CN107586799A - A kind of AFG1 of aspergillus parasiticus produces malicious fermentation process - Google Patents
A kind of AFG1 of aspergillus parasiticus produces malicious fermentation process Download PDFInfo
- Publication number
- CN107586799A CN107586799A CN201711014844.1A CN201711014844A CN107586799A CN 107586799 A CN107586799 A CN 107586799A CN 201711014844 A CN201711014844 A CN 201711014844A CN 107586799 A CN107586799 A CN 107586799A
- Authority
- CN
- China
- Prior art keywords
- afg1
- aspergillus parasiticus
- medium
- culture medium
- solid fermentation
- 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
- 238000000855 fermentation Methods 0.000 title claims abstract description 69
- 230000004151 fermentation Effects 0.000 title claims abstract description 69
- 241000228230 Aspergillus parasiticus Species 0.000 title claims abstract description 49
- 102100034212 AFG1-like ATPase Human genes 0.000 title claims abstract description 43
- 101000780581 Homo sapiens AFG1-like ATPase Proteins 0.000 title claims abstract description 43
- 239000002609 medium Substances 0.000 claims abstract description 58
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 239000007787 solid Substances 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 9
- 239000012138 yeast extract Substances 0.000 claims abstract description 7
- 235000002639 sodium chloride Nutrition 0.000 claims description 48
- 150000003839 salts Chemical class 0.000 claims description 47
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 45
- 235000010755 mineral Nutrition 0.000 claims description 45
- 239000011707 mineral Substances 0.000 claims description 45
- 239000002131 composite material Substances 0.000 claims description 34
- 241000894006 Bacteria Species 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 11
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- 235000012054 meals Nutrition 0.000 claims description 9
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 8
- 244000105624 Arachis hypogaea Species 0.000 claims description 8
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 8
- 235000018262 Arachis monticola Nutrition 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 235000020232 peanut Nutrition 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical class [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 235000007079 manganese sulphate Nutrition 0.000 claims description 7
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical class [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 235000010344 sodium nitrate Nutrition 0.000 claims description 7
- 239000004317 sodium nitrate Substances 0.000 claims description 7
- 230000033228 biological regulation Effects 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 244000061456 Solanum tuberosum Species 0.000 claims description 5
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 claims description 4
- 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 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 4
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 3
- 229960005091 chloramphenicol Drugs 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 229940041514 candida albicans extract Drugs 0.000 abstract description 3
- 235000013339 cereals Nutrition 0.000 abstract description 3
- 239000003053 toxin Substances 0.000 abstract description 3
- 231100000765 toxin Toxicity 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000007605 air drying Methods 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241000228212 Aspergillus Species 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 3
- 238000004500 asepsis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 229930195730 Aflatoxin Natural products 0.000 description 2
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 239000005409 aflatoxin Substances 0.000 description 2
- 239000002115 aflatoxin B1 Substances 0.000 description 2
- 229930020125 aflatoxin-B1 Natural products 0.000 description 2
- QRARGUIFAGCOOA-UHFFFAOYSA-N aspertoxin Chemical compound O1C2=C(C3(C=COC3O3)O)C3=CC(OC)=C2C(=O)C2=C1C=CC=C2OC QRARGUIFAGCOOA-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000005660 chlorination reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 231100000041 toxicology testing Toxicity 0.000 description 2
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 101710089042 Demethyl-4-deoxygadusol synthase Proteins 0.000 description 1
- 241001411320 Eriogonum inflatum Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 241000209094 Oryza Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000009286 beneficial 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
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- -1 hydrogen Sodium hydroxide Chemical class 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000029219 regulation of pH Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to fermentation engineering field, and in particular to a kind of AFG1 of aspergillus parasiticus produces malicious fermentation process.A kind of AFG1 of aspergillus parasiticus produces malicious fermentation process, and this method comprises the following steps：A, the resurrection that aspergillus parasiticus spore is carried out using enriched medium is bred, and b, solid fermentation culture is carried out using solid fermentation culture medium, is contained the yeast extract for accounting for culture medium gross weight 1 3% in the component of enriched medium and solid fermentation culture medium.AFG1 of the present invention toxin producing medium solves the problems such as AFG1 concentration is low, uneven, time-consuming in the cereal of current natural infection mould.
Description
Technical field
The invention belongs to fermentation engineering field, and in particular to a kind of AFG1 of aspergillus parasiticus produces malicious fermentation process.
Background technology
Aflatoxin (Aflatoxins, AFs) is by some of which productions such as aspergillus parasiticus, aspergillus flavus and collection peak aspergillus
The mycetogenetic secondary metabolite of poison, has the three-induced effects such as carcinogenic, teratogenesis and mutagenesis.According to the literature, identified
AFs up to more than 20 plant, wherein it is most commonly seen be AFs be aflatoxin B1 (AFB1), it is aflatoxin B 2 (AFB2), yellow
Aspertoxin G1 (AFG1), aflatoxin G 2 (AFG2).AFs can pollute the cereal such as corn, wheat, soybean and cottonseed and oil plant
Agricultural product, it can also remain in the agricultural byproducts such as some DDGS, wheat bran, Soybean Meal and Cottonseed Meal.When the agricultural product of AFs pollutions
During food as people, the health of people can be endangered, slight causes sub-health status, can seriously cause hepatic disease, causes dead
Die；And during the agricultural byproducts feeding animals of AFs pollutions, often cause breeding performonce fo animals to decline, premunition reduction, can be led when serious
It is lethal to die.Therefore, AFs pollution problem causes world many countries government and food safety monitoring machine in food and feed
The attention of structure, set the AFs limit standards in food and feed.AFs scientific research also enjoys the concern of domestic and foreign scholars,
Corresponding experiment is carried out in the animals such as pig, milk cow and birds.
However, AFG1 maximum concentrations are only 344ppb (Feng Jianlei, 2004) in the fermentation medium of domestic report, therefore
When carrying out the research of AFG1 related sciences, the AFG1 samples from external import expensive (452/mg) are both needed to, experimentation cost is high, or
Need to carry out commodity reservation, made troubles to related science research.
In summary, AFG1 fermentation medium and application process is developed, can be the AFG1 toxicological tests and AFs of animal
Food security research provide experiment material.
The content of the invention
The invention provides a kind of AFG1 of aspergillus parasiticus to produce malicious fermentation process, for solving current natural infection mould
The problems such as AFG1 concentration is low, uneven, time-consuming in cereal.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of AFG1 of aspergillus parasiticus produces malicious fermentation process, and this method comprises the following steps：
A, the resurrection that aspergillus parasiticus spore is carried out using enriched medium is bred, and described enriched medium each component is by weight
Amount percentages include：10-25% potatos, 4-10% glucose, 1-3% agar, 1-3% yeast extracts, 0.5% grandidierite
Thing salt, 0.2-0.5g/L chloramphenicol, surplus are water；
B, solid fermentation culture is carried out using solid fermentation culture medium, described solid fermentation culture medium each component is by weight
Percentages include：41-70% hominy grits, 24-45% peanut meals, 4-10% sucrose, 0.4-1% composite mineral salts, 1-3% ferment
Female medicinal extract, after having prepared in proportion, regulation solid fermentation moisture content in medium is 10-20%；
The component of composite mineral salt described in described enriched medium and solid fermentation culture medium is by percentage to the quality
For：40-60% sodium nitrate, 5-15% magnesium sulfate, 5-15% potassium chloride, 15-30% dipotassium hydrogen phosphates, 3-8% zinc sulfate, 3-
5% manganese sulfate, the aqueous ferrous sulfates of 0.2-0.8% five, above-mentioned each mineral salt raw material is well mixed, after crushed 80 mesh sub-sieves
Mix again, obtain composite mineral salt.
Preferably, the aspergillus parasiticus is posting for China General Microbiological culture presevation administrative center (CGMCC) offer
Raw Aspergillus (Aspergillus parasiticus) CGMCC NO.：3.0124.
Preferably, described enriched medium and solid fermentation culture medium, are with hydrochloric acid or sodium hydroxide regulation pH value
After 5.5-7.0, it is placed in 2L conical flasks, covers tampon, sterilizing 20min is carried out under conditions of 121-123 DEG C, 0.12MPa.
Preferably, the chloramphenicol composition in described enriched medium after sterilizing terminates, treats that enriched medium cools down
Add and mix after to less than 40 DEG C.
Inventor proves through substantial amounts of experiment display, parasitic under same culture conditions using enriched medium of the present invention
Time needed for aspergillus spore recovery only needs 2 days, and the recovery time than conventional medium shortens 4 days, and spore quantity improves 3
Times.At present, when carrying out aspergillus parasiticus Zengjing Granule, conventional medium component is：Potato, glucose, agar and water, should
Culture medium increasing bacterium speed is slow, and unit culture medium area spore quantity is few, and enriched medium efficiency provided by the invention substantially carries
It is high.
Preferably, the hominy grits and peanut meal described in described solid fermentation culture medium, through crushing, granularity reaches 20-
40 targets are accurate.The solid fermentation culture medium institute carbohydrate containing (hominy grits), protein (peanut meal), mineral matter (grandidierite
Thing salt) and the nutriment such as somatomedin (yeast extract) meet that aspergillus parasiticus efficiently produces the condition of poison just, and ratio is fitted
When achievable aspergillus parasiticus efficiently produces AFG1.When carrying out solid fermentation culture, the culture medium used in prior art is only to adjust
The crushing maize of moisture, required fermented incubation time is apparently higher than solid fermentation culture medium of the invention, and prior art is adopted
AFG1 concentration is substantially less than the solid fermentation culture medium A FG1 concentration of the present invention in culture medium.
Preferably, the optimum ratio of composite mineral salt is：40-53.5% sodium nitrate, 11.5-15% magnesium sulfate, 11-
14.5% potassium chloride, 17-22% dipotassium hydrogen phosphates, 3-6% zinc sulfate, 3-4% manganese sulfates, 0.5% 5 aqueous ferrous sulfate.
The sterilizing requirement of culture medium：Described enriched medium and solid fermentation culture medium, prepares (its in proportion respectively
In, the chloramphenicol composition in enriched medium treats that enriched medium is cooled to 40 DEG C or so and adds mixing after sterilizing terminates),
After being 5.5-7.0 with hydrochloric acid or sodium hydroxide regulation pH value, it is placed in 2L conical flasks, covers tampon, at 121-123 DEG C,
0.12MPa, sterilize 20min, and each culture medium prepares end-of-job.
Enriched medium flat panel production：In Biohazard Safety Equipment, the enriched medium to have sterilized is upside down in by sterilizing
Diameter 10cm culture dishes in, natural cooling.Then, enriched medium is placed in 37 DEG C of insulating boxs and cultivates 24h, asepsis growth
Person can use.
The inventive method uses above-mentioned solid fermentation culture medium, and the strain for selection of fermenting (comes from China for aspergillus parasiticus bacterium
General Microbiological Culture preservation administrative center, CGMCC NO.3.0124), zymotechnique is using conventional solid fermentation process.Press
According to culture medium provided by the invention and cultural method, AFG1 concentration in the solid air dry matter of toxin producing medium is reachable
28956ppb, fermentation titer compared with prior art, have significant progressive.And this method fermentation efficiency is high, producing cost
It is low, the blank of China AFG1 production field is filled up, and can greatly reduce the research cost in AFG1 toxicological study field.
The beneficial effects of the invention are as follows：The enriched medium that the present invention develops, no bacteria pollution interference, mould bring back to life propagation
It hurry up, conidium vitality is high, improves operating efficiency.The solid fermentation culture medium that the present invention develops, nutrition is balanced, and fungus growth is prosperous
Contain, cultivation cycle is short, it is only necessary to which 10-12 days, relatively common corn culture medium, incubation time shortened 5-7 days, and AFG1 concentration is notable
Improve.Solid fermentation culture medium provided by the invention, mainly soaked using hominy grits, peanut meal, sucrose, composite mineral salt and yeast
Cream, raw material are easy to get, inexpensively.The relative AFG1 from external import, the AFG1 prices that culture medium provided by the invention is produced can drop
Low more than 90%.Fermentation process provided by the invention, it is simple and easy, it is workable.
Embodiment
Below by specific embodiment, technical scheme is described in further detail.It should be appreciated that this hair
Bright implementation is not limited to the following examples, and any formal accommodation and/or change made to the present invention will all fall
Enter the scope of the present invention.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, and all equipment and raw material etc. are equal
It is commercially available or the industry is conventional.
Various embodiments of the present invention, the parasitism provided with China General Microbiological culture presevation administrative center (CGMCC) are bent
Mould (Aspergillus parasiticus) CGMCC NO.：3.0124 it is illustrated.
The preparation of embodiment 1-4 enriched medium
A kind of enriched medium for aspergillus parasiticus bacterium strain, after each component shown in table 1 is mixed, with hydrochloric acid and hydrogen
Sodium hydroxide solution regulation enriched medium pH value is 5.5-7.0.
Table 1
The preparation of embodiment 5-8 composite mineral salts
Proportioning in table 2 takes sodium nitrate, magnesium sulfate, potassium chloride, dipotassium hydrogen phosphate, zinc sulfate, manganese sulfate and five water
Ferrous sulfate, above-mentioned mineral salt is well mixed, it is standby after mixing again after crushed 80 mesh sub-sieves, obtain Zengjing Granule
Composite mineral salt described in base and solid fermentation culture medium.
Table 2
Now by taking the Zengjing Granule based formulas of the composite mineral salt formula of embodiment 6 and embodiment 2 as an example, it is compound to prepare 100g
Mineral salt and 1L enriched medium.
Composite mineral salt (100g):Respectively 45.0g sodium nitrate, 11.5g magnesium sulfate, 13.0g chlorinations are weighed with assay balance
Potassium, 20.0g dipotassium hydrogen phosphates, 6.0g zinc sulfate, 4.0g manganese sulfates, the aqueous ferrous sulfates of 0.5g five are well mixed in beaker, then
Whole mixed mineral salt are put into supercentrifuge, 10min is crushed, after whole samples are by 80 mesh sub-sieves, mix, put again
Put retain in brown bottle wide-mouth bottle it is standby.
Enriched medium (1L)：Potato cleans peeling, weighs 200g potatos and is cut into small pieces, and adds 800mL boilings to boil 20-
30 minutes, until can be poked by glass bar, with 4 layers of filtered through gauze into beaker, filter residue discarded, and into beaker, moisturizing is extremely
1000mL, then toward 60g glucose is added in beaker, 15g yeast extracts, 5g composite mineral salts, heating, 15g agar is added, after
Continuous heating stirring mixes, and after agar has dissolved, stirs, and supplies moisture again to 1000 milliliters after slightly cooling down, uses 5mol/
The salt acid for adjusting pH value of L concentration is in the range of 5.5-7.0.PH regulations finish, and are fitted into the conical flask that capacity is 2L, cover cotton
Plug, at a temperature of 121-123 DEG C, 0.12MPa, sterilize 20min.
In Biohazard Safety Equipment, after adding 0.5g/L chloramphenicol into 40 DEG C of enriched medium of sterilizing, then it is upside down in
In the diameter 10cm culture dishes of sterilization treatment, cooling wild Oryza species are solidifiable.Then, enriched medium is placed on 37 DEG C of incubators
24h is cultivated, asepsis growth person can be used, and enriched medium is prepared and finished.
Preparation for the slant medium of aspergillus parasiticus culture presevation
According to aspergillus parasiticus enriched medium formula table, culture presevation culture medium needed for preparation.The increasing bacterium prepared is trained
Base is supported, 0.5g/L chloramphenicol is added under 60 DEG C of non-curdled appearances of culture medium, is upside down in the test tube of sterilizing, volume is about test tube
The 1/3 of capacity, test tube is tilted, prepared by the slant medium of natural cooling, i.e. culture presevation finishes.
The preparation of embodiment 9-12 aspergillus parasiticus solid fermentation culture mediums
According to the formula of table 3, solid fermentation culture medium needed for preparation.
Table 3
Raw material prepares：By hominy grits and peanut meal through crushing, it is desirable to the quasi- sub-sieve of 20 targets can be crossed, but 40 targets can not be crossed
Quasi- sub-sieve, i.e. granularity are between 20-40 mesh.
Now by taking the composite mineral salt formula of embodiment 6 and the component of embodiment 10 as an example, prepare 100g composite mineral salts and
1L enriched medium.
Composite mineral salt (100g):Respectively 45.0g sodium nitrate, 11.5g magnesium sulfate, 13.0g chlorinations are weighed with assay balance
Potassium, 20.0g dipotassium hydrogen phosphates, 6.0g zinc sulfate, 4.0g manganese sulfates, the aqueous ferrous sulfates of 0.5g five are well mixed in beaker, then
Whole mixed mineral salt are put into supercentrifuge, 10min is crushed, after whole samples are by 80 mesh sub-sieves, mix, put again
Put retain in brown bottle wide-mouth bottle it is standby.
Solid fermentation culture medium (1kg).Weigh 500g hominy grits, 389g peanut meals, 80g sucrose, 6g composite mineral salts,
25g yeast extract, 100ml distilled water is separately measured, above-mentioned raw materials are well mixed, be placed in 3000ml conical flasks, cover tampon,
At 121-123 DEG C, 0.12MPa, sterilize 30min.In Biohazard Safety Equipment, sterilized solid medium is upside down in and sterilized
Conical flask in, tiling thickness is 1cm, or useful load is 60g/500ml triangular flasks, covers sterilized bottle stopper.So far, post
Raw aspergillus fermentation solid culture medium, which is prepared, to be finished.
The AFG1 of the aspergillus parasiticus of embodiment 13 produces malicious fermented and cultured
1st, the Multiplying culture method of aspergillus parasiticus strain
Strain is brought back to life：The aspergillus parasiticus freeze-dried powder 0.5mL physiological saline solutions that will be preserved in ampoule bottle, then with once
Property asepsis injector draw 4mL lysates, uniform point sample is on the enriched medium that embodiment 2 prepares, then uses sterile glass
Bacterium solution is smeared and is evenly distributed by rod.The enriched medium for having connect aspergillus parasiticus is placed in 35 DEG C of constant incubators and cultivates 46h, i.e.,
Substantial amounts of mycelia can be grown, reaches test requirements document, strain brings back to life and finished.
Distinguish composite mineral salt in the enriched medium of alternate embodiment 1,3,4 using the composite mineral salt formula of embodiment 6 to match somebody with somebody
Enriched medium made from side, it is respectively 43h, 54h and 58h that aspergillus parasiticus, which brings back to life required time,.
Under similarity condition, the composite mineral salt formula of embodiment 5,7,8, the difference enriched medium of alternate embodiment 1 are used
Middle composite mineral salt formula, it is respectively 42h, 47h and 51h that aspergillus parasiticus, which brings back to life required time,.
Using the composite mineral salt formula of embodiment 5,7,8, composite mineral salt in the enriched medium of alternate embodiment 2 is distinguished
Formula, it is respectively 45h, 44h and 55h that aspergillus parasiticus, which brings back to life required time,.
Using the composite mineral salt formula of embodiment 5,7,8, composite mineral salt in the enriched medium of alternate embodiment 3 is distinguished
Formula, it is respectively 41h, 46h and 52h that aspergillus parasiticus, which brings back to life required time,.
Use the composite mineral salt formula of embodiment 5,7,8, the difference enriched medium kind composite mineral salt of alternate embodiment 4
Formula, it is respectively 40h, 43h and 48h that aspergillus parasiticus, which brings back to life required time,.
2nd, aspergillus parasiticus fungi preservation：In Biohazard Safety Equipment, using aseptic inoculation pin, after sterilizing cooling, picking is above-mentioned
The bacterium solution of dissolving, rule on the slant medium of aspergillus parasiticus culture presevation in Z-shaped, cover sterilized ventilative tampon,
It is subsequently placed in 35 DEG C of constant incubators and cultivates 40h.It is to be generated grow more mycelia after, be placed in 4 DEG C of antistaling cabinets and preserve, often
Two months, according to this method passage once.
3rd, aspergillus parasiticus bacterium is in fermentation medium top fermentation
Aspergillus parasiticus bacterium Multiplying culture：Aseptic inoculation ring is used in Biohazard Safety Equipment, with a little normal saline flushing
The aspergillus parasiticus bacterium brought back to life is stated, and collects mycelia and spore liquid, the then intensive line on multiple enriched medium, is placed in 35
46h is cultivated in DEG C constant incubator.Cover with the enriched medium flat board of mycelia, it is possible to provide ferment required spore and mycelium.
Aspergillus parasiticus bacterium mycelia and the collection of spore：In Biohazard Safety Equipment, rinsed with 5mL sterile salines and increase bacterium training
Base flat board is supported, then carefully scrapes mycelia with sterile glass rod, mycelia and spore are collected in 250ml conical flasks, with sterile life
Salt solution dilution aspergillus parasiticus bacterium solution is managed, until spore density is 1.2 × 106cfu/mL。
2mL aspergillus parasiticuses mycelia and spore liquid are drawn with disposable syringe, bacterium solution is slowly sprayed on to embodiment 6 and prepared
In good solid medium, while it is well mixed using sterile glass rod, covers the ventilative tampon of sterilizing, be placed in relative humidity
85-90%, cultivate in the constant incubator that 35 DEG C of temperature.
The yeastiness of aspergillus parasiticus bacterium in conical flask, and incubator running situation are observed and recorded daily.
At interval of 3 days, fermentation medium is spun upside down using sterile glass rod in Biohazard Safety Equipment, paves fermentation training
Base is supported, makes the abundant ingress of air of culture medium, while sprays 5mL physiological saline, with the loss of moisture during afterfermentation.Cover
Sterile tampon, it is placed in 35 DEG C of constant incubators and continues to cultivate.
By the fermented and cultured of 10-12 days, culture medium thoroughly fermented in conical flask, and culture medium caking, mycelia color is in light
Yellow, fermented and cultured terminate.By culture in 121-123 DEG C, 0.12MPa, sterilize 20min, is steamed using freeze drier low pressure
Dry dry, dries pulverizing, the culture containing AFG1 are prepared and finished.
The content of AFG1 in fermentation medium is detected with high performance liquid chromatography (GB/T 5009.23-2006).Using reality
The composite mineral salt of example 6 and the solid fermentation culture medium of embodiment 10 are applied, fermented and cultured 12 days, AFG1 concentration is reachable in air-drying sample
To 28956ppb.
Using the solid fermentation culture medium of embodiment 9,11,12, fermentation process is carried out under above-mentioned the same terms, air-dries training
It is respectively 25781ppb, 23541ppb and 21234ppb to support AFG1 concentration in base.
It is compound in the solid fermentation culture medium of alternate embodiment 9 respectively using the composite mineral salt formula of embodiment 5,7,8
Mineral salt formula, fermentation process under the same conditions, in air-drying sample AFG1 concentration be respectively 23671ppb, 25553ppb and
22738ppb。
It is multiple in the solid fermentation culture medium of alternate embodiment 10 respectively using the composite mineral salt formula of embodiment 5,7,8
Close mineral salt formula, fermentation process under the same conditions, in air-drying sample AFG1 concentration be respectively 25381ppb, 21089ppb and
25337ppb。
It is multiple in the solid fermentation culture medium of alternate embodiment 11 respectively using the composite mineral salt formula of embodiment 5,7,8
Close mineral salt formula, fermentation process under the same conditions, in air-drying sample AFG1 concentration be respectively 21609ppb, 25855ppb and
21708ppb。
It is multiple in the solid fermentation culture medium of alternate embodiment 12 respectively using the composite mineral salt formula of embodiment 5,7,8
Close mineral salt formula, fermentation process under the same conditions, in air-drying sample AFG1 concentration be respectively 19691ppb, 25358ppb and
28031ppb。
Conclusion：The production poison concentration of prior art is that 344 ± 11.5ppb AFG1 of the present invention air-dry in the solid of toxin producing medium
Up to 28956ppb, the production poison concentration fermentation titer of 344 ± 11.5ppb AFG1 compared with prior art has to be shown concentration in thing
The progress of work.This method fermentation efficiency is high, and producing cost is low, has filled up the blank of China AFG1 production field, and can be very big
Reduce the research cost in AFG1 toxicological study field.
Embodiment described above is a kind of preferable scheme of the present invention, not the present invention is made any formal
Limitation, there are other variants and remodeling on the premise of without departing from the technical scheme described in claim.
Claims (6)
1. a kind of AFG1 of aspergillus parasiticus produces malicious fermentation process, it is characterised in that：This method comprises the following steps：
A, the resurrection that aspergillus parasiticus spore is carried out using enriched medium is bred, described enriched medium each component by weight hundred
Divide includes than meter：10-25% potatos, 4-10% glucose, 1-3% agar, 1-3% yeast extracts, 0.5% composite mineral salt, 0.2-
0.5g/L chloramphenicol, surplus are water；
B, solid fermentation culture, described solid fermentation culture medium each component percentage by weight are carried out using solid fermentation culture medium
Include than meter：41-70% hominy grits, 24-45% peanut meals, 4-10% sucrose, 0.4-1% composite mineral salts, 1-3% yeast extracts, press
After ratio has been prepared, regulation solid fermentation moisture content in medium is 10-20%；
The component of composite mineral salt is by percentage to the quality described in described enriched medium and solid fermentation culture medium：
40-60% sodium nitrate, 5-15% magnesium sulfate, 5-15% potassium chloride, 15-30% dipotassium hydrogen phosphates, 3-8% zinc sulfate, 3-5% manganese sulfates,
The aqueous ferrous sulfates of 0.2-0.8% five, above-mentioned each mineral salt raw material is well mixed, mixes, obtains again after crushed 80 mesh sub-sieves
To composite mineral salt.
2. the AFG1 of aspergillus parasiticus according to claim 1 produces malicious fermentation process, it is characterised in that：The aspergillus parasiticus is
China General Microbiological culture presevation administrative center（CGMCC）The aspergillus parasiticus bacterium of offer（Aspergillus parasiticus）CGMCC NO.：3.0124.
3. the AFG1 of aspergillus parasiticus according to claim 1 produces malicious fermentation process, it is characterised in that：
Described enriched medium and solid fermentation culture medium, after being 5.5-7.0 with hydrochloric acid or sodium hydroxide regulation pH value, it is placed in
In 2L conical flasks, tampon is covered, sterilizing 20min is carried out under conditions of 121-123 DEG C, 0.12MPa.
4. the AFG1 of aspergillus parasiticus according to claim 3 produces malicious fermentation process, it is characterised in that：Described increasing bacterium training
The chloramphenicol composition in base is supported after sterilizing terminates, adds and mixes after enriched medium is cooled to below 40 DEG C.
5. the AFG1 of aspergillus parasiticus according to claim 1 produces malicious fermentation process, it is characterised in that：Described solid fermentation
For hominy grits and peanut meal described in culture medium through crushing, granularity reaches 20-40 targets standard.
6. the AFG1 of aspergillus parasiticus according to claim 1 produces malicious fermentation process, it is characterised in that：Composite mineral salt it is excellent
Matching ratio is：40-53.5% sodium nitrate, 11.5-15% magnesium sulfate, 11-14.5% potassium chloride, 17-22% dipotassium hydrogen phosphates, 3-6% sulphur
Sour zinc, 3-4% manganese sulfates, 0.5% 5 aqueous ferrous sulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711014844.1A CN107586799B (en) | 2014-12-04 | 2014-12-04 | A kind of malicious fermentation process of AFG1 production of aspergillus parasiticus |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711014844.1A CN107586799B (en) | 2014-12-04 | 2014-12-04 | A kind of malicious fermentation process of AFG1 production of aspergillus parasiticus |
| CN201410727872.8A CN104498557B (en) | 2014-12-04 | 2014-12-04 | The toxin producing medium of AFG1 a kind of and the AFG1 of aspergillus parasiticus produce malicious fermentation process |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410727872.8A Division CN104498557B (en) | 2014-12-04 | 2014-12-04 | The toxin producing medium of AFG1 a kind of and the AFG1 of aspergillus parasiticus produce malicious fermentation process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107586799A true CN107586799A (en) | 2018-01-16 |
| CN107586799B CN107586799B (en) | 2019-04-26 |
Family
ID=52940007
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711014844.1A Expired - Fee Related CN107586799B (en) | 2014-12-04 | 2014-12-04 | A kind of malicious fermentation process of AFG1 production of aspergillus parasiticus |
| CN201410727872.8A Expired - Fee Related CN104498557B (en) | 2014-12-04 | 2014-12-04 | The toxin producing medium of AFG1 a kind of and the AFG1 of aspergillus parasiticus produce malicious fermentation process |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410727872.8A Expired - Fee Related CN104498557B (en) | 2014-12-04 | 2014-12-04 | The toxin producing medium of AFG1 a kind of and the AFG1 of aspergillus parasiticus produce malicious fermentation process |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN107586799B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877219A (en) * | 2021-01-29 | 2021-06-01 | 江西科技师范大学 | High-concentration cholesterol culture medium and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603011A (en) * | 2009-06-18 | 2009-12-16 | 北京师范大学 | The method and the special strain therefore thereof that prepare the two glucosides of epipodophyllotoxin |
| CN102851334A (en) * | 2012-07-03 | 2013-01-02 | 浙江大学 | Fermentation medium and fermentation method of aflatoxin B1 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101389240B1 (en) * | 2009-01-14 | 2014-04-24 | 올텍 법인회사 | Clay Interlaced Yeast compositions and methods of utilizing the same |
-
2014
- 2014-12-04 CN CN201711014844.1A patent/CN107586799B/en not_active Expired - Fee Related
- 2014-12-04 CN CN201410727872.8A patent/CN104498557B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603011A (en) * | 2009-06-18 | 2009-12-16 | 北京师范大学 | The method and the special strain therefore thereof that prepare the two glucosides of epipodophyllotoxin |
| CN102851334A (en) * | 2012-07-03 | 2013-01-02 | 浙江大学 | Fermentation medium and fermentation method of aflatoxin B1 |
Non-Patent Citations (1)
| Title |
|---|
| SHEILA OKOTH ET AL: "Toxigenic Potential of Aspergillus Species Occurring on Maize Kernels from Two Agro-Ecological Zones in Kenya", 《TOXINS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877219A (en) * | 2021-01-29 | 2021-06-01 | 江西科技师范大学 | High-concentration cholesterol culture medium and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107586799B (en) | 2019-04-26 |
| CN104498557A (en) | 2015-04-08 |
| CN104498557B (en) | 2018-01-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105053593A (en) | Traditional Chinese medicine residues prepared poultry feed additive and preparation method thereof | |
| CN104521551A (en) | Selenium-rich shiitake mushroom production process | |
| CN110278965A (en) | A kind of microbial bacterial agent and preparation method thereof for preventing and treating citrus bacterial canker disease | |
| CN102552335B (en) | Traditional Chinese medicine health care product, its preparation method and its application | |
| CN104017739B (en) | Complex micro organism fungicide, its preparation method and its application in high protein potato dreg fodder is produced | |
| CN101801212B (en) | Use of cordyceps species or extract thereof in the egg production | |
| CN101480226A (en) | Fungal fermentate feed addictive as well as preparation method and use thereof | |
| CN103976351B (en) | A kind of health food of develop immunitypty improving water flood and two-step fermentation preparation method thereof | |
| CN107114758A (en) | A kind of ferment cordyceps sinensis D-mannitol powder and preparation method thereof | |
| CN101508961A (en) | Method of preparing yeast rich in copper | |
| Ogidi et al. | Calcium bioaccumulation by Pleurotus ostreatus and Lentinus squarrosulus cultivated on palm tree wastes supplemented with calcium-rich animal wastes or calcium salts | |
| CN103299824B (en) | Method for preparing phellinus igniarius sclertium from fresh, tender and sweet corn | |
| CN103948023B (en) | The health food of a kind of develop immunitypty and improving water flood and two-step fermentation preparation method thereof | |
| CN105925493B (en) | A strain of tabasheer and its application in fermentation production of hypocrellin | |
| CN109123076A (en) | A kind of production method of livestock and poultry vitamin B2 auxotype probiotics | |
| CN104498557B (en) | The toxin producing medium of AFG1 a kind of and the AFG1 of aspergillus parasiticus produce malicious fermentation process | |
| CN101653189A (en) | Natural plant small molecular group substance concentrate and production method thereof | |
| CN103932179B (en) | A kind of health food and two-step fermentation preparation method thereof releasing the pressure and protect liver | |
| CN106635834B (en) | The Thelephora ganbajun mycelium zinc polysaccharide and application that one plant of wizened bacteria strain and its fermentation obtain | |
| CN102851334B (en) | Fermentation medium and fermentation method of aflatoxin B1 | |
| CN104531800B (en) | The toxin producing medium of AFG2 a kind of and the AFG2 of aspergillus parasiticus produce malicious fermentation process | |
| CN105586267B (en) | Produce the ganoderma lucidum mutagenic strain of ganoderma lucidum mycelium | |
| CN104694400B (en) | A kind of method that solid state fermentation prepares High color values monascus product | |
| CN109770056A (en) | A kind of Chinese medicine residue degradation bacteria and its preparing the application in poultry fodder additive | |
| CN104970354B (en) | A kind of reducing blood lipid and the functional food and its two-step fermentation preparation method of hypotensive |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190426 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |