CN110283748A - Bacillus cereus fermentation culture medium and raising bacillus cereus produce the fermentation process for killing rice leaf roller species activity - Google Patents
Bacillus cereus fermentation culture medium and raising bacillus cereus produce the fermentation process for killing rice leaf roller species activity Download PDFInfo
- Publication number
- CN110283748A CN110283748A CN201910587899.4A CN201910587899A CN110283748A CN 110283748 A CN110283748 A CN 110283748A CN 201910587899 A CN201910587899 A CN 201910587899A CN 110283748 A CN110283748 A CN 110283748A
- Authority
- CN
- China
- Prior art keywords
- bacillus cereus
- fermentation
- culture medium
- rice leaf
- leaf roller
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000193755 Bacillus cereus Species 0.000 title claims abstract description 164
- 238000000855 fermentation Methods 0.000 title claims abstract description 117
- 230000004151 fermentation Effects 0.000 title claims abstract description 117
- 239000001963 growth medium Substances 0.000 title claims abstract description 64
- 241000008892 Cnaphalocrocis patnalis Species 0.000 title claims abstract description 55
- 230000000694 effects Effects 0.000 title claims abstract description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 38
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000002609 medium Substances 0.000 claims abstract description 28
- 239000000203 mixture Substances 0.000 claims abstract description 23
- 239000001888 Peptone Substances 0.000 claims abstract description 22
- 108010080698 Peptones Proteins 0.000 claims abstract description 22
- 235000019319 peptone Nutrition 0.000 claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 claims abstract description 20
- 239000011780 sodium chloride Substances 0.000 claims abstract description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 19
- 235000015278 beef Nutrition 0.000 claims abstract description 18
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 18
- 239000000284 extract Substances 0.000 claims abstract description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 18
- 239000012138 yeast extract Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229920002261 Corn starch Polymers 0.000 claims abstract description 15
- 239000008120 corn starch Substances 0.000 claims abstract description 15
- 229940099112 cornstarch Drugs 0.000 claims abstract description 15
- 150000003839 salts Chemical class 0.000 claims abstract description 12
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 8
- -1 salts sodium chloride Chemical class 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 21
- 241000894006 Bacteria Species 0.000 claims description 16
- 239000002054 inoculum Substances 0.000 claims description 12
- 241000726221 Gemma Species 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- 210000004209 hair Anatomy 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 235000012907 honey Nutrition 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 230000012010 growth Effects 0.000 abstract description 21
- 239000000126 substance Substances 0.000 abstract description 15
- 238000009395 breeding Methods 0.000 abstract description 10
- 230000001488 breeding effect Effects 0.000 abstract description 10
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 2
- 241000894007 species Species 0.000 description 13
- 241000193830 Bacillus <bacterium> Species 0.000 description 12
- 230000001018 virulence Effects 0.000 description 12
- 238000010586 diagram Methods 0.000 description 10
- 241000209094 Oryza Species 0.000 description 9
- 235000007164 Oryza sativa Nutrition 0.000 description 9
- 235000009566 rice Nutrition 0.000 description 9
- 239000000575 pesticide Substances 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 210000004894 snout Anatomy 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000002028 Biomass Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
- 239000000306 component Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000000749 insecticidal effect Effects 0.000 description 4
- 239000012533 medium component Substances 0.000 description 4
- 235000019799 monosodium phosphate Nutrition 0.000 description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000002917 insecticide Substances 0.000 description 3
- 229940099596 manganese sulfate Drugs 0.000 description 3
- 239000011702 manganese sulphate Substances 0.000 description 3
- 235000007079 manganese sulphate Nutrition 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 244000089795 Clausena lansium Species 0.000 description 2
- 235000008738 Clausena lansium Nutrition 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
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 244000037433 Pongamia pinnata Species 0.000 description 2
- 235000004599 Pongamia pinnata Nutrition 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000000853 biopesticidal effect Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000036632 reaction speed Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000003519 ventilatory effect Effects 0.000 description 2
- 206010063409 Acarodermatitis Diseases 0.000 description 1
- 101710186708 Agglutinin Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241001660259 Cereus <cactus> Species 0.000 description 1
- 240000001817 Cereus hexagonus Species 0.000 description 1
- 241000426497 Chilo suppressalis Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241001498622 Cixius wagneri Species 0.000 description 1
- 241000098277 Cnaphalocrocis Species 0.000 description 1
- 241000098289 Cnaphalocrocis medinalis Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LLQPHQFNMLZJMP-UHFFFAOYSA-N Fentrazamide Chemical compound N1=NN(C=2C(=CC=CC=2)Cl)C(=O)N1C(=O)N(CC)C1CCCCC1 LLQPHQFNMLZJMP-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101710146024 Horcolin Proteins 0.000 description 1
- 235000006662 Lansium Nutrition 0.000 description 1
- 241001156382 Lansium Species 0.000 description 1
- 101710189395 Lectin Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- 101710179758 Mannose-specific lectin Proteins 0.000 description 1
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 1
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 241000565347 Pongamia Species 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 240000002044 Rhizophora apiculata Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000447727 Scabies Species 0.000 description 1
- 241001249129 Scirpophaga incertulas Species 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 241000130764 Tinea Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 239000000910 agglutinin Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 208000005687 scabies Diseases 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000010998 test method Methods 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
- 238000004879 turbidimetry Methods 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 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
-
- 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
-
- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- 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
- C12R2001/085—Bacillus cereus
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention, which discloses Bacillus cereus fermentation culture medium and improves bacillus cereus, produces the fermentation process for killing rice leaf roller species activity, and Bacillus cereus fermentation culture medium is made of the following components: carbon source, nitrogen source, inorganic salts and pure water；The carbon source is the mixture of blackstrap or cornstarch and blackstrap；The nitrogen source is the mixture of one or more of yeast extract, beef extract, peptone, ammonium chloride；The inorganic salts sodium chloride；The pH value range of the fermentation medium is 5.5~8.0.The selection of Bacillus cereus fermentation culture medium can provide the carbon source and nitrogen source of sufficient nutrition for the growth of bacillus cereus, enable bacillus cereus faster and better growth and breeding, improve the number of viable of bacillus cereus.The fermentation process for killing rice leaf roller species activity is produced the present invention also provides a kind of raising bacillus cereus, it is ensured that the activity that rice leaf roller substance is killed in bacillus cereus production is maintained at higher level.
Description
[technical field]
The present invention relates to technical field of microbial fermentation, especially Bacillus cereus fermentation culture medium and improve waxy bud
Spore bacillus produces the fermentation process for killing rice leaf roller species activity.
[background technique]
Pest occurs in turn during south rice is planted, with the harm of planthopper, rice leaf roller, striped rice borer, yellow rice borer
Property it is maximum, in order to reduce the incidence of rice grub, prevent the abuse of chemical pesticide, developing novel biological pesticide is to solve hand
One of section.Compared with traditional chemical pesticide, biological pesticide has to people and animals and non-target organism safety, and environment compatibility is good,
It is not easy to produce resistance, is easy to protect bio-diversity, the advantages that source is wide.Therefore, the development and application of high-performance bio pesticide are to people
Class health, environmental protection and the sustainable development of agricultural have extremely important meaning.Chinese medicinal plant source pesticide has become at present
One of the hot spot of biorational pesticide research exploitation.Pulse family in Chinese medicinal plant be the big botanical pesticide resource in China three it
One, leguminous plant has in the active insecticidal components such as flavone compound, alkaloid compound, desinsection agglutinin.So having
Necessity separation leguminous plant endophyte finds new biological pesticide resource.
Southern liquid medicine Calusena lansium (Pongamia pinnata (Linn.) Pierre) is pulse family Pongamia plant, and a kind of
Typical mangrove associates are recorded in " China's book on Chinese herbal medicine ", harvest when the fruit matures in the fall, lay Indian beech seed and dry, bitter
Mild toxicity cold in nature, can dispelling wind and eliminating dampness, detoxify desinsection, cure mainly rheumatic arthralgia, tinea scabies, running sore.Bacillus cereus (SeB3) is from water
One plant of Radical Injury in Wampee Seeds position separation acquisition is to rice grub rice leaf roller (Cnaphalocrocis medinalis
Guenee it) with the bacterial strain of stronger insecticidal activity, is analyzed through 16SrDNA sequential system auxology, the bacterial strain will be carried out and be accredited as
Bacillus cercus (B.cereus).
By Bacillus cereus SeB3 exploitation at commercialized new bio pesticide product, it is big to first have to solution bacterial strain
The difficult problem of amount breeding, while ensuring that its metabolite insecticidal activity is maintained at higher level, to further realize SeB3 research and development
It lays the foundation at efficient, wide spectrum, nuisanceless, inexpensive new bio insecticide.
[summary of the invention]
The purpose of the present invention is to provide a kind of Bacillus cereus fermentation culture mediums, solve asking for bacterial strain mass propagation hardly possible
Topic；The fermentation process for killing rice leaf roller species activity is produced the present invention also provides a kind of raising bacillus cereus, it is ensured that waxy
The activity that rice leaf roller substance is killed in bacillus production is maintained at higher level.
To solve the problems, such as that above-mentioned Bacillus cereus strain mass propagation is difficult, the present invention provides a kind of bacillus cereus
Fermentation medium is made of the following components:
Carbon source, nitrogen source, inorganic salts and pure water；
The carbon source is the mixture of blackstrap or cornstarch and blackstrap；
The nitrogen source is the mixture of one or more of yeast extract, beef extract, peptone, ammonium chloride；
The inorganic salts are sodium chloride；The pH value range of the fermentation medium is 5.5~8.0.
It is made as a kind of currently preferred Bacillus cereus fermentation culture medium of the component of following mass ratio: 2%
Blackstrap, 0.5~1.5% beef extract, 0.5% sodium chloride and surplus pure water；The pH value of the fermentation medium be 6.5~
7.5。
Preferably, the pH value of the fermentation medium is 7.0.
It is made as a kind of currently preferred Bacillus cereus fermentation culture medium of the component of following mass ratio: 2%
The mixture of blackstrap and cornstarch, 1% beef extract, 0.5% sodium chloride and surplus pure water, the blackstrap and corn
The mass ratio of starch is 1:1, and the pH value of the fermentation medium is 6.5~7.5.
It is made as a kind of currently preferred Bacillus cereus fermentation culture medium of the component of following mass ratio: 2%
Blackstrap, the mixture of 1% yeast extract and peptone, 0.5% sodium chloride and surplus pure water, the yeast extract
Mass ratio with peptone is 1:1, and the pH value of the fermentation medium is 6.5~7.0.
The waxy gemma bar of the species activity unit volume for the killing rice leaf roller that Bacillus cereus fermentation generates
The death rate for the rice leaf roller that bacteria culture fluid kills indicates that the rice that the bacillus cereus culture solution of unit volume kills is vertical
The death rate of leaf roll snout moth's larva is higher, then the species activity for the killing rice leaf roller that Bacillus cereus fermentation generates is higher.
Advantages of the present invention: Bacillus cereus fermentation culture medium of the invention, which is selected, to be the life of bacillus cereus
It is long that the carbon source and nitrogen source of sufficient nutrition are provided, enable bacillus cereus faster and better growth and breeding, improves waxy
The number of viable of bacillus, reaches 5.73 × 109CFU·mL-1~7.15 × 109CFU·mL-1, solve bacillus cereus
The problem of bacterial strain mass propagation hardly possible.
Higher level is maintained to solve the insecticidal activity that rice leaf roller active material is killed in above-mentioned bacillus cereus production
The problem of, the present invention, which provides a kind of raising bacillus cereus and produces, kills the fermentation process of rice leaf roller species activity, including with
Lower step:
1. the activation of strain: by the strain transfer of the bacillus cereus of preservation to slant medium, 37 DEG C of cultures 24 are small
When, it is spare；
2. the preparation of seed liquor: accessing the seed culture medium that liquid amount is 50ml in the triangular flask of 250ml, step is added
1. the strain that activation is completed in, is cultivated 18 hours under the conditions of 37 DEG C of cultivation temperature, revolving speed 180r/min；
3. shaking flask culture: taking 1ml step respectively, 2. the middle seed liquor for completing culture, access fill 50ml fermentation medium
It in the triangular flask of 200ml, sets in shaking table, the shaken cultivation 12~24 under the conditions of cultivation temperature is 30 DEG C, revolving speed is 160r/min
Hour.
As a preference of the present invention, step 3. in incubation time be 18~22 hours.
As a preference of the present invention, step 3. in the initial pH value of shaking flask culture be 6.5~7.5.
As a preference of the present invention, step 3. in the temperature of shaking flask culture be 35~42 DEG C.
As a preference of the present invention, step 3. described in seed liquor inoculum concentration be 5%~9%, the dress of the triangular flask
Liquid measure is 20%~40%.
The active fermentation process that rice leaf roller substance is killed in raising bacillus cereus production of the invention has the advantage that
The active fermentation process that rice leaf roller substance is killed in raising bacillus cereus production of the invention passes through to bacillus cereus
The condition of culture is screened, and then by comparing the influence under the conditions of each for the death rate of rice leaf roller, determination is mentioned
High bacillus cereus produces the fermentation process for killing rice leaf roller species activity, has been determined to be suitable for waxy gemma bar in the method
Bacterium output kills each condition of activity of the substance of rice leaf roller, it is ensured that the bacillus cereus of production produces and kills rice leaf roller
The activity of substance is maintained at higher level, so that the death rate that the substance that bacillus cereus produces kills rice leaf roller is maintained at
62%~95.8%.
[Detailed description of the invention]
Fig. 1 is influence diagram of the incubation time to bacillus cereus biomass of embodiment 34.
Fig. 2 is the incubation time of embodiment 34 to rice leaf roller death rate influence diagram.
Fig. 3 is the initial pH of embodiment 35 to 40 to bacillus cereus biomass influence diagram.
Fig. 4 is the initial pH of embodiment 35 to 40 to rice leaf roller death rate influence diagram.
Fig. 5 is the cultivation temperature of embodiment 41 to 45 to bacillus cereus biomass influence diagram.
Fig. 6 is influence diagram of the cultivation temperature to the rice leaf roller death rate of embodiment 41 to 45.
Fig. 7 is influence diagram of the liquid amount to bacillus cereus biomass of embodiment 46 to 50.
Fig. 8 is influence diagram of the liquid amount to the rice leaf roller death rate of embodiment 46 to 50.
Fig. 9 is influence diagram of the inoculum concentration to bacillus cereus biomass of embodiment 51 to 55.
Figure 10 is influence diagram of the inoculum concentration to the rice leaf roller death rate of embodiment 51 to 55.
[specific embodiment]
Following will be combined with the drawings in the embodiments of the present invention, carries out clear, complete description to technical solution of the present invention,
Obviously, the described embodiments are merely a part of the embodiments of the present invention.The material and test method that the present invention uses
1.1, material
1.1.1 the bacterial strain (SeB3) of bacillus cereus picks up from Guangdong pure water Radical Injury in Wampee Seeds in the coating woods of mountain forest seabeach,
Be for experiment the rice leaf roller of growth 7 days that insect provides from Inst. of Plant Protection, Guangdong Prov. Academy of Agricultural Sciences.
1.1.2 culture medium
Seed culture medium (W/V): peptone 1%, yeast extract 0.5%, sodium chloride 1%, natural ph.Basal fermentation
Culture medium (W/V): sucrose 1%, peptone 1%, disodium hydrogen phosphate 0.2%, sodium dihydrogen phosphate 0.2%, pH value 7.0.
1.1.3 main agents
Carbon source: blackstrap, sucrose, glucose, starch, cornstarch, maltose；
Nitrogen source: yeast extract, tryptone, beef extract, urea, ammonium chloride；
Inorganic salts: manganese sulfate, magnesium sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ferrous sulfate, sodium chloride, calcium chloride；It is pure
Water.
1.1.4 instrument and equipment
C1000 Touch PCR instrument (Shanghai Bole's life medical product Co., Ltd), the desk-top freezing of Allegra X-15R
Centrifuge (Beckman Coulter Inc.), Innova 44R shaking table (U.S. NBS), Rotary Evaporators RE-52 (Shanghai match ink science and technology
Biological Development Co., Ltd), Precision high-performance microbiological incubator (Co., Ltd in silent your science and technology of winged generation of match) surpasses
Low temperature refrigerator (U.S. Thermo), ZHS-100CH constant incubator (Shanghai Zhe figure), high-pressure steam sterilizing pan (Sanyo MLS-
3750)。
Above-mentioned culture medium, reagent and instrument are the conventional products that can be obtained by commercially available purchase.
1.2 method
1.2.1 cultural method
1. actication of culture: by the strain transfer to slant medium of the bacillus cereus of preservation, 37 DEG C of cultures 24 are small
When, it is spare.
2. the preparation of seed liquor: accessing the seed culture medium that liquid amount is 50ml in the triangular flask of 250ml, a ring is added
Activation is 1. strain that step completes activation culture, is cultivated 18 hours under the conditions of 37 DEG C, revolving speed 180r/min.
3. shaking flask culture: taking 1ml step respectively, 2. the middle seed liquor for completing culture, access fill 50ml basal fermentation culture
It in the triangular flask of the 200ml of base, sets in shaking table, the shaken cultivation 12 under the conditions of cultivation temperature is 30 DEG C, revolving speed is 160r/min
Hour.
1.2.2 measuring method
Total viable count measurement uses dilution plate counting method；The measurement of thalli growth curve uses turbidimetry, measures bacterium solution
OD600nm；PH value analyzer measures bacterium solution pH value.
Fermentation liquid virulence determination uses impregnating process, fermentation liquid is concentrated 20 times, then with aqua sterilisa in the ratio of 7:20
Mixing is diluted to stand-by medical fluid, is packed into stand-by medical fluid 10ml in plastic cup disposably drinking, is accessed with suction pipe and have just enter into maturation
Phase is no more than rice leaf roller 30 of 48h, and each cup is a processing, and each processing is repeated 3 times, while setting sterile culture
Liquid is control, investigates dead borer population afterwards for 24 hours, calculates the death rate.
1.2.3 all statistical analysis herein are statisticallyd analyze and complete (SAS Institute with SAS9.4 software
Inc.2014)。
The present invention uses the material base of single factor test and orthogonal test research Bacillus cereus fermentation culture, that is, determines suitable
It closes Bacillus cereus fermentation culture medium and is suitable for the nutriments such as optimum carbon source, nitrogen source, the inorganic salts of bacillus cereus breeding
Condition, and then provide it is a kind of suitable for bacillus cereus breeding fermentation medium.
Embodiment 1 to 7: carbon source, with blackstrap, cornstarch, blackstrap and cornstarch, (blackstrap and corn form sediment respectively
The mass ratio of powder is 1:1), the basis of 1% glucose solution, soluble starch, maltose equivalent substitution cultural method 1.2.1
Sucrose in fermentation medium, other compositions are constant, and bacillus cereus culture medium is made, then with waxy gemma bar obtained
Bacterium culture medium replaces basal fermentation medium of the step of cultural method 1.2.1 3. in shaking flask culture to carry out fermented and cultured, and 12 is small
When after measure fermentation liquid in viable bacteria content, be recorded in table 1, to determine optimum carbon source.
Table 1: different carbon source cultivation results table
Note: viable count unit: 109CFU·mL- 1
From table 1 from it can be concluded that, the bacillus cereus culture medium culture that embodiment 2 is made as carbon source of blackstrap
Bacillus cereus number of viable highest, reach 6.82 × 109CFU·mL- 1, blackstrap is most suitable as bacillus cereus
The carbon source of culture medium；Secondly for embodiment 3 using blackstrap and cornstarch mixture [m (blackstrap): m (corn flour)=
1:1] as the bacillus cereus culture medium culture of carbon source production bacillus cereus number of viable it is higher, reach 6.35 ×
109CFU·mL- 1, also relatively it is suitable as the carbon source of bacillus cereus culture medium；Select the carbon source system of embodiment 2 and embodiment 3
Bacillus cereus number of viable can be improved in the culture medium of work, consequently facilitating it is difficult to solve Bacillus cereus strain mass propagation
The problem of.
Embodiment 8 to 15: nitrogen source is leached with 1% yeast extract, beef extract, 1% urea, ammonium chloride, yeast respectively
Mixture (mass ratio of yeast extract and peptone is 1:1), yeast extract and the peptone and chlorine of object and peptone
Change mixture (mass ratio of yeast extract and peptone and ammonium chloride is 2:2:1), yeast extract and the peptone of ammonium
And mixture (mass ratio of yeast extract and peptone and urea is 2:2:1) equivalent substitution cultural method of urea
1.2.1 the peptone in basal fermentation medium, other compositions are constant, and bacillus cereus culture medium is made, then with system
3. the basal fermentation medium in shaking flask culture carries out bacillus cereus culture medium the step of replacing cultural method 1.2.1
Fermented and cultured measures the viable bacteria content in fermentation liquid, is recorded in table 2, to determine optimum nitrogen source after 12 hours.
Table 2: different nitrogen sources cultivation results table
Note: viable count unit: 109CFU·mL- 1
As can be drawn from Table 2, growth of the bacillus cereus in organic nitrogen source is significantly better than that inorganic nitrogen-sourced with fermentation,
After being fermented using organic nitrogen source, bacillus cereus viable count is 5.36 × 109CFUmL-1 or more.Embodiment 8 uses 1%
The bacillus cereus number of viable highest of bacillus cereus culture medium culture that is made as nitrogen source of beef extract, waxy bud
Spore bacillus viable count reaches 5.73 × 109CFU·mL-1.It secondly is embodiment 9 using yeast extract, peptone and ammonium chloride
The waxy gemma bar that is made as nitrogen source of mixture (mass ratio of yeast extract and peptone and ammonium chloride be 2:2:1)
The bacillus cereus number of viable of bacterium culture medium culture is higher, and bacillus cereus viable count reaches 5.51 × 109CFU·mL-1.Then be embodiment 10 use yeast extract and peptone mixture (mass ratio of yeast extract and peptone is 1:
1) the bacillus cereus number of viable as the bacillus cereus culture medium culture of nitrogen source production is high, and bacillus cereus is living
Bacterium number reaches 5.48 × 109CFU·mL-1；Select embodiment 8, the culture medium of the nitrogen source of embodiment 9 and embodiment 10 production can be with
Bacillus cereus number of viable is improved, consequently facilitating solving the problems, such as that Bacillus cereus strain mass propagation is difficult.
Embodiment 16 to 23: inorganic salts, respectively with 0.5 ‰ manganese sulfate, 0.4% manganese sulfate, 0.4% magnesium sulfate,
0.4% ferrous sulfate, 0.4% sodium chloride, 0.4% calcium chloride substitute the basal fermentation medium of cultural method 1.2.1
In sodium dihydrogen phosphate (0.2%) and disodium hydrogen phosphate (0.2%), other compositions are constant, bacillus cereus culture medium is made,
Then the step of replacing cultural method 1.2.1 with bacillus cereus culture medium obtained, 3. the basal fermentation in shaking flask culture was trained
It supports base and carries out fermented and cultured, the viable bacteria content in fermentation liquid is measured after 12 hours, is reported in Table 3 below, be most suitable for determination waxy
The inorganic salts of bacillus breeding.
Table 3: different inorganic salts cultivation results tables
Note: viable count unit: 109CFU·mL- 1
As can be drawn from Table 3, embodiment 18 is the bacillus cereus culture medium that inorganic salts production is not added, and does blank control
Group, bacillus cereus viable count are 4.77 × 109CFU·mL-1, and the bacillus cereus culture medium of inorganic salts is added,
Its viable count is 4.77 × 109CFU·mL-1Left and right, sodium, magnesium, iron, calcium and low concentration manganese ion to bacillus cereus liter
Temperature growth and fermentation influence less, therefore select the higher NaCl of bacillus cereus viable count to be used as and be suitble to bacillus cereus
The inorganic salts of the bacillus cereus culture medium of growth；.
Embodiment 24 to 32: in the bacillus cereus medium component of embodiment 2: 1% blackstrap, 1% peptone,
On the basis of 0.2% disodium hydrogen phosphate, 0.2% sodium dihydrogen phosphate and surplus pure water, blackstrap is chosen 1%, 2%, 3% 3
It is horizontal；1% peptone of bacillus cereus medium component of embodiment 2 is substituted with beef extract, choose 0.5%, 1%,
1.5% 3 level；By 0.2% disodium hydrogen phosphate of bacillus cereus medium component, 0.2% biphosphate of embodiment 2
Sodium is substituted with sodium chloride, 0.1%, 0.3%, 0.5% 3 level is chosen respectively, according to nutritional ingredient shown in table 4 than orthogonal
The bacillus cereus culture medium of embodiment 24 to 32 is prepared, then replaces culture side with bacillus cereus culture medium obtained
The step of method 1.2.1,3. the basal fermentation medium in shaking flask culture carried out fermented and cultured, measured in fermentation liquid after 12 hours
Bacillus cereus viable bacteria content, is reported in Table 4 below, to screen the culture medium for being most suitable for bacillus cereus growth, and will system
Meter result is analyzed.
Table 4: the orthogonal culture experiment result statistical form of bacillus cereus medium component
Note: viable count unit: 109CFU·mL- 1。
I1For viable count corresponding horizontal in first row data and；I2For the number of horizontal viable count corresponding in secondary series
According to；I3For viable count corresponding horizontal in third column data and；
K1 is that the synthesis of 1 horizontal upper viable count data is average；K2 is that the synthesis of 2 horizontal upper viable count data is average；K3 is 3
The synthesis of viable count data is average in level；R is the very poor of the factor.
As shown in Table 4, the bacillus cereus culture medium of optimum bacillus cereus growth is the waxy of embodiment 28
Fermentation of bacillus culture medium is made of the water of 2% blackstrap, 1% beef extract, 0.5% sodium chloride and surplus, cultivates
Bacillus cereus viable count highest, reaches 7.15 × 109CFU·mL-1；Then relatively it is suitble to the waxy of bacillus cereus growth
Bacillus culture medium is the Bacillus cereus fermentation culture medium for being embodiment 29, by 2% blackstrap, 1.5% beef extract,
0.1% sodium chloride and the water of surplus composition, the bacillus cereus viable count cultivated is higher, reaches 6.4 × 109CFU·
mL-1；Then the bacillus cereus culture medium for being suitble to bacillus cereus growth is the bacillus cereus hair for being embodiment 27
Ferment culture medium is made of the water of 2% blackstrap, 0.5% beef extract, 0.3% sodium chloride and surplus, the waxy gemma cultivated
Bacillus viable count is high, reaches 6.33 × 109CFU·mL-1；The bacillus cereus training of embodiment 28, embodiment 29, embodiment 27
Base is supported, so that bacillus cereus viable count all maintains higher quantity, i.e., 6.0 × 109CFU·mL-1More than, thus just
In solving the problems, such as that Bacillus cereus strain mass propagation is difficult.
Bacillus cereus cultivation results in table 4 are subjected to range analysis, analysis the results are shown in Table 5.
Table 5: the thallus growth pattern orthogonal test analysis of variance table of bacillus cereus
Note: F0.05 (2,2)=19.0, F0.01 (2,2)=99.0, preceding formula indicate F value and in the case where freedom degree be (2,2),
Significance is respectively the F value under 0.05 and 0.01
By the range analysis of table 5 it is found that each factor pair viable count yield effect size are as follows: blackstrap > sodium chloride > beef
Cream shows that blackstrap is extremely significant difference to the variance analysis of orthogonal test, and sodium chloride is significant difference, and beef extract is not show
Difference is write, therefore, nitrogen source (beef extract) can take 0.5~1.5% concentration to ferment, to solve bacillus cereus bacterium
The problem of strain mass propagation hardly possible.
Embodiment 33: the bacillus cereus culture medium of the present embodiment is made of the component of following mass ratio: 2% useless sugar
Honey and the mixture of cornstarch, 1% beef extract, 0.5% sodium chloride and surplus pure water, the blackstrap and cornstarch
Mass ratio be 1:1, the pH value of the fermentation medium is 6.5~7.5, is replaced with bacillus cereus culture medium obtained and is trained
3. the basal fermentation medium in shaking flask culture carries out fermented and cultured to the step of supporting method 1.2.1, measures fermented and cultured 12 hours
The bacillus cereus number of viable of the bacillus cereus culture medium culture of the present embodiment reaches 6.85 × 10 afterwards9CFU·mL-1.Due to blackstrap higher cost, it is therefore desirable to further decrease training under the premise of not influencing bacillus cereus mass propagation
Feeding cost.By embodiment 3 it is found that the bacillus cereus training for using the mixture of blackstrap and cornstarch to make as carbon source
Base is supported, bacillus cereus mass propagation can also be made.Compared with blackstrap is made, cost is relatively low for cornstarch, the present embodiment
Bacillus cereus culture medium a part of material cost can be reduced using the mixture of blackstrap and cornstarch.
Embodiment 1 to 33 studies the gemma for providing and being suitable for bacillus cereus breeding by single factor test and orthogonal experiment
Bacillus propagating culture medium improves waxy gemma to achieve the purpose that enable bacillus cereus faster and better growth and breeding
The number of viable of bacillus makes the viable count of bacillus cereus reach 5.73 × 109CFU·mL-1~7.15 × 109CFU·mL-1, solve the problems, such as that bacterial strain mass propagation is difficult.
Embodiment 34: by bacillus cereus through 500mL shaking flask liquid fermentation and culture under the conditions of 35 DEG C, 220r/min,
The Bacillus cereus fermentation culture medium substituent group of the optimum bacillus cereus breeding of embodiment 28 is selected when shaking flask culture
Plinth fermentation medium took fermentation liquid every 2 hours, measures the OD600nm of bacterium solution, cultivated 24 hours meter total plate counts, drew life
Long curve, while the test of fermentation liquid virulence is done, as shown in Fig. 1, bacillus cereus enters fast-growth after 6 hours
Phase reaches plateau in 22 hours.It should be controlled in the actual production process according to the growth characteristic of bacillus cereus, if
Fermentation seed liquid is wanted, the tunning that can choose at the fast-growth later period 18 hours does seed, and large scale fermentation production is then
Can be in 22 hours progress secondary batchings, to obtain bigger bacillus cereus biomass, as shown in Fig. 2, the vertical volume of rice
The death rate of leaf snout moth's larva the lag phase, logarithmic growth phase and stationary phase of bacillus cereus be constantly it is raised, reached at 22 hours
To 88.3%, start to reduce later, this is almost the same with the growth curve rule of bacillus cereus.In large scale fermentation culture
The species activity for the killing rice leaf roller that Bacillus cereus fermentation generates in 12~24 hour period is higher, and 18~22 is small
When the period in the species activity of killing rice leaf roller that generates of Bacillus cereus fermentation be within the scope of highest, in contrast
The death rate that the substance that the Bacillus cereus fermentation answered generates kills rice leaf roller is 83%~88.3%, preferably 22 hours
The species activity highest for the killing rice leaf roller that the Bacillus cereus fermentation of left and right period generates, kills rice leaf roller
The death rate reaches 88.3%.
Embodiment 35 mainly passes through to embodiment 40:pH influences somatic cells membrane charge, membrane permeability and nutriment
Degree of ionization influences absorption of the thallus to nutrient.Since the pH during shake flask fermentation is difficult to control, fermentation liquid can only be controlled
Initial pH.It, will be the step of cultural method 1.2.1 3. in shaking flask culture by the culture in shaking flask on the basis of embodiment 34
The initial pH value of matter is adjusted to 5.5,6.0,6.5,7.0,7.5,8.0 respectively, cultivates 22 hours under shake flask fermentation condition of culture, so
Its viable count is surveyed with dilution plate counting method afterwards, while measuring fermentation liquid virulence, is followed successively by reality according to initial pH value is ascending
Apply example 35 to 40.As shown in attached drawing 3 and attached drawing 4, initial pH value equal well-grown of bacillus cereus in 5.5~8.0 ranges,
Illustrate that bacillus cereus is wider to the adaptability of pH value, wherein initial pH value bacillus cereus in 6.5~7.5 ranges is living
Bacterium number is more, and the corresponding species activity for killing rice leaf roller is also at a higher range, corresponding wax
The death rate that the substance that sample fermentation of bacillus generates kills rice leaf roller is 83%~90.2%, the initial pH of embodiment 39
Bacillus cereus viable count is most when value is 7.5, and the virulence experiment of fermentation liquid the results show that embodiment 38 initial pH
The death rate of rice leaf roller reaches 90.2 when value is 7.0, is later in rapid decrease trend.So comprehensively considering waxy gemma bar
The yield of bacterium growth characteristic and fermentation liquid insecticide active substance determines that fermentation initial pH value is 7.0, i.e. culture material in shaking flask
When initial pH is 7.0, the species activity highest for the killing rice leaf roller that Bacillus cereus fermentation produces kills rice leaf roller
The death rate reach 90.2%.
Embodiment 41 to 45: temperature mainly influences the growth of thallus by changing enzyme reaction speed.General culture medium temperature
Degree increases, and enzyme reaction speed increases, and growth metabolism is accelerated.Enzyme is easy to lose activity because of overheat, shows as thallus and is easy aging,
Influence ultimate output.Based on embodiment 28 and embodiment 38, chooses 25,30,35,40,45 DEG C and distinguish as shaking table temperature
Influence of the different temperatures to zymotic fluid viable count is measured, 22h is cultivated under shake flask fermentation condition of culture, with dilution plate counting method
Its viable count is surveyed, while doing the test of fermentation liquid virulence, is from low to high respectively embodiment 41 to 45 according to temperature.Such as 5 He of attached drawing
Shown in attached drawing 6, bacillus cereus can well grow under 25~40 DEG C i.e. condition of culture of embodiment 41 to 44, and viable bacteria
For number with the raising of cultivation temperature in the trend that is slowly increased, convenient cultivation temperature range is 35~42 DEG C, corresponding
The death rate that the substance that Bacillus cereus fermentation generates kills rice leaf roller is 80%~93.1%, and viable count is bright at 45 DEG C
It is aobvious to reduce, the viable count highest of bacillus cereus at 40 DEG C, and the virulence experiment of fermentation liquid shows the death of rice leaf roller
Rate is also to reach 93.1% in 40 DEG C of cultivation temperature of embodiment 44, so the cultivation temperature in the optimal fermentation of shaking flask culture is
At 40 DEG C, the species activity highest for the killing rice leaf roller that Bacillus cereus fermentation produces.
Embodiment 46 to 50: based on embodiment 28, embodiment 38 and embodiment 44, choose 20,40,60,80,
5 liquid amounts of 100ml measure influence of the different liquid amounts to zymotic fluid viable count respectively, train under shake flask fermentation condition of culture
It supports 22 hours, surveys its viable count with dilution plate counting method, and measure the virulence of fermentation liquid, from less to more successively according to liquid amount
For embodiment 46 to 50.As shown in attached drawing 7 and attached drawing 8, in shaken cultivation bacillus cereus, using the 20ml of embodiment 46
Viable count is most in culture medium when liquid amount, and fermentation liquid can cause the rice leaf roller death rate to reach 94.5% at this time, and it is vertical to kill rice
The species activity highest of leaf roll snout moth's larva, with the increase of liquid amount, viable count is on a declining curve, and the virulence of fermentation liquid is also decreased obviously
Virulence declines rapidly when especially liquid amount increases to 60mL from 40mL.The fermentation for showing bacillus cereus is aerobism hair
Ferment, experiment are to adjust ventilatory capacity by changing liquid amount, and the small then ventilatory capacity of liquid amount is big, and dissolved oxygen level is high in culture medium, more
It is suitble to the thalli growth of aerobic bacillus cereus.Liquid amount is when increasing to 40% by 20%, Bacillus cereus fermentation
The species activity of the killing rice leaf roller of production gradually decreases, but also keeps that in a certain range, rice leaf roller can be killed
The death rate be 62%~94.5%.Therefore, dissolved oxygen can be improved by increasing the revolving speed of fermenter stirrer in production
Level, Lai Tigao fermentation liquid insecticide active substance concentration, and the substance for killing rice leaf roller is made to keep higher activity.
Embodiment 51 to 55: based on embodiment 28, embodiment 38, embodiment 44 and embodiment 46, inoculum concentration difference
1%, 3%, 5%, 7% and 9% is chosen, is cultivated 22 hours under shake flask fermentation condition of culture, surveys it with dilution plate counting method
Viable count measures the virulence of fermentation liquid simultaneously, respectively is embodiment 51 to 55 from less to more according to inoculum concentration.Such as 9 He of attached drawing
Shown in attached drawing 10, in 1%~9% range of inoculum concentration, the viable count of bacillus cereus is continuously increased, the virulence of fermentation liquid
Enhance with the increase of inoculum concentration, in 5%~9% range of inoculum concentration, the virulence of Bacillus cereus fermentation liquid is compared with inoculum concentration
It is significantly improved when 1%~5%, the death rate for killing rice leaf roller reaches 86%~95.8%, i.e. Bacillus cereus fermentation
The generated species activity for killing rice leaf roller improves.The viable count highest when the inoculum concentration of embodiment 55 is 9%, rice are vertical
The death rate of leaf roll snout moth's larva reaches 95.8%.So killing rice vertical volume when inoculum concentration is 9%, caused by Bacillus cereus fermentation
The species activity highest of leaf snout moth's larva.
By embodiment 35 to embodiment 40 it can be concluded that the culture medium of suitable bacillus cereus growth should also have
Condition is that pH value range is 5.5-8.0, and the Medium's PH Value range of convenient bacillus cereus growth is 6.5~7.5, most
The pH value of suitable bacillus cereus culture medium is 7.0.
The fermentation process that rice leaf roller species activity is killed in raising bacillus cereus production of the invention passes through in optimum
On the basis of the Bacillus cereus fermentation culture medium of bacillus cereus breeding, filters out and be suitable for improving bacillus cereus production
It kills the fermentation process of rice leaf roller species activity: being that 7.0,40 DEG C of shaking flask cultivation temperature, liquid amount 20% (account in initial pH value
The volume ratio of culture vessel) Bacillus cereus fermentation culture medium etc. under the conditions of cultivate 22 hours, produce bacillus cereus
The activity of substance of killing rice leaf roller keep higher level, it is vertical that the substance that Bacillus cereus fermentation generates kills rice
The death rate of leaf roll snout moth's larva is 62%~95.8% and then kills pest.
For the ordinary skill in the art, it is possible to understand that the case where not departing from the principle and spirit of the invention
Under these embodiments can be carried out with a variety of change, modification, replacement and modification, the scope of the present invention by appended claims and its
Equivalent limits.
Claims (10)
1. Bacillus cereus fermentation culture medium, which is characterized in that be made of the following components:
Carbon source, nitrogen source, inorganic salts and pure water；
The carbon source is the mixture of blackstrap or cornstarch and blackstrap；
The nitrogen source is the mixture of one or more of yeast extract, beef extract, peptone, ammonium chloride；
The inorganic salts sodium chloride；The pH value range of the fermentation medium is 5.5~8.0.
2. Bacillus cereus fermentation culture medium according to claim 1, which is characterized in that by the component of following mass ratio
Be made: 2% blackstrap, 0.5~1.5% beef extract, 0.5% sodium chloride and surplus pure water；The pH of the fermentation medium
Value is 6.5~7.5.
3. Bacillus cereus fermentation culture medium according to claim 2, it is characterised in that: the pH of the fermentation medium
Value is 7.0.
4. Bacillus cereus fermentation culture medium according to claim 1, which is characterized in that by the component of following mass ratio
Be made: the mixture of 2% blackstrap and cornstarch, 1% beef extract, 0.5% sodium chloride and surplus pure water, the useless sugar
The mass ratio of honey and cornstarch is 1:1, and the pH value of the fermentation medium is 6.5~7.5.
5. Bacillus cereus fermentation culture medium according to claim 1, which is characterized in that by the component of following mass ratio
Be made: 2% blackstrap, the mixture of 1% yeast extract and peptone, 0.5% sodium chloride and surplus pure water, the ferment
The mass ratio of female extract and peptone is 1:1, and the pH value of the fermentation medium is 6.5~7.0.
6. a kind of waxy gemma bar of raising based on Bacillus cereus fermentation culture medium described in any one of claim 1 to 5
Bacterium produces the fermentation process for killing rice leaf roller species activity, which comprises the following steps:
1. the activation of strain: by the strain transfer of the bacillus cereus of preservation to slant medium, 37 DEG C are cultivated 24 hours, standby
With；
2. the preparation of seed liquor: in the triangular flask of 250ml access liquid amount be 50ml seed culture medium, be added step 1. in
The strain for completing activation, is cultivated 18 hours under the conditions of 37 DEG C of cultivation temperature, revolving speed 180r/min；
3. shaking flask culture: taking 1ml step respectively, 2. the middle seed liquor for completing culture, access fill 50ml fermentation medium
It in the triangular flask of 200ml, sets in shaking table, the shaken cultivation 12~24 under the conditions of cultivation temperature is 30 DEG C, revolving speed is 160r/min
Hour.
7. the fermentation process according to claim 6 for improving bacillus cereus production and killing rice leaf roller species activity,
Be characterized in that: step 3. in incubation time be 18~22 hours.
8. the fermentation process according to claim 6 or 7 for improving bacillus cereus production and killing rice leaf roller species activity,
It is characterized by: step 3. in the initial pH value of shaking flask culture be 6.5~7.5.
9. the fermentation process according to claim 8 for improving bacillus cereus production and killing rice leaf roller species activity,
Be characterized in that: step 3. in the temperature of shaking flask culture be 35~42 DEG C.
10. according to the described in any item hairs for improving bacillus cereus production and killing rice leaf roller species activity of claim 6 to 9
Fermenting process, it is characterised in that: step 3. described in the inoculum concentration of seed liquor be 5%~9%, the liquid amount of the triangular flask is
20%~40%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910587899.4A CN110283748A (en) | 2019-07-02 | 2019-07-02 | Bacillus cereus fermentation culture medium and raising bacillus cereus produce the fermentation process for killing rice leaf roller species activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910587899.4A CN110283748A (en) | 2019-07-02 | 2019-07-02 | Bacillus cereus fermentation culture medium and raising bacillus cereus produce the fermentation process for killing rice leaf roller species activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110283748A true CN110283748A (en) | 2019-09-27 |
Family
ID=68021591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910587899.4A Pending CN110283748A (en) | 2019-07-02 | 2019-07-02 | Bacillus cereus fermentation culture medium and raising bacillus cereus produce the fermentation process for killing rice leaf roller species activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110283748A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164155A (en) * | 2021-12-22 | 2022-03-11 | 清远一生自然生物研究院有限公司 | Fermentation medium and fermentation process of bacillus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463333A (en) * | 2009-01-09 | 2009-06-24 | 南京工业大学 | Culture and protection method of high-activity Bacillus cereus CMCC63305 strain |
| CN104988101A (en) * | 2015-08-03 | 2015-10-21 | 黑龙江省科学院微生物研究所 | Bacillus cereus and application of bacillus cereus as agricultural biological agent |
-
2019
- 2019-07-02 CN CN201910587899.4A patent/CN110283748A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463333A (en) * | 2009-01-09 | 2009-06-24 | 南京工业大学 | Culture and protection method of high-activity Bacillus cereus CMCC63305 strain |
| CN104988101A (en) * | 2015-08-03 | 2015-10-21 | 黑龙江省科学院微生物研究所 | Bacillus cereus and application of bacillus cereus as agricultural biological agent |
Non-Patent Citations (2)
| Title |
|---|
| 李晓璐等: "水黄皮内生菌的分离及其杀虫菌株的筛选", 《沈阳农业大学学报》 * |
| 胡晓娟等: "响应面法优化蓝藻溶藻菌CZBC1发酵培养工艺", 《南方农业学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164155A (en) * | 2021-12-22 | 2022-03-11 | 清远一生自然生物研究院有限公司 | Fermentation medium and fermentation process of bacillus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109266586B (en) | Bacillus belgii BMF03 and application and fermentation method thereof | |
| CN105886428A (en) | Streptomyces albidoflavus and applications thereof in microbial fertilizers | |
| CN101845409B (en) | Bacillus firmus and application thereof | |
| CN103146586A (en) | Trichoderma harzianum strain for controlling plant fungus diseases and application thereof | |
| CN105002121B (en) | One plant of simple bacillus and its application | |
| CN104611254B (en) | Aerobacillus polymyxa Donker KN 03 and its cultural method and purposes | |
| CN108277177B (en) | Streptomyces microflavus solid fermentation medium, preparation method and fermentation method thereof, fermentation product, biocontrol product and application | |
| CN107646878A (en) | Prevent and treat microbial inoculum of tobacco mosaic virus (TMV) and preparation method thereof | |
| CN104726378B (en) | The method for improving salt stress turfgrass defence enzyme activity using Salt-tolerant microbial agent is strengthened | |
| CN105219655A (en) | Flavus biocontrol strain and the application thereof of aflatoxin do not produced to sterilant resistance | |
| CN110157624A (en) | A kind of Paecilomyces lilacinus large-scale method for producing based on automation koji machine | |
| CN108531404B (en) | Culture method of Fusarium proliferatum and application thereof | |
| CN109867565A (en) | A kind of plant protection product and its application method | |
| CN110079471A (en) | One plant of hydrogen-oxidizing bacterium A06 and its separation method and application with growth-promoting functions | |
| CN109234213A (en) | A kind of Pseudomonas chlororaphis strain X8 and its application | |
| CN109306330A (en) | The fermentation culture method of biocontrol bacteria strain bacillus amyloliquefaciens | |
| CN110283748A (en) | Bacillus cereus fermentation culture medium and raising bacillus cereus produce the fermentation process for killing rice leaf roller species activity | |
| CN106434403B (en) | A kind of Pediococcus pentosaceus bacterial strain and application thereof | |
| CN104962491B (en) | The degradation bacteria strains of D of herbicide 2,4 a kind of and its microbial inoculum and the application of production | |
| CN105948844A (en) | Method for producing bacillus licheniformis bacterial fertilizer by using pig raising bedding of microbial fermentation bed | |
| CN107446867B (en) | A kind of Upland Red Soil symbiotic nitrogen fixation bacterium and its cultural method and application | |
| CN109486705A (en) | The pale false bacillus strain X21 of one kind and its application | |
| CN109699683A (en) | A kind of talcum matrix Java cordyceps sinensis spore preparation | |
| CN112521948B (en) | Fertilizing method for improving secondary salinization greenhouse soil | |
| CN104789493A (en) | Method for improving protective enzyme activity of grass of water stress lawn by adopting reinforced drought-resistant microbial agent |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190927 |
|
| RJ01 | Rejection of invention patent application after publication |