CN102851334A - Fermentation medium and fermentation method of aflatoxin B1 - Google Patents
Fermentation medium and fermentation method of aflatoxin B1 Download PDFInfo
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Abstract
The invention belongs to the technical field of fermentation engineering, and especially relates to a fermentation medium and a fermentation method for producing aflatoxin B1. The fermentation medium of aflatoxin B1 contains a yeast extract which accounts for 1-5% of the total weight of the medium; the fermentation medium comprises a recovery medium, a solid fermentation medium or a growth promotion nutrient solution. The fermentation method of aflatoxin B1 comprises two steps of strain recovery and solid fermentation culture; aspergillus flavus is employed for fermentation; the fermentation medium provided by the invention is employed for strain recovery and solid fermentation culture. According to the medium and culture procedure provided by the invention, the concentration in a solid air-dried substance of the fermentation medium of aflatoxin B1 is up to 7349 ppb; when the fermentation medium and the fermentation method of the invention are compared with the prior art with respect to fermentation titer, significant advancement is provided.
Description
Technical field
The invention belongs to the fermentation engineering field, particularly a kind of fermention medium and fermentation process that produces aflatoxin B1.
Background technology
Aflatoxin (Aflatoxins) is the class mycotoxins that flavus or Aspergillus parasiticus produce, and is severe toxicity and strong carcinogen, and being delimited by the cancer research mechanism of the World Health Organization (WHO) in 1993 is I class carcinogens.Present fixed aflatoxin has kind more than 20, and the aflatoxin of pollution grain mainly contains aflatoxin B1, B2, G1, G2 and M1 etc.The hazardness of aflatoxin is that people and animal livers tissue are had destruction, can cause liver cancer even acute poisoning dead when serious.In the grain of natural contamination, aflatoxin B1 toxicity is maximum, measures also at most, and carinogenicity is also the strongest.Aflatoxin B1 can be at corn, and peanut detects in peanut oil, rice, cottonseed, birds, beasts and eggs, meat, milk (milk preparation) and some dry fruits, and recall rate and content are higher in the grain and oil in especially hot and humid area and the goods.Eaten by the people if contain the food of aflatoxin B1, can cause acute aflatoxicosis or induced hepatocellular carcinoma.The feed that contains aflatoxin B1 is eaten by animal, often causes the animal acute poisoning or brings the inferior clinical symptoms such as disease resistance is low.In addition, breast also can be converted into the aflatoxin B1 in the feed aflatoxin M 1 in the dairy products with animal (as: milk cow), and then brings the food-safety problem of dairy products and milk-product.
In order to explore the content control method of aflatoxin B1 in food or the feed, need to develop the substratum that suitable flavus growth (recovery) is fast, vigor is high, the growth characteristics of research flavus and the malicious rule of product.In carrying out food or feed during the aflatoxin B1 content detection, the aflatoxin B1 standard substance that experiment needs, all from external sterling, one of reason is that aflatoxin B1 content is on the low side in the domestic flavus fermention medium, extract required cultivation base unit weight large, cost is high.In addition, further investigate the pathogenesis of aflatoxin B1, also must select the extract of the high culture of aflatoxin B1 content or aflatoxin B1, carry out experimentation on animals, set up various animal models, its intoxicating and detoxifcation are studied.
At present, the Some Domestic investigator carries out animal toxicology and studies employed aflatoxin B1, and is normal from external sterling, expensive (be 569RMB/mg such as Sigma company production aflatoxin B1 price), and experimentation cost is high.And in the flavus culture of present domestic report the high-content of aflatoxin B1 to be that the 3312ppb(village shakes grand, 2010), content of toxins is lower, the required culture of single experiment is many, and is also large to the interference of experiment.
In sum, be necessary to develop fermention medium and the fermentation process of aflatoxin B1, the growth characteristics of research flavus are explored the optimum controling method of aflatoxin B1 content in feed and the food and are carried out aflatoxin B1 to the intoxicating detoxifcation experimental study of animal.
Summary of the invention
The invention provides a kind of fermention medium that produces aflatoxin B1, the spore vigor that is used for solving present traditional substratum is low, growth (recovery) cycle is long and produce poison and measure low problem.
The present invention also provides a kind of fermentation process of producing aflatoxin B1.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of fermention medium of aflatoxin B1 contains the yeast extract that accounts for substratum gross weight 1～5% in the component of this substratum, described fermention medium is that recovery substratum, solid fermentation substratum or growth promote nutritive medium.
As preferably, described each components based on weight percentage of recovery substratum comprises: 5～20% sucrose, 1～5% yeast extract, 1～5% wheat bran vat liquor, 1.5% agar, 1～4g/L SODIUMNITRATE, 0.2～0.8g/L sal epsom, 0.2～0.8 g/L Repone K, 0.6～1.2 g/L dipotassium hydrogen phosphates, 0.01g/L five aqueous ferrous sulfates, surplus are water, the initial pH of substratum is 5.5～7.0.In order to realize the rapid fluid resuscitation of aspergillus spore, the contriver has designed by sucrose, yeast extract, wheat bran vat liquor, agar, NaNO
3Be mixed with new flavus recovery substratum with trace element, test showed and to utilize recovery substratum of the present invention, and the required time of aspergillus spore recovery only needs two days, than the recovery time shorten of traditional C zapek ' s substratum four days.
As preferably, described each components based on weight percentage of solid fermentation substratum comprises: 60～80% long-grained nonglutinous rices, 8～30% sucrose, 4～10% dregs of beans, 1～5% yeast extract, 1～5% wheat bran, after having prepared in proportion, regulating the solid fermentation moisture content in medium is 5～10%.As preferably, described long-grained nonglutinous rice, dregs of beans and wheat bran are crossed the accurate sub-sieve of 20～40 targets through pulverizing.This solid fermentation substratum can realize that flavus efficiently produces aflatoxin B1.
As preferably, described growth promotes each components based on weight percentage of nutritive medium to comprise: 5～20% sucrose, 1～5% yeast extract, 1～5% wheat bran vat liquor, 1～4g/L SODIUMNITRATE, 0.2～0.8g/L sal epsom, 0.2～0.8 g/L Repone K, 0.6～1.2 g/L dipotassium hydrogen phosphate, 0.01g/L five aqueous ferrous sulfates, surplus is water, and the initial pH of substratum is 5.5～7.0.This growth promotes nutritive medium to be used for the loss of afterfermentation culturing process nutritive substance and moisture, can guarantee Aspergillus flavus high vigor growth on the solid fermentation substratum.
As preferably, the preparation method of described wheat bran vat liquor is as follows: get 1 unit weight wheat bran and place clean beaker, then the 4 unit weight distilled water of annotating after wheat bran and water fully mixed, are placed in 37 ℃ of thermostat water baths, soak 10～12h, get supernatant liquor and be positioned in the centrifuge tube, 2000～3000rpm, centrifugal 5～10 minutes, the supernatant liquor in the centrifuge tube is drawn in the clean beaker, the wheat bran extract prepares complete again.The wheat bran extract of preparation requires now with the current.
The sterilization of substratum requires: described recovery substratum, solid fermentation substratum and growth promote nutritive medium, after preparing in proportion respectively (wherein the recovery substratum also need dissolve agar through heating by electric cooker), place Erlenmeyer flask at 121～123 ℃, 0.12MPa, sterilization 20min, each substratum preparation end-of-job.
The inversion of recovery substratum: in Biohazard Safety Equipment, the recovery substratum that sterilization is good is upside down in through in the diameter 10cm culture dish of sterilizing, naturally cooling.
A kind of fermentation process of aflatoxin B1 comprises bacterial classification recovery and two steps of solid fermentation cultivation, adopts the flavus bacterial classification to ferment, and adopts fermention medium provided by the invention to carry out bacterial classification recovery and solid fermentation cultivation.The method has adopted the substratum after the above-mentioned improvement, and the bacterial classification that fermentation is selected is Aspergillus flavus (from China Committee for Culture Collection of Microorganisms common micro-organisms center, numbering 3.4409), and zymotechnique adopts conventional technique.According to substratum provided by the invention and cultivation program, concentration can reach 7349ppb in the air-dry thing of aflatoxin B1 fermention medium solid, and fermentation titer compared to existing technology has significant progress.
The invention has the beneficial effects as follows: the flavus recovery substratum of the present invention development, mould-growth speed is fast, and the spore vigor is high, recovers or time of going down to posterity greatly shortens, and has improved working efficiency.The solid fermentation substratum of the present invention's development, nutritious, thalli growth is vigorous, and culture cycle is short, only needs 12～14 days, relative general rice substratum, incubation time shortens 3～4 days, and aflatoxin B1 concentration significantly improves.Solid fermentation substratum provided by the invention mainly adopts long-grained nonglutinous rice, wheat bran, dregs of beans, sucrose and yeast, and raw material is easy to get, cheapness.From the aflatoxin B1 of external import, the aflatoxin B1 price that substratum provided by the invention is produced can reduce more than 80% relatively.Fermentation process provided by the invention, simple, workable.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation and/or change that the present invention is made all will fall into protection domain of the present invention.
In the present invention, if not refer in particular to, all part, per-cents are weight unit, and all equipment and raw material etc. all can be buied from market or the industry is commonly used.
Various embodiments of the present invention with the flavus (Aspergillus flavus) 3.4409 that China Committee for Culture Collection of Microorganisms common micro-organisms center provides, are set forth.
The growth of embodiment 1-4 flavus promotes the preparation of nutritive medium
After the mixing of the each component shown in the table 1, the pH value of regulating mixing solutions with hydrochloric acid soln is 5.5～7.0.
Table 1
Now take embodiment 2 as example, preparation 1L growth promotes nutritive medium.Take by weighing 150g sucrose, 20g yeast extract, 20ml wheat bran vat liquor, 2gNaNO
3, 0.3gMgSO
4, 0.3gKCl, 0.8gK
2HPO
4, 0.01gFeSO
4.7H
2O, 810ml distilled water places the 2000ml Erlenmeyer flask, behind all raw materials of heating for dissolving, uses the salt acid for adjusting pH value of 5mol/L concentration in 5.5～7.0.PH regulator is complete, covers tampon, at 121～123 ℃, and 0.12MPa, sterilization 20min.After the cooling, growth promotes compounding to finish in Biohazard Safety Equipment.
The preparation method of described wheat bran vat liquor following (lower same): get 1 unit weight wheat bran and place clean beaker, then the 4 unit weight distilled water of annotating, after wheat bran and water fully mixed, be placed in 37 ℃ of thermostat water baths, soak 10～12h, get supernatant liquor and be positioned in the centrifuge tube, 2000～3000rpm, centrifugal 5～10 minutes, again the supernatant liquor in the centrifuge tube is drawn in the clean beaker, the wheat bran extract prepares complete.
The preparation of embodiment 5-8 flavus recovery substratum
A kind of flavus recovery substratum, after the mixing of the each component shown in the table 2, the pH value of regulating mixing solutions with hydrochloric acid soln is 5.5～7.0.
Table 2
Now take the flavus recovery culture medium prescription of embodiment 6 as example, preparation 1L recovery substratum.Take by weighing 150g sucrose, 20g yeast extract, 20ml wheat bran vat liquor, 2gNaNO
3, 0.3gMgSO
4, 0.3gKCl, 0.8gK
2HPO
4, 0.01gFeSO
4.7H
2O, the 15g agar powder, 795ml distilled water places the 2000ml Erlenmeyer flask, behind all raw materials of heating for dissolving, uses the salt acid for adjusting pH value of 5mol/L concentration in 5.5～7.0.PH regulator is complete, covers tampon, at 121～123 ℃, and 0.12MPa, sterilization 20min.In Biohazard Safety Equipment, 60 ℃ of the recovery substratum of sterilization are not solidified, be upside down in the diameter 10cm culture dish through sterilization.Naturally cooling, namely the recovery substratum prepares complete.
The preparation of the slant medium of flavus bacterial classification preservation.
According to flavus recovery culture medium prescription table, prepare required culture presevation substratum.With the recovery substratum for preparing, when 60 ℃ of substratum do not solidify, be upside down in the test tube of sterilization, volume is about 1/3 of test tube capacity, the inclination test tube, naturally cooling, namely strain store medium prepares complete.
The preparation of embodiment 9-12 flavus solid fermentation substratum
According to the prescription of table 3, prepare required substratum.
Table 3
Raw material is prepared: long-grained nonglutinous rice, dregs of beans and wheat bran are pulverized, and granularity requirements can be crossed the accurate sub-sieve of 20 targets, but can not cross the accurate sub-sieve of 40 targets, namely between 20～40 orders.
Now take the component of embodiment 10 as example, the solid fermentation substratum of preparation 1kg.Take by weighing 120g sucrose, the 20g yeast extract, the 30g wheat bran, the 750g long-grained nonglutinous rice, the 80g dregs of beans, other measures 80ml distilled water, and above-mentioned raw materials is mixed, and places the 3000ml Erlenmeyer flask, covers tampon, at 121～123 ℃, 0.12MPa, sterilization 20min.In Biohazard Safety Equipment, sterilized solid medium is upside down in the sterilized Erlenmeyer flask, tiling thickness is 1cm, or loading capacity is the 50g/500ml triangular flask, covers sterilized bottle stopper.So far, the preparation of flavus fermentation solid substratum is complete.
Embodiment 13The fermentation culture of flavus
1, the recovery cultural method of flavus:
Ampoule is opened: the alcohol absorbent cotton with 75% carries out disinfection to the ampoule outside surface, with spirit lamp the top is heated, and drips a little sterilized water in heated tip, makes it to break, and then strikes down the ampoule top of breaking with tweezers.
The lyophilized powder dissolving: use aseptic straw to draw 0.3ml physiological saline, splash in the ampoule, gently vibration makes the dissolving of freeze-drying thalline be suspension.
The bacterial classification recovery: draw 0.5ml with disposable sterilized injector, evenly point sample is on the recovery culture medium flat plate that embodiment 6 prepares, and then aseptic glass stick is smeared bacterium liquid and is evenly distributed.Place 35 ℃ of constant incubators to cultivate 48h the recovery substratum that connects flavus, can grow a large amount of mycelia, reach test requirements document, recover complete.
The recovery substratum that adopts respectively embodiment 5,7,8 to make, flavus recovery reach and satisfy the requirement of experiment required time and be respectively 52h, 46h and 43h.
2, bacterial classification is preserved: in Biohazard Safety Equipment, use the aseptic inoculation pin, after the sterilization cooling, a small amount of bacterium liquid of picking makes aforementioned
The slant medium of flavus bacterial classification preservationBe the Z-shaped line on the inclined-plane, cover sterilized ventilative tampon, then place 35 ℃ of constant incubators to cultivate 36h.After growing more mycelia, be placed in 4 ℃ of antistaling cabinets and preserve, per two months, go down to posterity once according to present method.
3, flavus is in the fermention medium top fermentation
The Aspergillus flavus multiplication culture: use the aseptic inoculation ring, scraping mycelia on the agar plate that flavus has recovered, then intensive line on a plurality of recovery substratum places 35 ℃ of constant incubators to cultivate 48h.Cover with the agar plate of mycelia, as providing fermentation required spore.
The flavus fermentation preparation of spore liquid: in Biohazard Safety Equipment, draw the 2ml stroke-physiological saline solution, be expelled on the recovery flavus agar plate, then use the careful scraping mycelia of aseptic glass stick, draw bacterium liquid, place the 250ml Erlenmeyer flask, then use the stroke-physiological saline solution dilution, until aspergillus spore density is 1.0 * 10 in the bacterium liquid
6Cfu/ml.
Draw 2.5ml aspergillus spore liquid with disposable syringe, the solid medium that slow point sample prepares in embodiment 10 uses aseptic glass stick to mix simultaneously, covers the ventilative tampon of sterilization, places 35 ℃ of constant incubators to cultivate.Simultaneously, place a basin tap water in incubator, towel one end is placed water, an end is suspended on the rack, forms cascade, and the relative humidity in the control incubator is as 80～90%.
Humidity in the every day detection record incubator, and flavus fermentation state in the Erlenmeyer flask.
Every interval 3 days; in Biohazard Safety Equipment, use aseptic glass stick that fermention medium is stirred; spray simultaneously the 2.5ml growth and promote nutritive medium (being made by embodiment 2); with the loss of nutritive substance and moisture in the afterfermentation process, guarantee Aspergillus flavus high vigor growth and shorten incubation time 2～3 days on the solid fermentation substratum.After paving fermention medium, cover aseptic tampon, place 35 ℃ of constant incubators to continue to cultivate.
Through 12～14 days fermentation culture, substratum thoroughly fermented in the Erlenmeyer flask, and the mycelia color is deep green, and fermentation culture finishes.With culture in 121～123 ℃, 0.12MPa, 20 min that sterilize, then oven dry is pulverized, the culture that contains aflatoxin B1 prepares complete.
Detect the content of aflatoxin B1 in the fermention medium with high performance liquid chromatography (GB/T 5009.23-2006).Adopt the solid fermentation substratum of embodiment 10, fermentation culture 12 days, aflatoxin B1 concentration in the final air-dry sample of fermention medium is 7351ppb.
Adopt embodiment 9,11,12 solid fermentation substratum, carry out fermentative processing under above-mentioned the same terms, aflatoxin B1 concentration in the final air-dry sample of fermention medium is respectively 7151ppb, 7210ppb and 7349ppb.
Embodiment 14The fermentation culture of flavus
Concrete grammar is with embodiment 13, the recovery substratum that adopts is that embodiment 6 makes, the solid medium that adopts is made by embodiment 10, difference is: the flavus fermenting process adopts respectively embodiment 1,3,4 growth to promote that nutritive medium sprays, carry out fermentative processing under above-mentioned the same terms, aflatoxin B1 concentration in the final air-dry sample of fermention medium is respectively 7320ppb, 7210ppb and 7346ppb.
Above-described embodiment is a kind of better scheme of the present invention, is not that the present invention is done any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim puts down in writing.
Claims (7)
1. the fermention medium of an aflatoxin B1 is characterized in that: contain the yeast extract that accounts for substratum gross weight 1～5% in the component of this substratum, described fermention medium is that recovery substratum, solid fermentation substratum or growth promote nutritive medium.
2. the fermention medium of a kind of aflatoxin B1 according to claim 1, it is characterized in that: described each components based on weight percentage of recovery substratum comprises: 5～20% sucrose, 1～5% yeast extract, 1～5% wheat bran vat liquor, 1.5% agar, 1～4g/L SODIUMNITRATE, 0.2～0.8g/L sal epsom, 0.2～0.8 g/L Repone K, 0.6～1.2 g/L dipotassium hydrogen phosphates, 0.01g/L five aqueous ferrous sulfates, surplus are water, the initial pH of substratum is 5.5～7.0.
3. the fermention medium of a kind of aflatoxin B1 according to claim 1, it is characterized in that: described each components based on weight percentage of solid fermentation substratum comprises: 60～80% long-grained nonglutinous rices, 8～30% sucrose, 4～10% dregs of beans, 1～5% yeast extract, 1～5% wheat bran, after having prepared in proportion, regulating the solid fermentation moisture content in medium is 5～10%.
4. the fermention medium of a kind of aflatoxin B1 according to claim 3 is characterized in that: described long-grained nonglutinous rice, dregs of beans and wheat bran are crossed the accurate sub-sieve of 20～40 targets through pulverizing.
5. the fermention medium of a kind of aflatoxin B1 according to claim 1, it is characterized in that: described growth promotes each components based on weight percentage of nutritive medium to comprise: 5～20% sucrose, 1～5% yeast extract, 1～5% wheat bran vat liquor, 1～4g/L SODIUMNITRATE, 0.2～0.8g/L sal epsom, 0.2～0.8 g/L Repone K, 0.6～1.2 g/L dipotassium hydrogen phosphate, 0.01g/L five aqueous ferrous sulfates, surplus is water, and the initial pH of substratum is 5.5～7.0.
6. according to claim 2 or the fermention medium of 5 described a kind of aflatoxin B1, the preparation method who it is characterized in that described wheat bran vat liquor is as follows: get 1 unit weight wheat bran and place clean beaker, then the 4 unit weight distilled water of annotating, after wheat bran and water fully mixed, be placed in 37 ℃ of thermostat water baths, soak 10～12h, getting supernatant liquor is positioned in the centrifuge tube, 2000～3000rpm, centrifugal 5～10 minutes, the supernatant liquor in the centrifuge tube is drawn in the clean beaker, the wheat bran extract prepares complete again.
7. the fermentation process of an aflatoxin B1 comprises bacterial classification recovery and two steps of solid fermentation cultivation, adopts the flavus bacterial classification to ferment, and it is characterized in that: adopt fermention medium as claimed in claim 1 to carry out bacterial classification recovery and solid fermentation cultivation.
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| CN107557402A (en) * | 2014-12-04 | 2018-01-09 | 武汉轻工大学 | A kind of AFG2 of aspergillus parasiticus produces malicious fermentation process |
| CN107586799A (en) * | 2014-12-04 | 2018-01-16 | 武汉轻工大学 | A kind of AFG1 of aspergillus parasiticus produces malicious fermentation process |
| CN113234604A (en) * | 2021-04-30 | 2021-08-10 | 裕菁科技(上海)有限公司 | Preparation method of mixed culture medium for mycotoxin production |
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| CN101265456A (en) * | 2008-05-08 | 2008-09-17 | 江西农业大学 | Raw material wine-brewing direct adding composite bacterium preparation and preparing method thereof |
| CN102321631A (en) * | 2011-08-30 | 2012-01-18 | 贵州大学 | Construction and application of artificially-synthesized chicken interferon gene sequence and recombinant engineering bacterium thereof |
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| CN1597923A (en) * | 2004-08-13 | 2005-03-23 | 山东省食品发酵工业研究设计院 | Aspergillus flavus and process for producing calcium tartaric using the same |
| CN101265456A (en) * | 2008-05-08 | 2008-09-17 | 江西农业大学 | Raw material wine-brewing direct adding composite bacterium preparation and preparing method thereof |
| CN102321631A (en) * | 2011-08-30 | 2012-01-18 | 贵州大学 | Construction and application of artificially-synthesized chicken interferon gene sequence and recombinant engineering bacterium thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107557402A (en) * | 2014-12-04 | 2018-01-09 | 武汉轻工大学 | A kind of AFG2 of aspergillus parasiticus produces malicious fermentation process |
| CN107586799A (en) * | 2014-12-04 | 2018-01-16 | 武汉轻工大学 | A kind of AFG1 of aspergillus parasiticus produces malicious fermentation process |
| CN107557402B (en) * | 2014-12-04 | 2019-04-26 | 武汉轻工大学 | A kind of malicious fermentation process of AFG2 production of aspergillus parasiticus |
| CN107586799B (en) * | 2014-12-04 | 2019-04-26 | 武汉轻工大学 | A kind of malicious fermentation process of AFG1 production of aspergillus parasiticus |
| CN113234604A (en) * | 2021-04-30 | 2021-08-10 | 裕菁科技(上海)有限公司 | Preparation method of mixed culture medium for mycotoxin production |
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