CN104531800B - The toxin producing medium of AFG2 a kind of and the AFG2 of aspergillus parasiticus produce malicious fermentation process - Google Patents
The toxin producing medium of AFG2 a kind of and the AFG2 of aspergillus parasiticus produce malicious fermentation process Download PDFInfo
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Abstract
The present invention relates to the AFG2 of the toxin producing medium of AFG2 a kind of and aspergillus parasiticus to produce malicious fermentation process.Containing the yeast extract for accounting for culture medium gross weight 3 5% in the component of the culture medium, fermentation medium is enriched medium and solid fermentation culture medium.AFG2 of the present invention toxin producing medium solves the problems such as AFG2 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 the toxin producing medium of AFG2 a kind of and aspergillus parasiticus
AFG2 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 (AFG2), 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, AFG2 maximum concentrations are only 46ppb (Feng Jianlei, 2004) in the fermentation medium of domestic report, therefore
When carrying out the research of AFG2 related sciences, the AFG2 samples from external import expensive (930RMB/mg), experimentation cost are both needed to
Height, or need to carry out commodity reservation, made troubles to related science research.In summary, AFG2 fermentation medium is developed
And application process, experiment material can be provided for the food security research of the AFG2 toxicological tests and AFs of animal.
The content of the invention
The invention provides a kind of AFG2 toxin producing medium, for solving AFG2 in the cereal of current natural infection mould
The problems such as concentration is low, uneven, time-consuming.
Present invention also offers a kind of AFG2 of aspergillus parasiticus to produce malicious fermentation process.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of AFG2 toxin producing medium, the yeast leaching for accounting for culture medium gross weight 3-5% is contained in the component of the culture medium
Cream, described fermentation medium are enriched medium and solid fermentation culture medium.
Preferably, described enriched medium each component includes by weight percentage：25-30% potatos, 10-
15% glucose, 1-3% agar, 3-5% yeast extracts, 0.4% composite mineral salt, 0.3g/L penicillin, 0.3g/L streptomysins,
Surplus is water.Inventor proves through substantial amounts of experiment display, using enriched medium of the present invention, under same culture conditions, posts
Time needed for raw aspergillus spore recovery only needs 2 days, and the recovery time than conventional medium shortens 4 days, and spore density improves
10 times.At present, when carrying out aspergillus parasiticus Zengjing Granule, conventional medium component is：Potato, glucose, agar and water,
Culture medium rush aspergillus parasiticus growth efficiency is low, and time-consuming, is easily bacterial contamination, and enriched medium efficiency provided by the invention
Significantly improve.
Preferably, described solid fermentation culture medium each component includes by weight percentage：40-70% early rices,
20-45% Cottonseed Meals, 6-12% sucrose, 0.2-0.5% composite mineral salts, 3-5% yeast extracts, after having prepared in proportion, adjust
It is 15-20% to save solid fermentation moisture content in medium.Preferable scheme is 41.6-51.6% early rices, 37.9-44% cottonseeds
The dregs of rice, 6-10.9% sucrose, 0.5% composite mineral salt, 3-4% yeast extracts.Further, described early rice and Cottonseed Meal warp
Crush, granularity reaches 20-40 targets standard.Solid fermentation culture medium institute carbohydrate containing (early rice), the protein (cottonseed
The dregs of rice), the nutriment such as mineral matter (composite mineral salt) and somatomedin (yeast extract) meet that aspergillus parasiticus efficiently produces just
The condition of poison, and ratio is appropriate, and aspergillus parasiticus can be achieved and efficiently produce AFG2.When carrying out solid fermentation culture, prior art institute
Culture medium is only the crushing maize that have adjusted moisture, i.e., common corn, composition is single, and nutritional ingredient is uneven, required
Fermented incubation time it is longer, the present inventor also once attempts to be cultivated using single crushing maize culture medium, and effect is also very
Difference.AFG2 concentration is substantially less than the solid fermentation culture medium A FG2 concentration of the present invention in the culture medium that prior art uses.
Preferably, the preparation method of composite mineral salt is as follows：The component of the composite mineral salt is by percentage to the quality
For：42-48% sodium nitrate, 10-16% magnesium sulfate, 10-15% potassium chloride, 18-24% dipotassium hydrogen phosphates, 2.7-5.7% sulfuric acid
Zinc, 2-5% manganese sulfates, the aqueous ferrous sulfates of 0.2-0.8% five；Above-mentioned each mineral salt raw material is well mixed, crushed 80 mesh point
Mixed again after sample sieve, obtain composite mineral salt.
The sterilizing requirement of culture medium：Described enriched medium and solid fermentation culture medium, prepares (its in proportion respectively
In, the penicillin and streptomysin composition in enriched medium treat that enriched medium is cooled to 40 DEG C or so and added after sterilizing terminates
Enter to mix), after being 5.5-7.0 with hydrochloric acid or sodium hydroxide regulation pH value, it is placed in 2L conical flasks, covers tampon, in 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.
A kind of AFG2 fermentation mediums application process, including strain bring back to life propagation and two steps of solid fermentation culture, adopt
Fermented with aspergillus parasiticus strain, carrying out strain using fermentation medium provided by the invention brings back to life propagation and solid fermentation training
Support.This method uses above-mentioned solid fermentation culture medium, and the strain for selection of fermenting (comes from China General Microbiological for aspergillus parasiticus bacterium
Culture presevation administrative center, CGMCC NO.3.0124), zymotechnique is using conventional solid fermentation process.Carried according to the present invention
The culture medium and cultural method of confession, concentration is up to 914ppb in the solid air dry matter of toxin producing medium by AFG2, compared to existing skill
The fermentation titer of art, have significant progressive.And this method fermentation efficiency is high, producing cost is low, has filled up China AFG2's
The blank of production field, and can greatly reduce the research cost in AFG2 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 12-13 days, relatively common rice medium, incubation time shortened 5-7 days, and AFG2 concentration is notable
Improve.Solid fermentation culture medium provided by the invention, mainly soaked using early rice, Cottonseed Meal, sucrose, composite mineral salt and yeast
Cream, raw material are easy to get, inexpensively.The relative AFG2 from external import, the AFG2 prices that culture medium provided by the invention is produced can drop
Low more than 80%.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 44.0g sodium nitrate, 14.0g magnesium sulfate, 12.0g chlorinations are weighed with assay balance
Potassium, 20.0g dipotassium hydrogen phosphates, 5.7g zinc sulfate, 4.0g manganese sulfates, the aqueous ferrous sulfates of 0.3g 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, mixes again,
Be placed in brown bottle wide-mouth bottle retain it is standby.
Enriched medium (1L)：Potato is first cleaned into peeling, then weighs 270g potatos and is cut into small pieces, adds 800mL water
20-30 minutes are boiled, until can be poked by glass bar, with 4 layers of filtered through gauze into beaker, filter residue discards, the moisturizing into beaker
To 1000mL, then toward addition 120g glucose in beaker, 35g yeast extracts, 4g composite mineral salts, heating, add 15g fine jades
Fat, continue heating stirring and mix, after agar has dissolved, stir, moisture is supplied again to 1000 milliliters after slightly cooling down, use
The salt acid for adjusting pH value of 5mol/L concentration is in 5.5-7.0.PH regulations finish, and are fitted into the conical flask that capacity is 2L, cover tampon,
At 121-123 DEG C, 0.12MPa, sterilize 20min.
In Biohazard Safety Equipment, penicillin and streptomysin are added into 40 DEG C of enriched medium of sterilizing, is cultivating it
After concentration in base each reaches 0.3g/L, then it is upside down in the diameter 10cm culture dishes of sterilization treatment, cools down wild Oryza species
That is solidifiable.Then, enriched medium is placed in 37 DEG C of constant incubators and cultivates 24h, asepsis growth person can use, and increase bacterium
Culture 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, penicillin and streptomysin are added under 60 DEG C of non-curdled appearances of culture medium, its concentration in the medium is each reached
0.3g/L, it is upside down in the test tube of sterilizing, volume is about the 1/3 of test tube capacity, tilts test tube, natural cooling, i.e. culture presevation
Slant medium prepare finish.
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：Long-grained nonglutinous rice and Cottonseed Meal are crushed, it is desirable to the quasi- sub-sieve of 20 targets can be crossed, but 40 mesh standard scores can not be crossed
Sample sieves, i.e., granularity is 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 44.0g sodium nitrate, 14.0g magnesium sulfate, 12.0g chlorinations are weighed with assay balance
Potassium, 20.0g dipotassium hydrogen phosphates, 5.7g zinc sulfate, 4.0g manganese sulfates, the aqueous ferrous sulfates of 0.3g 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, mixes again,
Be placed in brown bottle wide-mouth bottle retain it is standby.
Solid fermentation culture medium (1kg).Weigh 455g long-grained nonglutinous rices, 400g Cottonseed Meals, 100g sucrose, 5g composite mineral salts, 40g
Yeast extract, 150ml distilled water is separately measured, above-mentioned raw materials are well mixed, be placed in 3000ml conical flasks, cover tampon,
121-123 DEG C, 0.12MPa, sterilize 30min.In Biohazard Safety Equipment, sterilized solid medium is upside down in sterilized
In conical flask, tiling thickness is 1cm, or useful load is 60g/500ml triangular flasks, covers sterilized bottle stopper.So far, it is parasitic
Aspergillus fermentation solid culture medium is prepared and finished.
The AFG2 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：In Biohazard Safety Equipment, the aspergillus parasiticus freeze-dried powder 0.5mL physiological saline that will be preserved in ampoule bottle
Dissolving, then draw 4mL lysates, the enriched medium that uniform point sample prepares in embodiment 2 with disposable sterilized injector
On, then bacterium solution is smeared with sterile glass rod and is evenly distributed.The enriched medium for having connect aspergillus parasiticus is placed in 35 DEG C of constant temperature trainings
Support in case and cultivate 44h, you can grow substantial amounts of mycelia, reach test requirements document, strain brings back to life and finished.
Composite mineral salt formula in the enriched medium of alternate embodiment 1,3,4 is distinguished using the composite mineral salt formula of example 6
Obtained enriched medium, it is respectively 42h, 48h and 52h 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 44h, 48h 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 56h 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 46h, 49h and 43h 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 45h, 42h and 51h 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 42h.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
Continuous culture 2d in DEG C constant incubator.Cover with the enriched medium flat board of mycelia, it is possible to provide ferment required spore and mycelia
Body.
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, collects mycelia and spore in sterilized 250ml conical flasks,
Aspergillus parasiticus bacterium solution is diluted with sterile saline, until spore density is 1.2~1.4 × 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 rice
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, crushing, the culture containing AFG2 are prepared and finished.
The content of AFG2 in fermentation medium is detected with high performance liquid chromatography (GB/T 5009.23-2006).Using reality
Apply the composite mineral salt of example 6 and the solid fermentation culture medium of embodiment 10, fermented and cultured 12d, AFG2 concentration is reachable in air-drying sample
To 914ppb.
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 900ppb, 910ppb and 899ppb to support AFG2 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 AFG2 concentration be respectively 908ppb, 902ppb and
789ppb。
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 AFG2 concentration be respectively 907ppb, 910ppb and
908ppb。
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 AFG2 concentration be respectively 902ppb, 904ppb and
872ppb。
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 AFG2 concentration be respectively 893ppb, 876ppb and
911ppb。
Conclusion：The production poison concentration of prior art is 46 ± 3.6ppb, and AFG2 of the present invention air-dries in the solid of toxin producing medium
Concentration has up to 914ppb, 46 ± 3.6ppb compared with prior art, AFG2 of the present invention production poison concentration fermentation titer in thing
It is significant progressive.The inventive method fermentation efficiency is high, and producing cost is low, has filled up the blank of China AFG2 production field, and
The research cost in AFG2 toxicological study field can greatly be reduced.
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 (2)
- A kind of 1. AFG2 toxin producing medium, it is characterised in that：Contain in the component of the culture medium and account for culture medium gross weight 3-5% Yeast extract, described culture medium is enriched medium or solid fermentation culture medium；Described enriched medium each component is pressed Percentage by weight meter includes：25-30% potatos, 10-15% glucose, 1-3% agar, 3-5% yeast extracts, 0.4% complex mineral Salt, 0.3g/L penicillin, 0.3g/L streptomysins, surplus are water, pH value 5.5-7.0；Described solid fermentation culture medium each group Divide includes by weight percentage：40-70% early rices, 20-45% Cottonseed Meals, 6-12% sucrose, 0.2-0.5% composite mineral salts, 3-5% yeast extracts, after having prepared in proportion, regulation solid fermentation moisture content in medium is 15-20%, pH value 5.5-7.0；Institute The component for stating composite mineral salt is by percentage to the quality：42-48% sodium nitrate, 10-16% magnesium sulfate, 10-15% potassium chloride, 18- 24% dipotassium hydrogen phosphate, 2.7-5.7% zinc sulfate, 2-5% manganese sulfates, the aqueous ferrous sulfates of 0.2-0.8% five；Above-mentioned each mineral salt is former Material is well mixed, is mixed again after crushed 80 mesh sub-sieves, obtains composite mineral salt.
- 2. AFG2 according to claim 1 toxin producing medium, it is characterised in that：Described early rice and Cottonseed Meal is advance It is crushed to granularity and reaches 20-40 mesh.
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| 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 |
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| CN104087539B (en) * | 2014-07-11 | 2016-06-15 | 湖南豫园生物科技有限公司 | Streptomyces microflavus solid fermentation culture medium, preparation method and fermentation process thereof |
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| CN102548568A (en) * | 2009-01-14 | 2012-07-04 | 全技术公司 | Clay interlaced yeast compositions and methods of utilizing the same |
| 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 |
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