CN102851334B - Fermentation medium and fermentation method of aflatoxin B1 - Google Patents
Fermentation medium and fermentation method of aflatoxin B1 Download PDFInfo
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- CN102851334B CN102851334B CN201210225758.6A CN201210225758A CN102851334B CN 102851334 B CN102851334 B CN 102851334B CN 201210225758 A CN201210225758 A CN 201210225758A CN 102851334 B CN102851334 B CN 102851334B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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, in the mechanism of the cancer research by the World Health Organization (WHO) in 1993, delimits as I class carcinogens.Current 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 to destruction, when serious, can cause even acute poisoning death of liver cancer.In the grain of natural contamination, aflatoxin B1 toxicity maximum, measure 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 in the grain and oil in especially hot and humid area and goods, recall rate and content are higher.If the food that contains aflatoxin B1 is eaten by the people, 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 feed the aflatoxin M 1 in 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 feed, need to develop and be applicable to the substratum that flavus growth (recovery) is fast, vigor is high, study the growth characteristics of flavus and produce malicious rule.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 in domestic flavus fermention medium, aflatoxin B1 content is on the low side, extract required cultivation base unit weight large, cost is high.In addition, further investigate the pathogenesis of aflatoxin B1, also must select culture that aflatoxin B1 content is high or the extract of aflatoxin B1, carry out experimentation on animals, set up various animal models, its intoxicating and removing toxic substances are studied.
At present, the Some Domestic investigator carries out the aflatoxin B1 of animal toxicology institute use, and normal from external sterling, expensive (as Sigma company production aflatoxin B1 price is 569RMB/mg), experimentation cost is high.And in the flavus culture of current 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, 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, explore the optimum controling method of aflatoxin B1 content in feed and food and carry out the intoxicating removing toxic substances experimental study of aflatoxin B1 to animal.
Summary of the invention
The invention provides a kind of fermention medium that produces aflatoxin B1, low for the spore vigor that solves current traditional substratum, 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, 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.
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 is water, and 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, aspergillus spore required time of recovering only to need 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, through pulverizing, are crossed the accurate sub-sieve of 20～40 target.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 the loss of nutritive medium for 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 be placed in clean beaker, then the 4 unit weight distilled water of annotating, after wheat bran and water are fully mixed, be placed in 37 ℃ of thermostat water baths, soak 10～12h, get supernatant liquor and be positioned in centrifuge tube, 2000～3000rpm, centrifugal 5～10 minutes, the supernatant liquor in centrifuge tube is drawn in clean beaker, the wheat bran extract prepares complete again.The wheat bran extract of preparation requires now with the current.
The sterilizing of substratum requires: described recovery substratum, solid fermentation substratum and growth promote nutritive medium, after preparing in proportion respectively (wherein the recovery substratum also needs to dissolve agar through heating by electric cooker), be placed in Erlenmeyer flask at 121～123 ℃, 0.12MPa, sterilizing 20min, each substratum preparation end-of-job.
The inversion of recovery substratum: in Biohazard Safety Equipment, by sterilizing, good recovery substratum is upside down in the diameter 10cm culture dish through sterilizing, naturally cooling.
A kind of fermentation process of aflatoxin B1, comprise bacterial classification recovery and two steps of solid fermentation cultivation, adopts the flavus bacterial classification to be fermented, 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 above-mentioned improvement, and the bacterial classification that fermentation is selected is Aspergillus flavus (from China Committee for Culture Collection of Microorganisms's common micro-organisms center, numbering 3.4409), and zymotechnique adopts conventional technique.According to substratum provided by the invention and cultivation program, in the air-dry thing of aflatoxin B1 fermention medium solid, concentration can reach 7349ppb, and fermentation titer compared to existing technology, have 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 adopt 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 that the present invention is made and/or change 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, the flavus provided with China Committee for Culture Collection of Microorganisms's common micro-organisms center (Aspergillus flavus) 3.4409, set forth.
the growth of embodiment 1-4 flavus promotes the preparation of nutritive medium
After each component shown in table 1 is mixed, the pH value of regulating mixing solutions with hydrochloric acid soln is 5.5～7.0.
Table 1
Now take embodiment 2 as example, and preparation 1L growth promotes nutritive medium.Take 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, be placed in the 2000ml Erlenmeyer flask, after 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, sterilizing 20min.After cooling in Biohazard Safety Equipment, growth promotes that compounding completes.
The preparation method of described wheat bran vat liquor following (lower same): get 1 unit weight wheat bran and be placed in clean beaker, then the 4 unit weight distilled water of annotating, after wheat bran and water are fully mixed, be placed in 37 ℃ of thermostat water baths, soak 10～12h, get supernatant liquor and be positioned in centrifuge tube, 2000～3000rpm, centrifugal 5～10 minutes, then the supernatant liquor in centrifuge tube is drawn in 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 each component shown in table 2 is mixed, the pH value of regulating mixing solutions with hydrochloric acid soln is 5.5～7.0.
Table 2
The flavus recovery culture medium prescription of embodiment 6 of now take is example, preparation 1L recovery substratum.Take 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, be placed in the 2000ml Erlenmeyer flask, after 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, sterilizing 20min.In Biohazard Safety Equipment, 60 ℃ of the recovery substratum of sterilizing are not solidified, be upside down in the diameter 10cm culture dish through sterilizing.Naturally cooling, 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.By the recovery substratum prepared, when 60 ℃ of substratum do not solidify, be upside down in the test tube of sterilizing, volume is about 1/3 of test tube capacity, the inclination test tube, naturally cooling, strain store medium prepares complete.
the preparation of embodiment 9-12 flavus solid fermentation substratum
According to the formula 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 target, but can not cross the accurate sub-sieve of 40 target, between 20～40 orders.
The component of embodiment 10 of now take is example, the solid fermentation substratum of preparation 1kg.Take 120g sucrose, the 20g yeast extract, the 30g wheat bran, the 750g long-grained nonglutinous rice, the 80g dregs of beans, separately measure 80ml distilled water, and above-mentioned raw materials is mixed, and is placed in the 3000ml Erlenmeyer flask, covers tampon, at 121～123 ℃, 0.12MPa, sterilizing 20min.In Biohazard Safety Equipment, sterilized solid medium is upside down in 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: with 75% alcohol absorbent cotton, the ampoule outside surface is carried out disinfection, with spirit lamp, top is heated, drip a little sterilized water in heated tip, make it to break, then with tweezers, strike down the ampoule top of breaking.
Lyophilized powder dissolves: use aseptic straw to draw 0.3ml physiological saline, splash in ampoule, vibration gently, dissolve the freeze-drying thalline and be suspension.
The bacterial classification recovery: draw 0.5ml with disposable sterilized injector, on the recovery culture medium flat plate that evenly point sample prepares in embodiment 6, then aseptic glass stick is smeared bacterium liquid to be evenly distributed.The recovery substratum that connects flavus is placed in to 35 ℃ of constant incubators and cultivates 48h, 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 reaches and meets 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 sterilizing is cooling, a small amount of bacterium liquid of picking, make aforementioned
the slant medium of flavus bacterial classification preservationbe the Z-shaped line on inclined-plane, cover sterilized ventilative tampon, then be placed in 35 ℃ of constant incubators and cultivate 36h.After growing more mycelia, be placed in 4 ℃ of antistaling cabinets and preserve, every two months, according to present method, go down to posterity once.
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 of having recovered in flavus, then intensive line on a plurality of recovery substratum, be placed in 35 ℃ of constant incubators and cultivate 48h.Cover with the agar plate of mycelia, as providing fermentation required spore.
The preparation of spore liquid for the flavus fermentation: in Biohazard Safety Equipment, draw the 2ml stroke-physiological saline solution, be expelled on recovery flavus agar plate, then use the careful scraping mycelia of aseptic glass stick, draw bacterium liquid, be placed in the 250ml Erlenmeyer flask, then use the stroke-physiological saline solution dilution, until in bacterium liquid, aspergillus spore density is 1.0 * 10
6cfu/ml.
Draw 2.5ml aspergillus spore liquid with disposable syringe, the solid medium that slowly point sample prepares in embodiment 10 is used aseptic glass stick to mix simultaneously, covers the ventilative tampon of sterilizing, is placed in 35 ℃ of constant incubators and cultivates.Simultaneously, place a basin tap water in incubator, towel one end is placed in to water, an end is suspended on rack, forms cascade, and the relative humidity of controlling in incubator of take is 80～90%.
Humidity in every day detection record incubator, and flavus fermentation state in Erlenmeyer flask.
At interval of 3 days; in Biohazard Safety Equipment, use aseptic glass stick that fermention medium is stirred; spray the 2.5ml growth simultaneously and promote nutritive medium (being made by embodiment 2); with the loss of nutritive substance and moisture in the afterfermentation process, guarantee that Aspergillus flavus high vigor on the solid fermentation substratum grows and shorten incubation time 2～3 days.After paving fermention medium, cover aseptic tampon, be placed in 35 ℃ of constant incubators and continue to cultivate.
Through the fermentation culture of 12～14 days, in Erlenmeyer flask, substratum thoroughly fermented, and the mycelia color is deep green, and fermentation culture finishes.By culture in 121～123 ℃, 0.12MPa, sterilizing 20 min, then dry to pulverize, the culture that contains aflatoxin B1 prepares complete.
Detect the content of aflatoxin B1 in fermention medium by 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 the solid fermentation substratum of embodiment 9,11,12, 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 adopted is that embodiment 6 makes, the solid medium adopted is made by embodiment 10, difference is: the flavus fermenting process adopts respectively the growth of embodiment 1,3,4 to promote that nutritive medium is sprayed, 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 preferably scheme of the present invention, not the present invention is done to 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 (3)
1. the fermentation process of an aflatoxin B1, comprise bacterial classification recovery and two steps of solid fermentation cultivation, adopts the flavus bacterial classification to be fermented, and it is characterized in that comprising the steps:
A, employing recovery substratum carry out the bacterial classification recovery,
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 phosphate, 0.01g/L five aqueous ferrous sulfates, surplus is water, and the initial pH of substratum is 5.5～7.0;
B, employing solid fermentation substratum carry out the solid fermentation cultivation,
Described each components based on weight percentage of solid fermentation substratum comprises: 60～80% long-grained nonglutinous rices, and 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%.
2. fermentation process according to claim 1 is characterized in that: in described solid fermentation substratum, described long-grained nonglutinous rice, dregs of beans and wheat bran, through pulverizing, are crossed the accurate sub-sieve of 20～40 target.
3. fermentation process according to claim 1, it is characterized in that: the preparation method of described wheat bran vat liquor is as follows: get 1 unit weight wheat bran and be placed in clean beaker, then the 4 unit weight distilled water of annotating, after wheat bran and water are fully mixed, be placed in 37 ℃ of thermostat water baths, soak 10～12h, get supernatant liquor and be positioned in centrifuge tube, 2000～3000rpm, centrifugal 5～10 minutes, the supernatant liquor in centrifuge tube is drawn in clean beaker, the wheat bran extract prepares complete again.
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