor or metabolite thereof, or the microbial agent in preparation of products for inhibiting bacteria or fungi.
In one embodiment of the invention, the product includes, but is not limited to, a pharmaceutical product, a feed additive, a cosmetic product, a skin care product.
In one embodiment of the invention, the bacteria include, but are not limited to, propionibacterium acnes, staphylococcus aureus, listeria, enterococcus faecium, pseudomonas putida; such fungi include, but are not limited to, candida albicans, aspergillus oryzae, aspergillus flavus, aspergillus versicolor, rhizomucor minutissimus, mucor umbrella, aspergillus costaricensis, cladosporium subcinereum.
The invention also provides an antibacterial lipopeptide, which is prepared by the following steps: inoculating bacillus bailii (Bacillus velezensis) ST7-6 into a culture medium for fermentation to prepare a fermentation liquid, and separating and extracting the fermentation liquid to obtain the antibacterial lipopeptide.
In one embodiment of the present invention, the antibacterial lipopeptide is obtained by culturing and fermenting the bacillus belicus (Bacillus velezensis) ST7-6 by the following method.
(1) Preparation of Bacillus bailii (Bacillus velezensis) ST7-6 seed solution:
and (3) carrying out liquid culture on single colony on the LB solid culture medium for 16-18 hours at 37 ℃ and 220rpm by using an inoculating loop strip to the LB liquid culture medium, so as to obtain seed liquid.
(2) Fermentation of Bacillus belicus (Bacillus velezensis) ST 7-6:
inoculating the seed solution into the liquid LB culture medium according to the strain proportion of 2% -5% (v/v), fermenting at 30-37 ℃ for 48-96 h and rotating speed of a shaking table at 160-220 rpm.
(3) Preparation of Bacillus bailii (Bacillus velezensis) ST7-6 antibacterial lipopeptid:
centrifuging the fermentation liquor at 8000rpm for 15min to obtain a fermentation supernatant, regulating the pH of the fermentation supernatant to 2.0,4 ℃ by using 6mol/L hydrochloric acid, standing overnight in a refrigerator, centrifuging the fermentation supernatant at 8000rpm for 15min, discarding the supernatant, leaving a precipitate, adding methanol, performing vortex vibration for 10min, performing ultrasonic treatment for 15min, and extracting for 4-8 h. And centrifuging again, and taking the supernatant to pass through a 0.22 mu m filter membrane to obtain the antibacterial lipopeptide produced by bacillus bailii.
In one embodiment of the invention, the liquid LB medium formulation: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1L, pH 6.8.8 of distilled water.
In one embodiment of the invention, the antimicrobial lipopeptides may have a better antagonism against skin pathogens.
In one embodiment of the present invention, the skin pathogenic bacteria mainly include propionibacterium acnes, candida albicans, and staphylococcus aureus.
In one embodiment of the invention, the antagonism effect is achieved by measuring the size of the inhibition zone by a double-layer flat oxford cup experiment
In one embodiment of the invention, the antimicrobial lipopeptides have a better inhibition effect on food-related strains.
The food spoilage bacteria include Aspergillus oryzae, aspergillus flavus, aspergillus versicolor, listeria, rhizomucor minutissima, mucor umbrella, enterococcus faecium, pseudomonas putida, etc.
In one embodiment of the invention, antagonism is achieved by measuring the size of the zone of inhibition by a coated flat-plate oxford cup method.
The invention provides the bacillus belicus Bacillus velezensis ST-6 and the application of the antibacterial lipopeptide thereof in cosmetics and skin care products.
The invention also provides application of the bacillus bailii Bacillus velezensis ST-6 and the antibacterial lipopeptid thereof in food preservation, antibacterial drugs and the like.
Advantageous effects
(1) According to the invention, bacillus beljalis Bacillus velezensis ST-6 with broad-spectrum antibacterial effect is obtained by separating and screening from the surface of pickled cabbage by an oxford cup method, the strain fermentation liquor has obvious antibacterial effect on propionibacterium acnes, the diameter of a antibacterial circle reaches 22.25mm, and the antibacterial lipopeptide can be prepared from the supernatant of the strain fermentation liquor.
(2) The antibacterial lipopeptide has broad-spectrum antibacterial effect. For food related strains such as Aspergillus oryzae, aspergillus flavus, aspergillus versicolor, listeria, rhizomucor minutissima, mucor umbrella, enterococcus faecium and Pseudomonas putida; skin pathogenic bacteria: propionibacterium acnes, candida albicans and staphylococcus aureus all have obvious antibacterial effects.
(3) The bacillus belicus is safe in source and wide in application, and the lipopeptide has the unique advantages of low toxicity, good biodegradability, useful physicochemical properties, biological activity and the like, can be used as a food preservative or an additive of cosmetics and skin care products to play a role in bacteriostasis, and has wide application value in various aspects of cosmetics, skin care products, food industry and the like.
Preservation of biological materials
Bacillus belicus (Bacillus velezensis) ST7-6, taxonomic designation Bacillus velezensis, was deposited at the Cantonese microorganism strain collection at 18, 4, 2022 under accession number GDMCC No:62393, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
Drawings
Fig. 1: diameter of zone of inhibition of propionibacterium acnes by different strains.
Fig. 2: strain morphology.
Fig. 3: the bacillus belicus ST7-6 antibacterial lipopeptide is analyzed by thin layer chromatography, wherein the A plate is a chromatography plate before in-situ acid hydrolysis, and the B plate is a chromatography plate after in-situ acid hydrolysis.
Fig. 4: PCR detection of lipopeptide synthesis gene of Bacillus bailii ST7-6.
Fig. 5: antibacterial effect of bacillus bailii ST7-6 antibacterial lipopeptid on propionibacterium acnes.
Detailed Description
The present invention will be further described with reference to specific embodiments in order to make the above objects, features and advantages of the present invention more comprehensible.
The media described in the examples below are as follows:
LB liquid medium: 10g/L of peptone (Oxoid company, england), 5g/L of yeast powder (Oxoid) and 10g/L of sodium chloride.
LB plate: 10g/L of peptone (Oxoid company in England), 5g/L of yeast powder (Oxoid), 10g/L of sodium chloride and 20g/L of agar powder.
YM medium: 5g/L of peptone (Oxoid Co., UK), 3g/L of yeast powder (Oxoid), 3g/L of malt extract, 10g/L of glucose, 20g/L of agar and pH 6.2.
PDA plate: 6g/L of potato powder, 20g/L of glucose, 0.1g/L of chloramphenicol and 20g/L, pH 5.8.8 of agar.
Example 1: isolation, screening and identification of Bacillus bailii strain Bacillus velezensis ST-6
The bacillus subtilis Bacillus velezensis ST-6 with broad-spectrum antibacterial effect is obtained by separating and screening from the surfaces of pickled Chinese cabbage. The main biological characteristics are that the solid LB culture medium presents milky small dots and the edges are rough and irregular.
The fermentation liquor and the produced antibacterial lipopeptid have broad-spectrum antibacterial activity. The method comprises the following specific steps:
1. isolation of strains
The method comprises the following specific steps:
(1) Food pickled Chinese cabbage is selected, the ultrasonic table is used for sampling and shearing, the pickled Chinese cabbage is placed into a 50ml sterile centrifuge tube, and 10g of pickled Chinese cabbage is weighed by a weighing balance.
(2) Adding the glass beads and 25ml of sterile physiological saline, shaking at 220rpm and a shaking table at 37 ℃ for 30min, and uniformly mixing to obtain a sample suspension.
(3) Taking 1ml of sample suspension, carrying out 10-time gradient dilution to 10 -1 、10 -2 、10 -3 、10 -4 。
(4) 100. Mu.L of each gradient dilution was applied to LB agar plate medium with a pipette, and each gradient was cultured in a 37℃incubator for 48 hours in parallel.
(5) Single colonies were picked, streaked and isolated for later screening.
2. Screening of strains
The method comprises the following specific steps:
(1) Screening test strains by adopting an oxford cup double-layer flat plate method and using propionibacterium acnes as indicator bacteria. Inoculating single colony on LB plate into liquid LB culture medium according to 1% (v/v) ratio, shake culturing at 37deg.C and 220rpm for 24 hr to obtain fermentation liquor;
(2) Pouring 10ml of melted solid RCM culture medium into a culture dish, clamping oxford cups on a flat plate by using sterile forceps after solidification, dissolving the solid culture in a microwave oven, adding bacterial suspension of propionibacterium acnes according to the proportion of 5% (v/v) when the temperature is reduced to about 50 ℃, and adding about 10ml of solid culture medium containing propionibacterium acnes, wherein the bacterial concentration of the bacterial suspension of propionibacterium acnes is as follows: 10 8 cfu/ml, pouring the cfu/ml into the upper layer of the solidified solid culture medium, and clamping out the oxford cup after the culture medium is solidified to form a sample hole.
(3) 100 mu L of fermentation liquor of the strain to be tested is respectively injected into the holes, and the concentration of the strain in the fermentation liquor is 10 8 cfu/ml, 3 replicates per treatment were set. Anaerobic culture is carried out at 37 ℃ for 48 hours.
(4) Taking out the oxford cup plate from the incubator, measuring the diameter of the bacteriostasis zone of the microorganism by using a ruler, and taking the average value as the diameter of the bacteriostasis zone of the microorganism fermentation broth.
As shown in figure 1, 5 strains capable of obviously antagonizing Propionibacterium acnes are named as strain A, strain B, strain C, strain D and strain E according to the diameter of the inhibition zone, wherein the diameter of the inhibition zone of the fermentation liquor of the strain B reaches 22.25mm, and the inhibition effect on Propionibacterium acnes is best. Thus, strain B was used for the next step of strain identification.
3. Identification of strains
The method comprises the following specific steps:
(1) The strain B obtained in step 2 was amplified with the strain 16SrRNA gene using the universal bacterial primers 27F and 1492R. Wherein the sequence of the general bacterial primer 27F is 5'-AGAGTTTGATCMTGGCTCAG-3', and the sequence of the general bacterial primer 1492R is 5'-GGTTACCTTGTTACGACTT-3'.
(2) PCR was performed using 30. Mu.L of the reaction mixture, and an enzyme Taq DNA Polymerase (Takara Co.) was selected for PCR at a pre-denaturation temperature of 95℃for 3min; 34 cycles of amplification stage, at 95℃for 30s;55 ℃ for 30s; the PCR product was obtained by performing 1min at 72℃for 40 s.
(3) And (3) analyzing the PCR product obtained in the step (2) through agarose gel electrophoresis and sending the PCR product to Songan company in Shanghai for sequencing.
(4) Sequences were aligned with previously published bacterial 16S rRNA sequences using BLAST in NCBI database with 99% homology and the nucleotide sequence shown in SEQ ID NO. 1. The strain B was in LB solid medium, and the results are shown in FIG. 2. The colony characteristics are as follows: milky white, round dots, rough edges.
The identified strain B is Bacillus velezensis, belongs to bacillus beijerinus and is named Bacillus velezensis ST-6.
Example 2: the preparation and antibacterial effect of the bacillus belicus strain Bacillus velezensis ST7-6 antibacterial lipopeptide are specifically as follows:
1. preparation of Bacillus belicus strain Bacillus velezensis ST-6 fermentation broth.
(1) Preparation of Bacillus bailii (Bacillus velezensis) ST7-6 seed solution:
and (3) carrying out liquid culture on single colony on the LB solid culture medium for 16-18 hours at 37 ℃ and 220rpm by using an inoculating loop strip to the LB liquid culture medium, so as to obtain seed liquid.
(2) Fermentation of Bacillus belicus (Bacillus velezensis) ST 7-6:
inoculating the seed solution into a liquid LB culture medium according to the strain proportion of 3% (v/v), wherein the fermentation temperature is 30 ℃, the fermentation time is 72h, and the rotation speed of a shaking table is 220rpm, so as to prepare the fermentation liquid.
2. Preparation of Bacillus bailii (Bacillus velezensis) ST7-6 antibacterial lipopeptides.
Centrifuging the fermentation liquor prepared in the step 1 for 15min at 8000rpm to obtain a fermentation supernatant, regulating the pH of the fermentation supernatant to 2.0,4 ℃ by using 6mol/L hydrochloric acid, standing for 12h, centrifuging the fermentation supernatant at 8000rpm for 15min, discarding the supernatant, leaving a precipitate, adding methanol (the methanol is added according to 10% by volume), performing vortex oscillation for 10min, performing ultrasonic treatment for 15min, and extracting for 4-8 h. And centrifuging again, filtering the supernatant with a 0.22 mu m filter membrane, and taking filtrate to obtain the antibacterial lipopeptide solution produced by bacillus belicus.
3. Identification and analysis of antibacterial lipopeptides
(1) Analysis of antibacterial lipopeptides by thin layer chromatography
And carrying out thin layer chromatography analysis by adopting an in-situ acid hydrolysis ninhydrin color development method. Two activated silica gel plates are marked as an A plate and a B plate by pencils, a transverse line is drawn at a position 1cm away from the bottom, capillaries are extended into bacillus berryis ST7-6 antibacterial lipopeptide samples at intervals of 0.5cm on the transverse line, some samples are dipped, and sample application is carried out on the silica gel plates. Pouring a developing agent which is not more than the transverse line at the bottom of the silica gel plate into a chromatographic cylinder, wherein the ratio of the developing agent is chloroform: methanol: water=65: 25:4 (v/v/v), spread for 40min in a chromatography jar until the spreading agent reaches 1cm from the top of the silica gel plate, take out the silica gel plate and place in a fume hood. After evaporation of the solvent, the a plate was directly developed with ninhydrin reagent (0.5% ninhydrin acetone solution). Placing the plate B into a high-temperature-resistant sealed bottle, placing about 2mL of concentrated hydrochloric acid in the bottle in advance in a small cup, fumigating for 2h in a 110 ℃ oven, carrying out in-situ acid hydrolysis, cooling in a fume hood after the hydrolysis, blowing off the hydrochloric acid, volatilizing a dry reagent, and finally developing color by using an ninhydrin reagent.
As a result, as shown in FIG. 3, after in situ acid hydrolysis of the B plate, a purple spot was observed which was not observed in the A plate numbered (5), indicating that the component represented by this spot did not contain free amino groups before acidolysis, and that the free amino groups were exposed after acidolysis to develop color by ninhydrin. The lipopeptides are generally cyclic structures, without free amino groups, which are opened after acidolysis and exposed. Thus the bacteriostatic substance produced by bacillus belicus ST7-6 is a lipopeptide and is a cyclic lipopeptide.
(2) PCR detection of lipopeptide synthesis genes
The lipopeptide synthesis related gene in bacillus bailii ST7-6 was detected by molecular biological means to further determine whether it was a lipopeptide. The genome of Bacillus belicus ST7-6 was extracted by using a bacterial genome extraction kit from Shanghai Biotechnology Co., ltd, and the synthetic gene of lipopeptide was amplified, and the primer design was as shown in Table 1.
Table 1: primer design for lipopeptide synthesis genes
The results are shown in FIG. 4. By PCR detection, the gene sfp, fenB, ituA, bacD corresponds to lanes, and agarose gel electrophoresis detects single bands, and the length corresponds to the size. Bacillus is described as containing genes related to lipopeptide synthesis, i.e., containing the lipopeptides encoded by these genes sfp, fenB, ituA, bacD.
4. Antibacterial effect of antibacterial lipopeptid on propionibacterium acnes
The antibacterial effect of bacillus beljalis ST7-6 antibacterial lipopeptides on propionibacterium acnes is detected by using an oxford cup double-layer flat plate method. Pouring 10mL of melted solid RCM culture medium into a culture dish, clamping oxford cups on a flat plate by using sterile forceps after solidification, dissolving RCM solid culture in a microwave oven, adding bacterial suspension of propionibacterium acnes according to the proportion of 5% (v/v) when the temperature is reduced to about 50 ℃, and adding about 10mL of the RCM melted solid culture medium containing propionibacterium acnes, wherein the bacterial concentration of the bacterial suspension of propionibacterium acnes is as follows: 10 8 cfu/mL, was poured into a petri dish containing 10mL of RCM medium before, and a solid medium upper layer was formed, resulting in an oxford cup double-layer plate. After the culture medium is solidified, clamping out the oxford cup to form a sample hole. After 100. Mu.L of the antibacterial lipopeptide solution of Bacillus bailii prepared in the step 2 of the example 2 was added into the well, the oxford cup double-layer plate was placed into an incubator, anaerobic culture was performed at 37℃for 48 hours, the oxford cup plate was taken out of the incubator, and the diameter of the inhibition zone of the microorganism was measured with a ruler. The antibacterial lipopeptid is tested for antibacterial effect on propionibacterium acnes by adopting an oxford cup double-layer flat plate method.
As shown in FIG. 5, the diameter of the antibacterial zone of the bacillus belicus ST7-6 antibacterial lipopeptide on propionibacterium acnes can reach 21.0mm.
Example 3: antibacterial spectrum of antibacterial lipopeptid of bacillus belicus strain Bacillus velezensis ST7-6
The bacillus bailii strain Bacillus velezensis ST-6 antibacterial lipopeptide can produce broad-spectrum antibacterial effect on fungi and gram-positive bacteria. To test the application of the bacterial strain antibacterial lipopeptid in the food industry, cosmetics and skin care products. The antibacterial effect of the antibacterial lipopeptid of the strain on various strains including aspergillus oryzae, aspergillus flavus, aspergillus versicolor, listeria, rhizomucor minutissima, mucor umbrella, aspergillus costaricensis, cladosporium subcinereum, candida albicans, staphylococcus aureus, enterococcus faecium and pseudomonas putida is measured.
The method comprises the following specific steps:
1. detection of inhibition effect of antibacterial lipopeptid on mold
(1) Respectively taking out Aspergillus oryzae, aspergillus flavus, aspergillus versicolor, rhizomucor minutissima, mucor umbrella, aspergillus costaricensis, cladosporium subcinereum and glycerol pipe of the strain stored in-80deg.C refrigerator, and thawing at room temperature for 20min.
(2) 100. Mu.L of the bacterial liquid stored in the glycerol tube was aspirated and the resulting mixture was spread on a PDA plate. Culturing at 30 ℃ for 72 hours until fungi grow on the whole plate and the growth state is good.
(3) Pouring 10mL of sterile physiological saline into the plate, scraping the PDA plate by using a coating rod to obtain a mixed solution, and filtering the mixed solution by using sterile gauze to obtain the spore liquid of the fungi.
(5) The fungal spore liquid was counted using a hemocytometer. The concentration of the spore liquid is regulated to be 1 multiplied by 10 6 cfu/mL。
100. Mu.L of spore liquid was taken and spread evenly on PDA plates. Place oxford cup on plate, draw 100. Mu.L of bacillus bailii antibacterial lipopeptide solution prepared in step 2 of example 2 with a pipette, add into oxford cup-like well, and culture at 30deg.C for 72h.
The diameter of the inhibition zone is measured by a ruler, and the average value is taken as the diameter of the inhibition zone of the antibacterial lipopeptide. The results are shown in Table 2:
table 2: antibacterial effect of antibacterial lipopeptid on mold
2. Antibacterial effect of antibacterial lipopeptid on other bacteria
(1) Taking out glycerol tubes of Candida albicans, listeria, staphylococcus aureus, enterococcus faecium and Pseudomonas putida stored in-80deg.C refrigerator, and thawing at room temperature for 20min.
(2) Respectively dipping a fungus liquid loop by an inoculating loop, and streaking candida albicans to YM culture medium; and (3) streaking staphylococcus aureus, listeria, enterococcus faecium and pseudomonas putida to an LB culture medium respectively. Candida albicans is cultured at 30 ℃ for 48 hours, and other strains are cultured at 37 ℃ for 24 hours.
(3) Pouring 10mL of melted solid culture medium of the corresponding strain into a culture dish, clamping oxford cups on a flat plate by using sterile forceps after solidification, melting the solid culture in a microwave oven, and adding pathogenic bacteria into the solid culture medium cooled to about 50 ℃ according to the proportion of 1% (v/v). The solid culture medium containing pathogenic bacteria (the concentration of bacteria is 10) 8 cfu/mL) 10mL was poured into the solidified upper layer of the solid medium, and after the medium solidified, the oxford cup was clamped out to form a sample well.
(4) 100ul of the antibacterial lipopeptide solution of Bacillus bailii Bacillus velezensis ST-6 prepared in the step 2 of example 2 is added into the sample well, and the culture is carried out according to the culture temperature of the corresponding strain (candida albicans is cultured for 48h at 30 ℃ and other strains are cultured for 24h at 37 ℃).
After the experiment is finished, the oxford cup plate is taken out from the incubator, the diameter of the inhibition zone is measured by a ruler, and the average value is taken as the diameter of the inhibition zone of the antibacterial lipopeptide. The results are shown in Table 3:
table 3: antibacterial effect of antibacterial lipopeptid on other bacteria
As can be seen from tables 2 and 3, the antibacterial lipopeptid of bacillus belicus ST7-6 has good antibacterial effect on different strains, and can be widely applied to food and cosmetics.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> antibacterial lipopeptid produced by bacillus bailii and application thereof in cosmetics and foods
<130> BAA220595A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1430
<212> DNA
<213> artificial sequence
<400> 1
cttcggcggc tggctccata aaggttacct caccgacttc gggtgttaca aactctcgtg 60
gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120
attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180
gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240
cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300
tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420
cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480
gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540
gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta 600
atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac 660
ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720
agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780
ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt ttccaatgac 840
cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg cgagcccttt 900
acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960
gttagccgtg gctttctggt taggtaccgt caaggtgccg ccctatttga acggcacttg 1020
ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 1080
ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta ggagtctggg 1140
ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgtcgcctt 1200
ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta agtggtagcc 1260
gaagccacct tttatgtctg aaccatgcgg ttcagacaac catccggtat tagccccggt 1320
ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380
ctaacatcag ggagcaagct cccatctgtc cgctcgactt gcatgtatag 1430
Claims (8)
1. Bacillus bailii strainBacillus velezensis) ST7-6, wherein bacillus beijerinckii ST7-6 is deposited with the collection of microorganism strains, cantonese province, under the accession number GDMCC No:62393, the preservation date is 2022, 4 and 18.
2. A microbial agent, which is characterized in that the microbial agent contains bacillus belicus ST7-6 or fermentation broth thereof according to claim 1; the fermentation liquor contains bacillus beijerinckii ST7-6.
3. An antibacterial drug comprising the bacillus belicus ST7-6 according to claim 1 or the microbial agent according to claim 2.
4. Use of bacillus beleimeris ST7-6 according to claim 1 or of a microbial agent according to claim 2 for inhibiting bacteria or fungi, characterized in that said use is not aimed at the diagnosis or treatment of diseases.
5. As claimed in4, wherein the bacteria are propionibacterium acnes, staphylococcus aureus, listeria, enterococcus faecium and pseudomonas putida; the fungi are candida albicans, aspergillus oryzae, aspergillus flavus, aspergillus versicolor, rhizomucor minutissima, mucor umbrella-shaped mucor,Aspergillus costaricensis、Cladosporium subcinereum。
6. Use of the bacillus belicus ST7-6 as claimed in claim 1 or the microbial agent as claimed in claim 2 for the preparation of a medicament for inhibiting bacteria or fungi.
7. An antibacterial lipopeptide is characterized in that the preparation method of the antibacterial lipopeptide comprises the following steps: inoculating bacillus belicus ST7-6 of claim 1 into a culture medium for fermentation to obtain a fermentation broth, and separating and extracting the fermentation broth to obtain the antibacterial lipopeptide; specifically, the fermentation broth is centrifuged at 8000rpm for 15min to obtain a fermentation supernatant, the pH of the fermentation supernatant is regulated to 2.0,4 ℃ by 6mol/L hydrochloric acid, the fermentation supernatant is placed overnight in a refrigerator, the fermentation supernatant is centrifuged at 8000rpm for 15min, the supernatant is discarded, the precipitate is left, methanol is added into the precipitate, vortex shaking is carried out for 10min, ultrasound is carried out for 15min, after extraction is carried out for 4-8 h, the supernatant is centrifuged again, and the supernatant is taken to pass through a 0.22 mu m filter membrane, thus obtaining the antibacterial lipopeptide produced by bacillus beijensis.
8. The use of the antimicrobial lipopeptide according to claim 7 for inhibiting bacteria or fungi, wherein the bacteria are propionibacterium acnes, staphylococcus aureus, listeria, enterococcus faecium, pseudomonas putida; the fungi are candida albicans, aspergillus oryzae, aspergillus flavus, aspergillus versicolor, rhizomucor minutissima, mucor umbrella-shaped mucor,Aspergillus costaricensis、Cladosporium subcinereumThe method comprises the steps of carrying out a first treatment on the surface of the The use is not for the diagnosis or treatment of diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210651979.3A CN115044505B (en) | 2022-06-09 | 2022-06-09 | Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210651979.3A CN115044505B (en) | 2022-06-09 | 2022-06-09 | Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115044505A CN115044505A (en) | 2022-09-13 |
| CN115044505B true CN115044505B (en) | 2024-03-01 |
Family
ID=83160508
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210651979.3A Active CN115044505B (en) | 2022-06-09 | 2022-06-09 | Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115044505B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116019858B (en) * | 2022-10-14 | 2023-10-10 | 大连三仪动物药品有限公司 | Compound preparation containing bacillus bailii, preparation method and application thereof |
| EP4382090A1 (en) | 2022-12-08 | 2024-06-12 | Evonik Operations GmbH | Cosmetical and pharmaceutical compositions containing bacillus strains or fermentation broths thereof |
| CN116082470B (en) * | 2022-12-12 | 2023-08-15 | 南京工业大学 | Bacillus bailii antibacterial lipopeptide and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283742A (en) * | 2019-06-17 | 2019-09-27 | 北京农业生物技术研究中心 | The Bei Laisi bacillus of one plant of broad-spectrum disease resistance and its application |
| WO2022030662A1 (en) * | 2020-08-05 | 2022-02-10 | (재) 전남바이오산업진흥원 | Novel bacillus velezensis l2 strain isolated from anchovy fermentation solution, and aekjeot prepared using same |
| CN115161234A (en) * | 2022-07-07 | 2022-10-11 | 安徽农业大学 | Bacillus belgii and application thereof in silage preservation |
-
2022
- 2022-06-09 CN CN202210651979.3A patent/CN115044505B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283742A (en) * | 2019-06-17 | 2019-09-27 | 北京农业生物技术研究中心 | The Bei Laisi bacillus of one plant of broad-spectrum disease resistance and its application |
| WO2022030662A1 (en) * | 2020-08-05 | 2022-02-10 | (재) 전남바이오산업진흥원 | Novel bacillus velezensis l2 strain isolated from anchovy fermentation solution, and aekjeot prepared using same |
| CN115161234A (en) * | 2022-07-07 | 2022-10-11 | 安徽农业大学 | Bacillus belgii and application thereof in silage preservation |
Non-Patent Citations (2)
| Title |
|---|
| Bioprospecting of a novel endophytic Bacillus velezensis FZ06 from leaves of Camellia assamica: Production of three groups of lipopeptides and the inhibition against food spoilage microorganisms;Fang-Zhou Li 等;Journal of Biotechnology;第323卷;第42-53页，参见摘要 * |
| 贝莱斯芽孢杆菌 Pm9 生物防治潜力 及全基因组分析;李永丽 等;河 南 农 业 大 学 学 报;第55卷(第6期);第1081-1088页，参见摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115044505A (en) | 2022-09-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115044505B (en) | Antibacterial lipopeptid produced by bacillus bailii and application of antibacterial lipopeptid in cosmetics and foods | |
| CN106906167B (en) | Bacillus amyloliquefaciens for biocontrol and application thereof | |
| CN110452848B (en) | Bacillus belgii and application thereof | |
| CN111019866B (en) | Endophytic bacillus of Pu' er tea tree leaves and application thereof | |
| CN107099484B (en) | Lysobacter enzymogenes and application thereof | |
| CN107338202B (en) | Bacillus amyloliquefaciens with broad-spectrum pathogenic bacterium inhibiting function and application thereof | |
| CN113717907B (en) | Bacillus belgii and application thereof | |
| CN109294954B (en) | Amylose streptomycete and application thereof | |
| CN116004429B (en) | Biocontrol strain for preventing and treating apple rot and application thereof | |
| CN118126845A (en) | Inula longissima endophytic fungus YAFEF048 strain and separation method and application thereof | |
| CN116355763B (en) | Symbiotic fungus for oil wheat and spruce and application thereof | |
| CN116426394B (en) | Jade Long Biaolan symbiotic fungus and application thereof | |
| CN101691542B (en) | Muscodor endophytic fungi and application thereof and fungicide | |
| WO2010115802A1 (en) | New actinomycetes strain compositions and their use for the prevention and/or the control of micro organism inducing plant diseases | |
| CN108315266B (en) | Paecilomyces cicadae and application thereof | |
| CN108715817B (en) | Streptomyces albidoflavus strain and application thereof | |
| CN108004154B (en) | Sporobolomyces yunnanensis 17wy1, microbial preparation thereof and application thereof in wheat powdery mildew prevention and treatment | |
| CN108130278B (en) | Purslane sclerotium mould and its preparing the application in anti-Ralstonia solanacearum drug | |
| CN114317317B (en) | Bacillus belicus strain capable of resisting salt and producing lipopeptid at high yield and application thereof | |
| CN113564074B (en) | Myxobacteria and application thereof in preparation of antibacterial drugs | |
| CN114958616A (en) | Cinnamomum camphora symbiotic fungus YAFEF008 and separation method thereof | |
| CN111154675B (en) | Bacillus acidocaldarius SYY15 and application thereof | |
| Lang et al. | Antimicrobial potential of the endophytic actinobacteria isolated from Harpagophytum procumbens: a Southern African medicinal plant | |
| Rante et al. | Antimicrobial activity of Streptomyces spp. sponge-associated isolated from Samalona Island of South Sulawesi, Indonesia | |
| CN114214205B (en) | Antrodia camphorata symbiotic fungus AcEF007 and separation method 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |