CN109984189A - A kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens - Google Patents
A kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens Download PDFInfo
- Publication number
- CN109984189A CN109984189A CN201910354887.7A CN201910354887A CN109984189A CN 109984189 A CN109984189 A CN 109984189A CN 201910354887 A CN201910354887 A CN 201910354887A CN 109984189 A CN109984189 A CN 109984189A
- Authority
- CN
- China
- Prior art keywords
- fresh
- bacillus
- cut
- vegetable
- atrophy
- 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.)
- Granted
Links
- 241000366676 Justicia pectoralis Species 0.000 title claims abstract description 113
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 62
- 235000012055 fruits and vegetables Nutrition 0.000 title claims abstract description 45
- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 26
- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 26
- 206010003694 Atrophy Diseases 0.000 title claims abstract description 24
- 230000037444 atrophy Effects 0.000 title claims abstract description 24
- 238000013329 compounding Methods 0.000 title claims abstract description 9
- 239000003755 preservative agent Substances 0.000 claims abstract description 43
- 230000002335 preservative effect Effects 0.000 claims abstract description 43
- 239000002131 composite material Substances 0.000 claims abstract description 40
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 15
- 239000006228 supernatant Substances 0.000 claims abstract description 15
- 239000000052 vinegar Substances 0.000 claims abstract description 11
- 235000021419 vinegar Nutrition 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000004321 preservation Methods 0.000 claims description 18
- 230000000844 anti-bacterial effect Effects 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 9
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 238000010612 desalination reaction Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000011591 potassium Substances 0.000 claims 1
- 229910052700 potassium Inorganic materials 0.000 claims 1
- 235000015096 spirit Nutrition 0.000 claims 1
- 239000011593 sulfur Substances 0.000 claims 1
- 229910052717 sulfur Inorganic materials 0.000 claims 1
- 241000220225 Malus Species 0.000 abstract description 45
- 235000021016 apples Nutrition 0.000 abstract description 39
- 241000894006 Bacteria Species 0.000 abstract description 35
- 241000588724 Escherichia coli Species 0.000 abstract description 18
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 14
- 241000607762 Shigella flexneri Species 0.000 abstract description 12
- 241000186779 Listeria monocytogenes Species 0.000 abstract description 11
- 244000106835 Bindesalat Species 0.000 abstract description 9
- 235000000318 Bindesalat Nutrition 0.000 abstract description 9
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 8
- 244000052769 pathogen Species 0.000 abstract description 7
- 230000001717 pathogenic effect Effects 0.000 abstract description 7
- 238000000855 fermentation Methods 0.000 abstract description 5
- 230000004151 fermentation Effects 0.000 abstract description 5
- 230000007423 decrease Effects 0.000 abstract description 4
- 238000012870 ammonium sulfate precipitation Methods 0.000 abstract 1
- 238000000502 dialysis Methods 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 31
- 240000008415 Lactuca sativa Species 0.000 description 28
- 235000003228 Lactuca sativa Nutrition 0.000 description 28
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 20
- 230000000694 effects Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 17
- 102000030523 Catechol oxidase Human genes 0.000 description 12
- 108010031396 Catechol oxidase Proteins 0.000 description 12
- 230000001408 fungistatic effect Effects 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 10
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 239000000126 substance Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 208000016261 weight loss Diseases 0.000 description 8
- 230000004580 weight loss Effects 0.000 description 8
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 229930003268 Vitamin C Natural products 0.000 description 7
- 235000013399 edible fruits Nutrition 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 235000019154 vitamin C Nutrition 0.000 description 7
- 239000011718 vitamin C Substances 0.000 description 7
- 241000228245 Aspergillus niger Species 0.000 description 6
- 239000000022 bacteriostatic agent Substances 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 235000021186 dishes Nutrition 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 3
- 241000228197 Aspergillus flavus Species 0.000 description 3
- 238000001712 DNA sequencing Methods 0.000 description 3
- 241000305071 Enterobacterales Species 0.000 description 3
- 241000192125 Firmicutes Species 0.000 description 3
- 241000726221 Gemma Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 238000007747 plating Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 241000193410 Bacillus atrophaeus Species 0.000 description 2
- 241001478240 Coccus Species 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
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 241001443915 Sapromyces Species 0.000 description 2
- 241000607768 Shigella Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000021022 fresh fruits Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical compound COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 239000001393 triammonium citrate Substances 0.000 description 2
- 235000011046 triammonium citrate Nutrition 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- CCBICDLNWJRFPO-UHFFFAOYSA-N 2,6-dichloroindophenol Chemical compound C1=CC(O)=CC=C1N=C1C=C(Cl)C(=O)C(Cl)=C1 CCBICDLNWJRFPO-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229960001867 guaiacol Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000009461 vacuum packaging Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to technical field of microbe application, provide a kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens.Bacillus licheniformis, atrophy bacillus, bacillus amyloliquefaciens with stronger bacteriostatic activity are filtered out from Shanxi mature vinegar vinegar fermented grain, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, number is respectively CGMCC 16909,16911,15732.Strain fermentation supernatant is made to the composite preservative of 1.0mg/mL after ammonium sulfate precipitation, dialysis, freeze-drying, with on Fresh-cut Apples and romaine lettuce are fresh-keeping, after the Fresh-cut Apples of sprinkling pathogen mixed liquor and romaine lettuce are handled with composite preservative as the result is shown, compared with the control group, there is apparent bacteriostasis, staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, Listeria monocytogenes clump count have significant decrease, reduce the spoilage rate of Fresh-cut Apples and romaine lettuce.
Description
Technical field
The invention belongs to technical field of microbe application, and in particular to one kind is by bacillus licheniformis, atrophy bacillus
With the fresh-cut fruit and vegetable antistaling agent of the produced bateriostatics compounding of bacillus amyloliquefaciens.
Background technique
Fresh-cut fruit and vegetable be fresh fruit and vegetable raw-material by selecting, cleaning, removing the peel, cutting, disinfection, the production work such as packaging
The instant fruit and vegetable product that skill is formed, also referred to as micro Process fruits and vegetables.Fresh-cut fruit and vegetable had not only maintained the original fresh state of fruits and vegetables, but also passed through
Processing makes product sanitation and hygiene, belongs to clean vegetables scope, natural, nutrition, fresh, conveniently, can meet people pursue natural, nutrition,
The demand of allegro life style etc..The market demand is gradually increased, and fresh-cut fruit and vegetable industry will welcome flourishing at full speed
Development.
Due to the original structural state of cutting processing damage fresh fruit of vegetables, significant changes, fruits and vegetables itself occur for physiology
The juice characteristic of generation can provide Reproduction Conditions to microorganism, and infecting microorganism mainly includes pathogenic bacterium such as large intestine bar
Bacterium, staphylococcus aureus, Bacterium enteritidis, Listeria monocytogenes etc., if fresh-cut fruit and vegetable is in processing, transport, sales process
Middle to be infected by pathogenic bacteria, fresh-cut fruit and vegetable product meeting fast rotting can also cause food source to influence product nutrition and economic value
Property disease and the health for endangering the public.Therefore, effective suppression should be implemented to fresh-cut fruit and vegetable product in production and storage
Bacterium measure is to ensure product quality.
Currently, chemical synthesis class bacteriostatic agent in fresh-cut fruit and vegetable is fresh-keeping using more, although chemically synthesized bacteriostatic agent
Rapidly, but such bacteriostatic agent lures carcinous, teratogenesis and easily causes food slow poisoning etc. there are some for cheap, sterilization
Unsafe problems, thus, the food sterilization and preservative for researching and developing natural safety are particularly important.Relative to chemical synthesis
For bacteriostatic agent, natural bacteriostatic agent has the characteristics that wide, non-toxic, the strong biocidal property of sphere of action.Wherein, microbial source antistaling agent
It is the research hotspot of current novel foodstuff bacteriostatic agent.Bacillus is that a kind of to generate a variety of important metabolites include Efficient antibacterial
The bacterium of substance, because it has many advantages, such as that growth and breeding is fast, be easy to survive, resistance is strong, nutritional requirement is simple, in food fresh keeping
Preservative exploitation aspect has a good application prospect.Publication No. be CN106939289 A announce understand starch gemma bacterial strain and
Corruption, which generates, after its product can adopt various fruits effectively inhibits, and can be used for preparing the spoilage organisms such as a kind of inhibition mould, rod method
And the biological and ecological methods to prevent plant disease, pests, and erosion antistaling agent with wider antimicrobial spectrum.The antibacterial exploitation of Bacillus at present is mostly single bacterial strain, and the present invention is old from Shanxi
It is isolated in mature vinegar vinegar fermented grain to staphylococcus aureus (gram-positive bacteria), Escherichia coli (Gram-negative bacteria), aspergillus niger
(disease fungus) has 3 plants of Bacillus of strong bacteriostatic activity respectively, and carries out composite usage in fresh-cut fruit and vegetable after preparing its bateriostatics
In fresh-keeping, and thereby it is made to have wider antibacterial application range and better fungistatic effect.
Summary of the invention
The purpose of the present invention is to provide a kind of safe and effective fresh-cut fruit and vegetable antistaling agent and preparation method thereof and users
Method can effectively inhibit in fresh-cut fruit and vegetable preservation because the rotten and brown stain as caused by enzymatic reaction caused by microbial reproduction is existing
As extending the shelf life of product to keep the quality of fresh-cut fruit and vegetable.
The present invention provides a kind of bacillus licheniformis CGMCC 16909 being isolated from Shanxi mature vinegar fermentation process,
The Substance that atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens CGMCC 15732 are generated is prepared compound
Antistaling agent, and applied Fresh-cut Apples and Fresh-cut Lettuce it is fresh-keeping in.Technical scheme is as follows:
Screening with high bacteriostatic activity Bacillus: acquiring the vinegar fermented grain sample in Shanxi mature vinegar acetic fermentation stage respectively, uses
MRS culture medium is diluted coating, finally isolates 50 plants of Bacillus, carries out having suppression to it using Oxford cup inhibition zone method
The active screening of bacterium, wherein Bacillus 297 is preferable to Gram-negative bacteria fungistatic effect, wherein to the fungistatic effect of Escherichia coli
Most significant, antibacterial circle diameter is 20.54 mm；Bacillus 1671 is preferable to gram-positive bacteria fungistatic effect, wherein to golden yellow
Staphylococcic fungistatic effect is most significant, and antibacterial circle diameter is 21.82 mm；Bacillus 2014 is to the antibacterial effect of fruits and vegetables Sapromyces
Fruit is preferable, and wherein the fungistatic effect of aspergillus niger is most significant, and antibacterial circle diameter is 23.48 mm.
The MRS culture medium the preparation method comprises the following steps: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g,
K2HPO42g, sodium acetate 2g, Triammonium citrate 2g, Tween 80 1g, MgSO40.2g, MnSO40.05g, distilled water 1000mL.
PH to 6.2 6.6 is adjusted, 121 DEG C of sterilizing 20min are spare.
16Sr DNA sequencing, primer are as follows: upstream: 5 '-CAGATGGGAGCTT are carried out to Bacillus 297,1671,2014
GCTCCCTG-3 ', downstream: 5 '-CGACTTCACCCAATCATCTG-3 ', identified Bacillus 297 are atrophy bacillus
(Bacillus atrophaeus), Bacillus 1671 be bacillus licheniformis (Bacillus licheniformis), Bacillus
2014 for bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and it is deposited in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, deposit number is atrophy bacillus CGMCC 16911, bacillus licheniformis
CGMCC 16909, bacillus amyloliquefaciens CGMCC 15732.
The preparation of fresh-cut fruit and vegetable composite preservative: by the bacillus licheniformis CGMCC 16909 of activation, atrophy bacillus
CGMCC 16911, bacillus amyloliquefaciens CGMCC 15732 press 5% inoculum concentration, are inoculated in MRS fluid nutrient medium respectively,
37 DEG C, after 160rpm/min is cultivated for 24 hours respectively, 4 DEG C, 10000 r/min are centrifuged 10 min, take supernatant；With the HCl of 6mol/L
Supernatant is tuned into neutrality by solution, and solid ammonium sulfate is added while stirring, and when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；
4000r/min collects precipitating after being centrifuged 20 min, is dissolved with the phosphate buffer of pH=7.2, desalination of dialysing, and vacuum refrigeration is dry
After dry, compound according to mass ratio 1:1:1 to get composite preservative.
The preparation method of phosphate buffer described above are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, chlorine
Change sodium 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
Vacuum freeze drying described above be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Apples it is fresh-keeping in, method particularly includes:
Apple is cut into small pieces (2cm × 2cm × 2cm) according to shape, by the apple after fresh-cut be immersed in 0.5% ascorbic acid and
It takes out and drains after 2min in 1.0% citric acid, be put into the disease for first uniformly spraying 250 μ L in sterilized petri dishes with the small watering can of 50mL
Opportunistic pathogen mixed liquor (concentration 104 Cfu/mL the composite preservative (concentration 1.0mg/mL) of 500 μ L), then is uniformly sprayed, later
Preservative film is covered after the drying of super-clean bench sterile wind, is placed in 4 DEG C of environment, to spoilage organisms golden yellow grape common during preservation
Coccus, Escherichia coli, Listeria monocytogenes, Bacterium enteritidis and shigella flexneri clump count are detected, and measure guarantor
The variation of the physical and chemical indexes such as the weight-loss ratio of Fresh-cut Apples, total phenol, vitamin C, MDA content, POD and PPO enzyme activity during hiding.
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Lettuce it is fresh-keeping in, method particularly includes:
It romaine lettuce is torn into piece is cleaned up in the chlorine water of 120mg/L and drained, is put into first uniform with the small watering can of 50mL in sterilized petri dishes
Spray the pathogen mixed liquor (concentration 10 of 250 μ L4 Cfu/mL), then uniformly (concentration is the composite preservative of 500 μ L of sprinkling
1.0mg/mL), it is put into after super-clean bench air-dries and is encapsulated with preservative film.Be placed in it is fresh-keeping under 4 DEG C of environment, observe composite preservative to fresh-cut
The fresh-keeping effect of romaine lettuce, to spoilage organisms staphylococcus aureus common during preservation, Escherichia coli, Listeria monocytogenes, intestines
Scorching salmonella and shigella flexneri clump count are detected, and measure the weight-loss ratio of Fresh-cut Lettuce, total phenol, dimension during preservation
The variation of the physical and chemical indexes such as raw element C, MDA content, POD and PPO enzyme activity.
Applying the fresh-cut fruit and vegetable composite fresh-keeping agent solution for being 1.0mg/mL in the fresh-keeping middle concentration of fresh-cut fruit and vegetable is with sterile
What water was configured to.
After the Fresh-cut Apples of sprinkling pathogen mixed liquor and romaine lettuce are handled with composite preservative as the result is shown, with control group phase
Than having apparent bacteriostasis, staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, single increasing Lee
The clump count of this special bacterium has significant decrease, reduces the spoilage rate of Fresh-cut Apples and romaine lettuce.
Detailed description of the invention
Fig. 1 is bacillus licheniformis CGMCC 16909, atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens
The colonial morphology and cellular morphology figure of CGMCC 15732, in figure: A is the colonial morphology of atrophy bacillus CGMCC 16911；B
For atrophy bacillus CGMCC 16,911 1000 × under cellular morphology figure；C is the bacterium of bacillus licheniformis CGMCC 16909
Fall form；D be bacillus licheniformis CGMCC 16,909 1000 × under cellular morphology figure；E is bacillus amyloliquefaciens CGMCC
15732 colonial morphology；F be bacillus amyloliquefaciens CGMCC 15,732 1000 × under cellular morphology figure；
Fig. 2 is atrophy bacillus CGMCC 16911, bacillus licheniformis CGMCC 16909, bacillus amyloliquefaciens CGMCC
15732 16S rDNA sequential system develops tree；
Fig. 3 is application of the fresh-cut fruit and vegetable composite preservative in Fresh-cut Apples are fresh-keeping, in figure: A is sterile water process the 0th day fresh
Cut apple；B is the 5th day Fresh-cut Apples of sterile water process；C is the 8th day Fresh-cut Apples of sterile water process；D is fresh-cut fruit and vegetable
Composite preservative handles the 0th day Fresh-cut Apples；E is the Fresh-cut Apples that fresh-cut fruit and vegetable composite preservative handles the 5th day；
F is the Fresh-cut Apples that fresh-cut fruit and vegetable composite preservative handles the 8th day；
Fig. 4 is the fresh-cut fruit and vegetable composite preservative clump count fresh-keeping to Fresh-cut Apples, and in figure: A is the total plate count of Fresh-cut Apples；
B is the E. coli clones number of Fresh-cut Apples；C is the S. aureus colonies number of Fresh-cut Apples；D is the good fortune of Fresh-cut Apples
Family name's Shigella clump count；E is the Bacterium enteritidis clump count of Fresh-cut Apples；F is the Listeria monocytogenes bacterium of Fresh-cut Apples
Fall number；
Fig. 5 is the fresh-cut fruit and vegetable composite preservative physical and chemical index fresh-keeping to Fresh-cut Apples, and in figure: A is the weight-loss ratio of Fresh-cut Apples；
B is the total phenol content of Fresh-cut Apples；C is the Vitamin C content of Fresh-cut Apples；D is the MDA content of Fresh-cut Apples；E is fresh-cut apple
The PPO enzyme activity of fruit；F is the POD enzyme activity of Fresh-cut Apples；
Fig. 6 is application of the fresh-cut fruit and vegetable composite preservative in Fresh-cut Lettuce is fresh-keeping, in figure: A is sterile water process the 0th day fresh
Cut romaine lettuce；B is the 5th day Fresh-cut Lettuce of sterile water process；C is the 7th day Fresh-cut Lettuce of sterile water process；D is fresh-cut fruit and vegetable
Composite preservative handles the 0th day Fresh-cut Lettuce；E is the Fresh-cut Lettuce that fresh-cut fruit and vegetable composite preservative handles the 5th day；
F is the Fresh-cut Lettuce that fresh-cut fruit and vegetable composite preservative handles the 7th day；
Fig. 7 is the fresh-cut fruit and vegetable composite preservative clump count fresh-keeping to Fresh-cut Lettuce, and in figure: A is the total plate count of Fresh-cut Lettuce；
B is the E. coli clones number of Fresh-cut Lettuce；C is the S. aureus colonies number of Fresh-cut Lettuce；D is the good fortune of Fresh-cut Lettuce
Family name's Shigella clump count；E is the Bacterium enteritidis clump count of Fresh-cut Lettuce；F is the Listeria monocytogenes bacterium of Fresh-cut Lettuce
Fall number；
Fig. 8 is the fresh-cut fruit and vegetable composite preservative physical and chemical index fresh-keeping to Fresh-cut Lettuce, and in figure: A is the weight-loss ratio of Fresh-cut Lettuce；
B is the total phenol content of Fresh-cut Lettuce；C is the Vitamin C content of Fresh-cut Lettuce；D is the MDA content of Fresh-cut Lettuce；E is raw for fresh-cut
The PPO enzyme activity of dish；F is the POD enzyme activity of Fresh-cut Lettuce.
Specific embodiment
Technical solution of the present invention is described further below by specific embodiment.
Embodiment 1: screening, identification and preservation with high bacteriostatic activity Bacillus
(1) there is the screening compared with high bacteriostatic activity Bacillus: acquiring the distiller's wort sample in Shanxi mature vinegar alcoholic fermentation stage respectively
With the vinegar fermented grain sample in acetic fermentation stage, coating is diluted using MRS culture medium, finally isolates 50 plants of Bacillus.It adopts
The screening with bacteriostatic activity is carried out to it with Oxford cup inhibition zone method, wherein Bacillus 297 is to the antibacterial effect of Gram-negative bacteria
Fruit is preferable, wherein the fungistatic effect to Escherichia coli is most significant, antibacterial circle diameter is 20.54 mm；Bacillus 1671 is to gram
Positive bacteria fungistatic effect is preferable, wherein the fungistatic effect to staphylococcus aureus is most significant, antibacterial circle diameter is 21.82 mm；
Bacillus 2014 is preferable to fruits and vegetables Sapromyces fungistatic effect, and wherein the fungistatic effect of aspergillus niger is most significant, and antibacterial circle diameter is
23.48 mm。
Above-mentioned Escherichia coli, staphylococcus aureus, aspergillus niger are bought to China General Microbiological culture presevation pipe
Reason center, the deposit number of Escherichia coli is CGMCC 1.1684, the deposit number of staphylococcus aureus is CGMCC
1.184, the deposit number of aspergillus niger is CGMCC 3.2915.
Above-mentioned MRS solid medium the preparation method comprises the following steps: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g,
K2HPO42g, sodium acetate 2g, Triammonium citrate 2g, Tween 80 1g, MgSO40.2g, MnSO40.05g, distilled water 1000mL.
PH to 6.2 6.6 is adjusted, 121 DEG C of sterilizing 20min are spare.
Above-mentioned screening has bacteriostatic activity Bacillus method particularly includes: isolate and purify 50 plants of Bacillus are connected to MRS
Fluid nutrient medium, 37 DEG C, 160 r/min cultivate 24 h, and 10000 r/min are centrifuged 10 min and collect Bacillus fermented supernatant fluid,
It is spare after being adjusted to neutrality with the NaOH of 6.0 mol/L respectively.By Escherichia coli, staphylococcus aureus, Fu Shi shiga
Bacterium, Listeria monocytogenes, Bacterium enteritidis, Friedlander's bacillus, Huo Shi enterobacteria, pseudomonas aeruginosa are respectively connected to
37 DEG C in BHI fluid nutrient medium, after 160 r/min cultivate 12 h, with 5 times (10 of normal saline dilution5Cfu/mL), pipette
The diluted bacterium solution of 0.1mL to BHI plate is coated, and after placing on BHI plate the Oxford cup to 1 h of refrigerator freezing of sterilizing
It takes out, Bacillus fermented supernatant fluid is pipetted into 200 μ L into Oxford cup, puts 37 DEG C of incubator cultures；By aspergillus niger, aspergillus flavus,
Mould point is connected to the PDA culture medium plate for being placed with Oxford cup, and Bacillus fermented supernatant fluid is pipetted 200 μ L into Oxford cup,
And put 30 DEG C of incubator cultures.
Above-mentioned shigella flexneri, Listeria monocytogenes, Friedlander's bacillus, Huo Shi enterobacteria, P. aeruginosa
Bacterium is bought to north and receives biotechnology center, and the deposit number of shigella flexneri is ATCC 12022, Listeria monocytogenes
Deposit number is ATCC 19114, the deposit number of Friedlander's bacillus is ATCC 10031, the preservation of Huo Shi enterobacteria is compiled
It number is ATCC 700323, the deposit number of pseudomonas aeruginosa is ATCC 9027；Salmonella is bought to Chinese medicine bacterium
Preservation administrative center, deposit number are CMCC 50041-16；Aspergillus flavus, mould are bought to China General Microbiological strain and are protected
Administrative center is hidden, the deposit number of aspergillus flavus is CGMCC 3.11969, the deposit number of mould is CGMCC 3.15687.
(2) there is the identification and preservation compared with high bacteriostatic activity Bacillus
Morphological Identification: in MRS plate culture after using oese picking Bacillus 297, Bacillus 1671, Bacillus 2014 a little
It crosses on base, isolates single colonie, the bacterium colony grown on culture medium is taken pictures, is recorded, then contaminated by gram
Look mirror inspection, observes its cell morphological characteristic.Bacillus 297 forms 4.42 mm of colony diameter, bacterium colony on MRS plating medium
Approximate circle, intermediate projections at flakes, milky, opaque, stickiness is larger；Bacillus 1671 is on MRS plating medium
4.02 mm of colony diameter is formed, rough surface, opaque, sticky, the more folds of protuberance, edge are irregular, bacterium colony is in shallow white；
Bacillus 2014 forms 4.58 mm of colony diameter on MRS plating medium, and bacterium colony is rounded, more sticky, rough surface is convex
It rises, is neat in edge, milky, opaque；Its cellular morphology: Gram's staining is in G+, in the shape of a rod, there are gemma, such as Fig. 1 in centre
It is shown.
16Sr DNA sequencing: extracting the genome of Bacillus 297, Bacillus 1671, Bacillus 2014 with kit, carries out
16Sr DNA sequencing and strain idenfication, primer are as follows: upstream: the downstream 5 '-CAGATGGGAGCTTGCTCCCTG-3 ': 5 '-
CGACTTCACCCAATCATCTG-3 ', using BLAST software through tetraploid rice, the results showed that Bacillus 297 is atrophy gemma
Bacillus (Bacillus atrophaeus), Bacillus 1671 be bacillus licheniformis (Bacillus licheniformis), bud
Spore bacterium 2014 be bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and systematic evolution tree is constructed, see Fig. 2.
And it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is atrophy bacillus
CGMCC 16911, bacillus licheniformis CGMCC 16909, bacillus amyloliquefaciens CGMCC 15732.
Embodiment 2: the preparation of fresh-cut fruit and vegetable composite preservative
By the bacillus licheniformis CGMCC 16909 of activation, atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens
CGMCC 15732 presses 5% inoculum concentration, is inoculated in MRS fluid nutrient medium respectively, 37 DEG C, 160rpm/min is cultivated for 24 hours respectively
Afterwards, 4 DEG C, 10000 r/min are centrifuged 10 min, take supernatant；Supernatant is tuned into neutrality with the HCl solution of 6mol/L, while stirring
It mixes side and solid ammonium sulfate is added, when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；4000r/min is collected after being centrifuged 20 min
Precipitating is dissolved with the phosphate buffer of pH=7.2, desalination of dialysing, multiple for 1:1:1 according to weight ratio after vacuum freeze drying
Match, is composite preservative after vacuum packaging.
The preparation method of phosphate buffer described above are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, chlorine
Change sodium 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
Vacuum freeze drying described above be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
Embodiment 3: application of the composite preservative in Fresh-cut Apples are fresh-keeping
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Apples it is fresh-keeping in, specific implementation method is such as
Under:
Apple is cut into small pieces (2cm × 2cm × 2cm) according to shape, the apple after fresh-cut is immersed in 0.5% ascorbic acid
It is drained with being taken out after 2min in 1.0% citric acid (color stabilizer), is put into sterilized petri dishes and is first uniformly sprayed with the small watering can of 50mL
Pathogen mixed liquor (the concentration 10 of 250 μ L4 Cfu/mL composite preservative (the concentration 1.0mg/ of 500 μ L), then is uniformly sprayed
ML), preservative film is covered after the drying of super-clean bench sterile wind later, be placed in 4 DEG C of environment, to spoilage organisms gold common during preservation
Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Bacterium enteritidis and shigella flexneri clump count are detected,
And measure the physical and chemical indexes such as weight-loss ratio, total phenol, vitamin C, MDA content, POD and PPO enzyme activity of Fresh-cut Apples during preservation
Variation.
As shown in figure 3, compared with the control group, the spoilage rate of the Fresh-cut Apples handled with composite preservative reduces；And
There is apparent bacteriostasis, wherein staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, single increasing Lee
The clump count of this special bacterium is significantly reduced (such as Fig. 4).As shown in Figure 5, the Fresh-cut Apples handled with composite preservative, it is weightless
Rate, MDA content climbing speed are slower, and vitamin C and total phenol content decline slower, peroxidase activity (POD), polyphenol oxidase
(PPO) living in downward trend after first rising, last enzyme activity is higher than sterile water process, has good fresh-keeping effect.
Pathogen mixed liquor described above specific the preparation method comprises the following steps: respectively by Escherichia coli, staphylococcus aureus,
Shigella flexneri, Bacterium enteritidis and Listeria monocytogenes are inoculated in BHI fluid nutrient medium, in 37 DEG C, 160 r/min
It cultivates 12 h and obtains cause of disease bacterium solution, supernatant is abandoned in 10000 r/min, l0 min centrifugation, heavy with 0.1% sterile peptone water washing
It forms sediment 2 times, and precipitating is resuspended with 0.1% sterile peptone water, be respectively prepared 104 The bacteria suspension of cfu/mL, and it is isometric
It is mixed into pathogen mixed liquor.
What composite preservative described above was prepared method particularly includes: composite preservative is configured to 1.0mg/ with sterile water
mL。
The measuring method of microorganism detection described above are as follows: Fresh-cut Apples aseptically weigh 10g sample, after shredding
It is incorporated in 90mL sterile saline, acutely shakes, gradient dilution is coated with after mixing, then to common spoilage organisms golden yellow Portugal
Grape coccus (mannitol sodium chloride agar medium), Escherichia coli (EMB agar medium), single monocytogenes
(will congratulates selection culture for (PALCAM agar culture medium), Bacterium enteritidis (maconkey agar culture medium), shigella flexneri
Base) it is counted.
The specific measuring method of physical and chemical index described above are as follows:
(1) measuring method of weight-loss ratio: during preservation, the daily same period takes the Fresh-cut Apples under the conditions of different disposal to claim
Weight, and calculate weight-loss ratio.Weight-loss ratio=(preservation initial stage fruit weight-preservation latter stage fruit weight)/preservation initial stage fruit weight
x100%
(2) measuring method of total phenol: using the colorimetric estimation of Folin-Ciocalteau method, weighs 5 g Fresh-cut Apples, is added 20
It is homogenized in the methanol solution ice bath of mL pre-cooling, 4 DEG C, 12000 r/min are centrifuged 20min, take 1mL supernatant in 25mL volumetric flask
In, mixing is vibrated after 1mL forint phenol solution is added, 7.5% sodium carbonate liquor of 10mL is added, is finally arrived with deionized water constant volume
25mL.25 DEG C of placement 1h, measure its light absorption value at 750 nm.
(3) ascorbic measuring method: using 2,6 dichloroindophenol titration measurings, weighs 5 g of Fresh-cut Apples, be added etc.
The 2%HCl solution of amount, is pounded homogenate.3g slurried sample (it is made to contain ascorbic acid 0.1-0.5mg) is weighed, small beaker is placed in
In, sample is moved into 100mL volumetric flask with 1% HCl solution, and be diluted to scale, is shaken up.Sample liquid is filtered, is discarded initially
Then several milliliters of filtrates are drawn 10mL filtrate with pipette rapidly, are placed in 50mL conical flask, with calibrated 2,6 dichloros
Indophenols titration, until solution pinkiness is until colour-fast in 15 seconds.
(4) measuring method of malonaldehyde (MDA) content: weighing 0.4g Fresh-cut Apples and be put into mortar, and a little quartz is added
The TCA of sand and 2mL 0.1%, inkstone are transferred in test tube at homogenate, then rinse mortar in two times with the TCA of 3mL 0.1%, are merged
The thiobarbituricacidα- solution of 5mL 0.5% is added in extracting solution in extracting solution, shakes up for test tube to be put into boiling water bath and develop the color instead
Answer 15min, to the time after be removed and placed in ice bath be cooled to room temperature immediately, after test tube is cooling, be transferred in 10mL centrifuge tube
3000r/min is centrifuged 15min, takes supernatant, surveys the light absorption value at 532nm and 600nm.
(5) measuring method of peroxidase activity (POD): the poly- second of crosslinking that 5g Fresh-cut Apples contain 2.5% with 20mL is weighed
The phosphate buffer (0.2mol/L pH 7.5) of alkene pyrrolidone mixes, and grinds rapidly under condition of ice bath, at 4 DEG C, 10000r/
Min is centrifuged 15mim, and supernatant is thick enzyme.1mL crude enzyme liquid is drawn, the phosphate buffer of 1mL pH 7.5, then plus 1mL is added
The buffer of catechol containing 1mmol/L after mixing, is returned to zero with distilled water, is immediately placed in 420nm and is gone out to measure absorbance value,
It is primary every 10s record, 2min is measured, 0.001 as 1 active unit is changed using sample absorbance value, unit is U/ (g ﹒
min)。
(6) measuring method of polyphenol oxidase enzyme activity (PPO): the poly- second of crosslinking that 5g Fresh-cut Apples contain 2.5% with 20mL is weighed
The phosphate buffer (0.2mol/L pH 7.5) of alkene pyrrolidone mixes, and grinds rapidly under condition of ice bath, at 4 DEG C, 10000r/m
It is centrifuged 15mim, supernatant is thick enzyme.Take the phosphoric acid buffer of 0.05% guaiacol solution 2mL(0.2mol/L pH 7.5
Solution is formulated), the thick zyme extract of 0.5mL is added, 5min is kept the temperature in 30 DEG C of water-baths, adds 1mL 0.08%
H2O2It mixes after solution, is returned to zero with distilled water, be immediately placed in measurement absorbance value at 470nm, measurement primary every 30s record
3min changes 0.001 as 1 active unit using sample absorbance value, and unit is U/ (g ﹒ min).
Embodiment 4: application of the composite preservative in Fresh-cut Lettuce is fresh-keeping
Prepare composite preservative according to above-mentioned technical proposal, applied Fresh-cut Lettuce it is fresh-keeping in, specific implementation method is such as
Under:
It romaine lettuce is torn into piece is cleaned up in the chlorine water (color stabilizer) of 120mg/L and drained, be put into sterilized petri dishes and first use 50mL
Small watering can uniformly spray the pathogen mixed liquor (concentration 10 of 250 μ L4 Cfu/mL the compound guarantor of 500 μ L), then is uniformly sprayed
It fresh dose (concentration 1.0mg/mL), is put into after super-clean bench air-dries and is encapsulated with preservative film.Be placed in it is fresh-keeping under 4 DEG C of environment, observe it is compound
Antistaling agent is to the fresh-keeping effect of Fresh-cut Lettuce, to spoilage organisms staphylococcus aureus common during preservation, Escherichia coli, Dan Zeng
Listeria, Bacterium enteritidis and shigella flexneri clump count are detected, and measure the mistake of Fresh-cut Lettuce during preservation
The variation of the physical and chemical indexes such as rate, total phenol, vitamin C, MDA content, POD and PPO enzyme activity again.
As shown in fig. 6, compared with the control group, the spoilage rate of the Fresh-cut Lettuce handled with composite preservative reduces；And
There is apparent bacteriostasis, wherein staphylococcus aureus, Escherichia coli, Bacterium enteritidis, shigella flexneri, single increasing Lee
The clump count of this special bacterium is significantly reduced (such as Fig. 7).As shown in Figure 8, the Fresh-cut Lettuce handled with composite preservative, it is weightless
It is slower that rate has just begun to ramp up rate, then very fast, and vitamin C, MDA and total phenol content decline slower, peroxidase activity
(POD), in downward trend after first rising, last enzyme activity is higher than sterile water process, has polyphenol oxidase enzyme activity (PPO)
Good fresh-keeping effect.
Claims (5)
1. a kind of fresh-cut fruit and vegetable by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens
Antistaling agent, it is characterised in that: the bacillus licheniformis, atrophy bacillus, bacillus amyloliquefaciens are from Shanxi mature vinegar vinegar
It separated in unstrained spirits, screen gained, the bacillus licheniformis deposit number is CGMCC 16909, atrophy bacillus deposit number
For CGMCC 16911, bacillus amyloliquefaciens deposit number is CGMCC 15732, depositary institution: Chinese microorganism strain preservation
Administration committee's common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date:
On December 10th, 2018, on May 7th, 2018.
2. one kind according to claim 1 is produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens
Bateriostatics compounding fresh-cut fruit and vegetable antistaling agent, it is characterised in that: the fresh-cut fruit and vegetable composite preservative the preparation method comprises the following steps: will live
Bacillus licheniformis CGMCC 16909, the atrophy bacillus CGMCC 16911, bacillus amyloliquefaciens CGMCC 15732 of change
It by 3% inoculum concentration, is inoculated in MRS fluid nutrient medium respectively, 37 DEG C, after 160rpm/min is cultivated for 24 hours respectively, 4 DEG C, 10000
R/min is centrifuged 10 min, takes supernatant；Supernatant is tuned into neutrality with the HCl solution of 6mol/L, solid sulfur is added while stirring
Sour ammonium, when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；4000r/min collects precipitating after being centrifuged 20 min, with pH=7.2
Phosphate buffer dissolves, desalination of dialysing, and after vacuum freeze drying, compounds according to mass ratio 1:1:1 to get composite preservative.
3. one kind according to claim 2 is produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens
The fresh-cut fruit and vegetable antistaling agent of bateriostatics compounding, it is characterised in that: the preparation method of the phosphate buffer are as follows: biphosphate
Potassium 0.24g, disodium hydrogen phosphate 1.42g, sodium chloride 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
4. one kind according to claim 2 is produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens
The fresh-cut fruit and vegetable antistaling agent of bateriostatics compounding, it is characterised in that: the vacuum freeze drying is in -40 DEG C, vacuum degree 100
Dry 40 h under the conditions of Pa.
5. it is a kind of it is described in claim 1 produced by bacillus licheniformis, atrophy bacillus and bacillus amyloliquefaciens it is antibacterial
Application of the fresh-cut fruit and vegetable antistaling agent of object compounding in fresh-cut fruit and vegetable, the concentration are 1.0mg/mL fresh-cut fruit and vegetable composite fresh-keeping
Agent solution is formulated with sterile water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910354887.7A CN109984189B (en) | 2019-04-29 | 2019-04-29 | Fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910354887.7A CN109984189B (en) | 2019-04-29 | 2019-04-29 | Fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109984189A true CN109984189A (en) | 2019-07-09 |
| CN109984189B CN109984189B (en) | 2022-07-01 |
Family
ID=67135518
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910354887.7A Active CN109984189B (en) | 2019-04-29 | 2019-04-29 | Fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109984189B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110269093A (en) * | 2019-07-19 | 2019-09-24 | 云南省农业科学院农产品加工研究所 | A kind of fresh-cut fruit and vegetable fresh-keeping liquid |
| CN113712077A (en) * | 2021-08-23 | 2021-11-30 | 南京工业大学 | Fruit preservative compounded by using bacillus fermentation product and application thereof |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715800A (en) * | 2009-12-18 | 2010-06-02 | 宁波大学 | Compound anti-rot and fresh-keeping method for strawberries after picking |
| CN102559533A (en) * | 2010-12-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Bacillus atrophaeus, and preparation and application of bacillus atrophaeus |
| CN103070226A (en) * | 2013-01-05 | 2013-05-01 | 山西农业大学 | Color-protecting and fresh-keeping method for fresh-cut potatoes |
| CN103173397A (en) * | 2013-04-02 | 2013-06-26 | 山东省农业科学院农产品研究所 | Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof |
| CN103589655A (en) * | 2013-05-20 | 2014-02-19 | 山西农业大学 | Preparation method for Bacillus amyloliquefaciens HRH 317 and antibacterial substances thereof |
| CN103966283A (en) * | 2013-01-30 | 2014-08-06 | 大连海洋大学 | Kelp endophytic bacillus licheniformis protein, application thereof to preservation and application method thereof |
| CN104430834A (en) * | 2014-12-30 | 2015-03-25 | 华南师范大学 | Application of bacillus amyloliquefaciens strain B014 in postharvest preservation of fruits and vegetables |
| CN104988102A (en) * | 2015-08-07 | 2015-10-21 | 四川农业大学 | Culture method of bacillus amyloliquefaciens and application thereof |
| CN105695366A (en) * | 2016-04-05 | 2016-06-22 | 中国农业科学院植物保护研究所 | Bacillus strain and application thereof |
| CN106939289A (en) * | 2017-02-23 | 2017-07-11 | 浙江大学 | A kind of biological and ecological methods to prevent plant disease, pests, and erosion Bacillus amyloliquefaciens strain and application thereof |
| CN107338202A (en) * | 2017-06-27 | 2017-11-10 | 浙江理工大学 | Suppress bacillus amyloliquefaciens and its application of pathogen function with wide spectrum |
| CN108077411A (en) * | 2017-12-20 | 2018-05-29 | 山西省农业科学院农产品贮藏保鲜研究所 | The preparation method and its application method of bacillus amyloliquefaciens Y-3 bio-preservatives |
| CN109090225A (en) * | 2018-07-09 | 2018-12-28 | 湖州吴兴道场城乡建设发展有限公司 | A kind of method of biological preservation crisp winter jujube |
| CN109090224A (en) * | 2018-07-09 | 2018-12-28 | 湖州吴兴道场城乡建设发展有限公司 | A kind of microorganism fruit antistaling agent |
-
2019
- 2019-04-29 CN CN201910354887.7A patent/CN109984189B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715800A (en) * | 2009-12-18 | 2010-06-02 | 宁波大学 | Compound anti-rot and fresh-keeping method for strawberries after picking |
| CN102559533A (en) * | 2010-12-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Bacillus atrophaeus, and preparation and application of bacillus atrophaeus |
| CN103070226A (en) * | 2013-01-05 | 2013-05-01 | 山西农业大学 | Color-protecting and fresh-keeping method for fresh-cut potatoes |
| CN103966283A (en) * | 2013-01-30 | 2014-08-06 | 大连海洋大学 | Kelp endophytic bacillus licheniformis protein, application thereof to preservation and application method thereof |
| CN103173397A (en) * | 2013-04-02 | 2013-06-26 | 山东省农业科学院农产品研究所 | Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof |
| CN103589655A (en) * | 2013-05-20 | 2014-02-19 | 山西农业大学 | Preparation method for Bacillus amyloliquefaciens HRH 317 and antibacterial substances thereof |
| CN104430834A (en) * | 2014-12-30 | 2015-03-25 | 华南师范大学 | Application of bacillus amyloliquefaciens strain B014 in postharvest preservation of fruits and vegetables |
| CN104988102A (en) * | 2015-08-07 | 2015-10-21 | 四川农业大学 | Culture method of bacillus amyloliquefaciens and application thereof |
| CN105695366A (en) * | 2016-04-05 | 2016-06-22 | 中国农业科学院植物保护研究所 | Bacillus strain and application thereof |
| CN106939289A (en) * | 2017-02-23 | 2017-07-11 | 浙江大学 | A kind of biological and ecological methods to prevent plant disease, pests, and erosion Bacillus amyloliquefaciens strain and application thereof |
| CN107338202A (en) * | 2017-06-27 | 2017-11-10 | 浙江理工大学 | Suppress bacillus amyloliquefaciens and its application of pathogen function with wide spectrum |
| CN108077411A (en) * | 2017-12-20 | 2018-05-29 | 山西省农业科学院农产品贮藏保鲜研究所 | The preparation method and its application method of bacillus amyloliquefaciens Y-3 bio-preservatives |
| CN109090225A (en) * | 2018-07-09 | 2018-12-28 | 湖州吴兴道场城乡建设发展有限公司 | A kind of method of biological preservation crisp winter jujube |
| CN109090224A (en) * | 2018-07-09 | 2018-12-28 | 湖州吴兴道场城乡建设发展有限公司 | A kind of microorganism fruit antistaling agent |
Non-Patent Citations (4)
| Title |
|---|
| YAOQI GUO: "Isolation and characterization of a Bacillus atrophaeus strain and its potential use in food preservation", 《FOOD CONTROL》 * |
| 付瑞敏等: "生物可食性活性膜对红富士苹果采后防腐保鲜的研究", 《食品科技》 * |
| 出晓铭等: "生防菌解淀粉芽孢杆菌抗菌蛋白的研究进展", 《包装与食品机械》 * |
| 刘丁等: "萎缩芽孢杆菌处理提高花生黄曲霉抗性的作用机制", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110269093A (en) * | 2019-07-19 | 2019-09-24 | 云南省农业科学院农产品加工研究所 | A kind of fresh-cut fruit and vegetable fresh-keeping liquid |
| CN113712077A (en) * | 2021-08-23 | 2021-11-30 | 南京工业大学 | Fruit preservative compounded by using bacillus fermentation product and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109984189B (en) | 2022-07-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102433282B (en) | Bacillus subtilis NB12, as well as culture method and application thereof | |
| CN104694430B (en) | Bacillus amyloliquefaciens plant subspecies ZFH 3 and its application | |
| CN104320980B (en) | Protective foods containing pediococcus acidilactici J9 and its preparation method | |
| CN108148794A (en) | A kind of the bacillus subtilis DYr3.3 and preparation method and application of broad-spectrum antibacterial activity | |
| CN111718881B (en) | Siamese bacillus and application thereof | |
| CN102342567B (en) | Pepper peeling method | |
| CN107788517B (en) | Method for enhancing enzyme efficacy by using lactobacillus bulgaricus | |
| CN101822288A (en) | Preparation method of vacuum freeze-dried product of marineyeast Rhodosporidium paludigenum Fell and Tallman | |
| CN109984189A (en) | A kind of fresh-cut fruit and vegetable antistaling agent by bacillus licheniformis, atrophy bacillus and the produced bateriostatics compounding of bacillus amyloliquefaciens | |
| CN100512651C (en) | Biocontrol fungus for preventing and controlling plant mycosis and its preparation process | |
| CN101914459A (en) | Peach fruit disease biological antiseptic preservative and application and used cryptococcus laurentii | |
| CN108795782B (en) | Method for preventing and treating pear postharvest diseases and storing and refreshing pear fruits by using abnormal Weikeham bacteria | |
| CN111264817B (en) | Application of direct vat set lactobacillus starter in preserved szechuan pickle pickling | |
| CN106244498B (en) | Preparation method of fruit and vegetable pesticide residue purification microbial inoculum | |
| Uzeh et al. | Incidence and survival of Escherichia coli O157: H7 and Listeria monocytogenes on salad vegetables | |
| CN115851479B (en) | Bacteria with antagonism to botrytis cinerea and application thereof | |
| CN109402023B (en) | Mildew-proof bacillus strain and application thereof in grain storage | |
| CN115141785B (en) | Bacillus subtilis and application thereof in cabbage planting | |
| CN104757544B (en) | A kind of dietary supplements with bacteriostasis and its preparation method and application | |
| CN111411056A (en) | Lactobacillus paracasei subspecies tenacious strain, screening method and application | |
| CN116536207A (en) | Bacillus atrophaeus WLKYSY-4, biological microbial inoculum and application thereof | |
| CN105733008A (en) | Preparation method of novel antibacterial biological preservative film | |
| CN112522119A (en) | Preparation method and application of combined microbial inoculum based on Klebsiella citrullina and Candida intermedia | |
| CN101053342A (en) | Bio-preserving fungus agent and its preparation method | |
| CN116103186B (en) | Enterobacter cloacae and application thereof in preventing and treating litchi frost epidemic diseases |
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 |