CN104830701A - Preparation method of trametes versicolor fermentation liquor and application of trametes versicolor fermentation liquor in degrading aflatoxin B1 - Google Patents
Preparation method of trametes versicolor fermentation liquor and application of trametes versicolor fermentation liquor in degrading aflatoxin B1 Download PDFInfo
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Abstract
The invention relates to microbial fermentation and in particular relates to a preparation method of trametes versicolor fermentation liquor and the application of the trametes versicolor fermentation liquor in degrading aflatoxin B1. The trametes versicolor PTrametes versicolor CICC 14001 is inoculated to a sterile culture medium containing a carbon source, a nitrogen source, inorganic salts and trace elements and fermented into the fermentation liquor under the condition of aeration; the trametes versicolor fermentation liquor prepared by use of the method can be applied to degrading the aflatoxin B1. The preparation method is used for solving the technical problems of the existing method, such as demand on specific conditions and materials, high labor intensity, incomplete detoxification, great damage to treated materials, demand on drastic conditions, secondary pollution always caused by generation of chemical residues, and has the advantages of simple and convenient process and high safety; besides, the trametes versicolor fermentation liquor is used for degrading the aflatoxin B1, and the degradation speed is high and the degradation rate is high.
Description
Technical field
The present invention relates to fermentable, refer to that a kind of preparation method of Trametes versicolor fermented liquid and Trametes versicolor fermented liquid are at aflatoxin degradation B especially
1in application.
Background technology
Aflatoxin (Afiatoxin, AFT) is the extremely strong microbial metabolism material of a class toxicity, to the mankind and animal injury very big, especially based on the destruction to humans and animals liver organization, liver cancer time serious, can be caused even dead.Aflatoxin mainly results from Aspergillus flavus and Aspergillus parasiticus bacterium, and some aspergillus, mould, Mucor, fusarium, head mold, streptomycete and actinomycetes etc. also can produce this toxin in addition.Aflatoxin is very wide in occurring in nature distribution, with more common in peanut, corn, soybean, grain and goods thereof.After aflatoxin enters body, the amount in liver, compared with being high in other histoorgans, illustrates that liver is maximum by aflatoxin harm.New research shows that aflatoxin is all relevant with the synthesis of its arrestin matter to humans and animals Health hazard.The synthesis of its interfere information RNA and DNA, and then interference cell protein synthesis, cause animal systemic injury.Further research shows that the damaging effect of aflatoxin to the mankind is divided into directly and indirect two kinds, and directly the grain group food taken in containing aflatoxin is direct effect to the murder by poisoning of people; Aflatoxin also the mode of food chain can shift (meat, milk etc.) and at people's cylinder accumulation, causing healthy harm is indirect action.
In several aflatoxin, AFB
1toxicity the strongest, maximum to the harm of people.The symptoms such as the clinical manifestation at initial stage is had a stomach upset, appetite stimulator, Nausea and vomiting, abdominal distension, hepatic region tenderness, severe patient generation hepatitis, oedema, liver cirrhosis, stupor, so that twitch dead.Aflatoxin, once enter in humans and animals body, can be collected at each organ (especially liver).It can not be decomposed, and long time can only be leaned on to drain, and therefore, aflatoxin in vivo retention time is grown and endangered large.Its harm has caused worldwide attention.
At present to aflatoxin prophylactico-therapeutic measures and detoxification, be mainly divided into three major types, comprise physics, chemistry and biological detoxication and disintoxication.Physical comprises the methods such as absorption, radiotreatment, thermal treatment, the extracting of washing liquid phase; Chemical method is mainly highly basic, strong acid and strong oxidizer process; Biological process is biological enzyme preparation degrades effect.Because physical method needs specified conditions and material, labour intensity is large, and materials are many, and condition is harsh, sometimes has destruction and loss effect to the nutritive ingredient of treated object, and detoxification is incomplete, and application is restricted, and is difficult to realize scale operation.Chemical method medicine used has comparatively havoc effect to process material, and requirement condition is violent, often produces chemical residual, causes secondary pollution.Biological detoxication method be a kind of efficient, without harm detoxification, because it divides toxolysin and noresidue in a mild condition, do not destroy nutritive ingredient, do not produce the reasons such as secondary pollution, be subject to people at present and pay attention to widely being also the Main way of research simultaneously.
Microbiological deterioration aflatoxin mainly adopts bacterium and fungi, and external great mass of data research display, fungi is many, and bacterium report is less.
Trametes versicolor (being commonly called as Coriolous Dersicolor (Fr.) Quel fungus) is one of fungi of most pharmaceutical use, is rich in krestin in its fermented liquid.Krestin has heat-clearing, removing toxic substances, anticancer and effect of protecting the liver, is good human body immunomodulator.In recent years, krestin has made multiple drug form and healthcare products circulate in market.Coriolous Dersicolor (Fr.) Quel fungus liquid submerged fermentation also can produce multiple zymin and protein, and wherein laccase, proteolytic enzyme, peroxidase, amylase etc. are Major Enzymes.At present, laccase aflatoxin degradation B
1research has been reported, and degradation rate is higher, but not yet has launch products.
Summary of the invention
The object of the present invention is to provide the preparation method of Trametes versicolor fermented liquid (rainbow conk) and Trametes versicolor (rainbow conk) fermented liquid at aflatoxin degradation B
1in application.
Overall technology design of the present invention is:
The preparation method of Trametes versicolor fermented liquid, is inoculated in Trametes versicolor PTrametes versicolor CICC14001 and carries out aerobic fermentation in the aseptic culture medium containing carbon source, nitrogenous source, inorganic salt and trace element and make fermented liquid;
Fermention medium is made up of the component of following mass percent:
Bagasse 1.9%-2.3%, wheat bran 0.1%-0.12%, soybean cake powder 0.1%-0.13%, glucose 0.5%-0.7%, fructose 0.5%-0.7%, ammonium sulfate 0.08%-0.11%, potassium primary phosphate 0.15%-0.2%, magnesium sulfate 0.04%-0.06%, vitamins B
10.00001%-0.00003%, methyl catechol 0.00013%, the volume ratio that trace element solution accounts for aseptic culture medium is 0.07%-0.12%, and surplus is sterilized water, pH=4.5;
The processing condition of aerobic fermentation are as follows:
Inoculum size is volume ratio=6-8: 100, temperature 29 DEG C-31 DEG C, air flow 1: 0.7-1.2vvm, and pressure is 0.03Mpa-0.05Mpa, and culture cycle is for being not less than 220 hours.
The control of fermentation termination, except time factor, should, in conjunction with 722 visible spectrophotometers, adopt Dan white matter Nong Du≤2.5mg/ml in Coomassie Brilliant Blue monitoring fermented liquid to determine fermentation termination.
The Trametes versicolor fermented liquid adopting aforesaid method to prepare is at degraded aflatoxin degradation B
1in application.
Concrete technical conceive of the present invention also has:
For the needs of suitability for industrialized production, preferred technical scheme is, described Trametes versicolor bacterial classification access aseptic culture medium before through enlarged culturing.
More preferred technical scheme is, described enlarged culturing comprises the preparation of slant strains, the preparation of first order seed and the preparation of secondary seed.
In the preparation of described slant strains, substratum adopts the one in potato culture or dextrose culture-medium, and temperature is 29 DEG C-31 DEG C, culture cycle 4-5 days.
The preparation of described first order seed comprises following processing step:
Slant strains being inoculated into by the inoculum size that every inclined-plane connects 3 bottles is equipped with in primary-seed medium triangular flask, under 30 DEG C of conditions, shaking culture is not less than 90 hours and makes first order seed, loading amount is that the triangular flask of 1000ml loads 200ml primary-seed medium, have a certain amount of mycelium suspended to estimate in primary-seed medium, uniformity sturdy with microscopy mycelial growth cultivates terminal for first order seed.
The preparation of described secondary seed comprises following processing step:
Volume ratio=10 according to first order seed and secondary seed medium: the ratio of 100, be equipped with after first order seed being accessed sterilizing in the fermentor tank of secondary seed medium, loading amount is volume ratio=60-70:100, it is 29 DEG C-31 DEG C in temperature, air flow is 1: 0.5-0.7vvm, pressure is that the aerated culture carrying out being not less than 72 hours under the condition of 0.03Mpa-0.05Mpa makes secondary seed, have a large amount of mycelium suspended to estimate in liquid nutrient medium, uniformity sturdy with microscopy mycelial growth cultivates terminal for secondary seed.
Described primary-seed medium and secondary seed medium adopt the raw material of following mass fraction to form:
Glucose 0.9%-1.1%, yeast extract paste 0.1%-0.12%, ammonium sulfate 0.08%-0.11%, potassium primary phosphate 0.15%-0.2%, magnesium sulfate 0.04%-0.06%, urea 0.012%-0.015%, the volume ratio that trace element solution accounts for seed culture medium is 0.07%-0.12%, surplus is water, pH=4.5.
Described trace element solution is adopted and is prepared with the following method: calcium chloride 100mg, ferrous sulfate 100mg, zinc sulfate 20mg, copper sulfate 25mg, is settled to 1000ml.
The liquid that the fermented liquid adopting method of the present invention to obtain obtains after adopting ordinary method to carry out solid-liquid separation is Trametes versicolor fermentation liquor preparation, the method of solid-liquid separation, including, but not limited to adopting the prior aries such as sheet frame separation, whizzer separation, does not all depart from technical spirit of the present invention.
Substantive distinguishing features acquired by the present invention and significant technical progress are:
1, present invention process is easy, ripe, only needs submerged fermentation equipment, and namely the liquid after solid-liquid separation can be used for removing toxic substances, preparation process three-waste free pollution.
2, the Trametes versicolor used in the present invention, not in pathogenic species scope, safe and reliable.
3, in the present invention, the main raw material of substratum is bagasse and soybean cake powder, has safe, nontoxic, green feature.
4, this Trametes versicolor fermented liquid is used for aflatoxin degradation B
1, degradation speed is fast, and degradation rate is high, mild condition, and selectivity is good, stable performance.For polluting AFB under natural condition of degrading
1the feed of (content≤100 μ g/kg), degradation rate>=77%.
5, the product adopting the present invention to prepare uses simple, and secondary can not be caused to endanger.
Embodiment
Below in conjunction with embodiment, the invention will be further described; but should not be construed as limitation of the invention; the content that protection scope of the present invention is recorded with claim is as the criterion, any according to the equivalent technical elements replacement done by specification sheets, does not all depart from protection scope of the present invention.
Embodiment 1
The preparation method of Trametes versicolor fermented liquid, is inoculated in Trametes versicolor PTrametes versicolor CICC14001 and carries out aerobic fermentation in the aseptic culture medium containing carbon source, nitrogenous source, inorganic salt and trace element and make fermented liquid;
Fermention medium is made up of the component of following mass percent:
Bagasse 1.9%, wheat bran 0.12%, soybean cake powder 0.1%, glucose 0.7%, fructose 0.5%, ammonium sulfate 0.1%, potassium primary phosphate 0.15%, magnesium sulfate 0.05%, vitamins B
10.00003%, methyl catechol 0.00013%, the volume ratio that trace element solution accounts for aseptic culture medium is 0.07%, and surplus is sterilized water, pH=4.5;
The processing condition of aerobic fermentation are as follows:
Inoculum size is volume ratio=8: 100, temperature 31 DEG C, air flow 1: 0.7vvm, and pressure is 0.04Mpa, and culture cycle is for being not less than 220 hours.
The control of fermentation termination, except time factor, should, in conjunction with 722 visible spectrophotometers, adopt Dan white matter Nong Du≤2.5mg/ml in Coomassie Brilliant Blue monitoring fermented liquid to determine fermentation termination.
Described Trametes versicolor bacterial classification access aseptic culture medium before through enlarged culturing.
Described enlarged culturing comprises the preparation of the preparation of slant strains, the preparation of first order seed and secondary seed.
In the preparation of described slant strains, substratum adopts the one in potato culture or dextrose culture-medium, and temperature is 30 DEG C, culture cycle 4 days.
The preparation of described first order seed comprises following processing step:
Slant strains being inoculated into by the inoculum size that every inclined-plane connects 3 bottles is equipped with in primary-seed medium triangular flask, under 30 DEG C of conditions, shaking culture makes first order seed in 90 hours, loading amount is that the triangular flask of 1000ml loads 200ml primary-seed medium, have a certain amount of mycelium suspended to estimate in primary-seed medium, uniformity sturdy with microscopy mycelial growth cultivates terminal for first order seed.
The preparation of described secondary seed comprises following processing step:
Volume ratio=10 according to first order seed and secondary seed medium: the ratio of 100, be equipped with after first order seed being accessed sterilizing in the fermentor tank of secondary seed medium, loading amount is volume ratio=70:100, it is 29 DEG C in temperature, air flow is 1: 0.7vvm, pressure is that the aerated culture carrying out being not less than 72 hours under the condition of 0.05Mpa makes secondary seed, have a large amount of mycelium suspended to estimate in liquid nutrient medium, uniformity sturdy with microscopy mycelial growth cultivates terminal for secondary seed.
Described primary-seed medium and secondary seed medium adopt the raw material of following mass fraction to form:
Glucose 1%, yeast extract paste 0.1%, ammonium sulfate 0.11%, potassium primary phosphate 0.15%, magnesium sulfate 0.06%, urea 0.012%, the volume ratio that trace element solution accounts for seed culture medium is 0.07%, and surplus is water, pH=4.5.
Described trace element solution is adopted and is prepared with the following method: calcium chloride 100mg, ferrous sulfate 100mg, zinc sulfate 20mg, copper sulfate 25mg, is settled to 1000ml.
The fermented liquid adopting the method for the present embodiment to obtain adopts ordinary method to carry out solid-liquid separation, comprising but be not limited to and adopt sheet frame separation, whizzer separation etc.
Embodiment 2
The difference of the present embodiment and embodiment 1 is:
Fermention medium is made up of the component of following mass percent:
Bagasse 2.1%, wheat bran 0.11%, soybean cake powder 0.11%, glucose 0.5%, fructose 0.7%, ammonium sulfate 0.08%, potassium primary phosphate 0.17%, magnesium sulfate 0.04%, vitamins B
10.00002%, methyl catechol 0.00013%, the volume ratio that trace element solution accounts for aseptic culture medium is 0.09%, and surplus is sterilized water, pH=4.5;
Inoculum size is volume ratio=7: 100, temperature 29 DEG C, air flow 1: 0.9vvm, and pressure is 0.03Mpa, and culture cycle is for being not less than 220 hours.
In the preparation of described slant strains, culture temperature is 29 DEG C, culture cycle 5 days.
In the preparation of described secondary seed:
Loading amount is volume ratio=60:100, is 30 DEG C in temperature, and air flow is 1: 0.5vvm, and pressure is that the aerated culture carrying out being not less than 72 hours under the condition of 0.03Mpa makes secondary seed.
Described primary-seed medium and secondary seed medium adopt the raw material of following mass fraction to form:
Glucose 0.9%, yeast extract paste 0.11%, ammonium sulfate 0.09%, potassium primary phosphate 0.17%, magnesium sulfate 0.05%, urea 0.013%, the volume ratio that trace element solution accounts for seed culture medium is 0.08%, and surplus is water, pH=4.5.
Embodiment 3
The difference of the present embodiment and embodiment 1 is:
Fermention medium is made up of the component of following mass percent:
Bagasse 2.3%, wheat bran 0.10%, soybean cake powder 0.13%, glucose 0.6%, fructose 0.6%, ammonium sulfate 0.11%, potassium primary phosphate 0.2%, magnesium sulfate 0.06%, vitamins B
10.00001%, methyl catechol 0.00013%, the volume ratio that trace element solution accounts for aseptic culture medium is 0.12%, and surplus is sterilized water, pH=4.5;
Inoculum size is volume ratio=6: 100, temperature 30 DEG C, air flow 1: 1.2vvm, and pressure is 0.05Mpa, and culture cycle is for being not less than 220 hours.
In the preparation of described slant strains, culture temperature is 31 DEG C, culture cycle 4.5 days.
The preparation of described secondary liquid seed comprises following processing step:
Volume according to first order seed: volume ratio=10 of secondary seed medium: first order seed is accessed the secondary seed medium after sterilizing by 100, loading amount is volume ratio=65:100, it is 28 DEG C in temperature, air flow is 1: 0.6vvm, and pressure is that the aerobic fermentation carrying out being not less than 72 hours under the condition of 0.04Mpa makes secondary liquid seed.
Described primary-seed medium and secondary seed medium adopt the raw material of following mass fraction to form:
Glucose 1.1%, yeast extract paste 0.12%, ammonium sulfate 0.08%, potassium primary phosphate 0.2%, magnesium sulfate 0.04%, urea 0.015%, the volume ratio that trace element solution accounts for seed culture medium is 0.12%, and surplus is water, pH=4.5.
The addition of the Trametes versicolor fermentation liquor preparation that applicant prepares with regard to above-described embodiment and aflatoxin degradation B
1ability carry out the toxin aqueous solution and corn feed detoxification experiment respectively.Process of the test and result as follows:
1, process of the test
After the Trametes versicolor fermentation liquor preparation getting corn feed 10 ‰ (W:W) adds the dilution of certain water gaging, more respectively with containing 97.7 μ g/kg AFBs
1corn feed mixing, with corn feed surface wettability as well, under being enclosed in 37 DEG C of conditions, be incubated more than 48 hours, according to AFB in GB/17480-2008 feed
1measuring method-enzyme-linked immunosorbent assay, to measure after degraded AFB in feed
1content, and calculate degradation rate.
2, test-results
Detoxification experiment 1
The Trametes versicolor fermentation liquor preparation utilizing the method for embodiment 1-3 to obtain is respectively 2 ‰, 6 ‰, 10 ‰ ratios by mass percentage and adds different concns AFB to
1in the aqueous solution of (content is at 5-120 μ g/L), fully mix, under 37 DEG C of conditions, be incubated more than 48 hours, for aflatoxin degradation B
1toxicity.Degraded toxin test result is as table 1.
AFB in table 1 Trametes versicolor fermentation liquor preparation degradation water solution
1result
Note: Biao Zhong result data unit is μ g/l.
Illustrated, at AFB by test-results in table 1
1content is in 11.0 μ g/l solution, adds 2 ‰ Trametes versicolor fermentation liquor preparations, to AFB
1degradation rate>=84.5%; At AFB
1content is in 54.3 μ g/l solution, adds 6 ‰ Trametes versicolor fermentation liquor preparations, to AFB
1degradation rate>=85.1%; At AFB
1content is in 97.7 μ g/l solution, adds 10 ‰ Trametes versicolor fermentation liquor preparations, to AFB
1degradation rate>=86.5%.In a word, AFB in the aqueous solution
1during the μ g/l of content≤100, add≤10 ‰ Trametes versicolor fermentation liquor preparations, contratoxin degradation rate can reach more than 80%.
Detoxification experiment 2
Equally, get 3 ‰ of corn feed respectively, 7 ‰, 10 ‰ ratios (W:W) Trametes versicolor fermentation liquor preparation, after adding the dilution of certain water gaging, more respectively with containing 11.0 μ g/kg, 54.3 μ g/kg, 97.7 μ g/kg AFBs
1corn feed mixing, with corn feed surface wettability as well, under being enclosed in 37 DEG C of conditions, be incubated more than 48 hours, measurement result is as table 2.
AFB in table 2 Trametes versicolor fermentation liquor preparation degrading maize feed
1measurement result
Note: Biao Zhong result data unit is μ g/kg.
Drawn by table 2 result: adopt Trametes versicolor fermentation liquor preparation, AFB in degrading maize feed
1content, when 11.0 μ g/kg, adds Trametes versicolor fermentation liquor preparation amount 3 ‰, its removing toxic substances rate>=80.9%; AFB
1content, when 54.3 μ g/kg, adds Trametes versicolor fermentation liquor preparation amount 7 ‰, its removing toxic substances rate>=65.0%; AFB
1content, when 97.7 μ g/kg, adds Trametes versicolor fermentation liquor preparation amount 10 ‰, its removing toxic substances rate>=77.0%
Adopt Trametes versicolor fermentation liquor preparation, aflatoxin degradation B
1toxicity, compared by detoxification experiment 1,2 and draw, detoxifying effect is better than detoxifying effect in corn feed in aqueous.
Claims (9)
1. the preparation method of Trametes versicolor fermented liquid, is characterized in that Trametes versicolor PTrametesversicolor CICC14001 to be inoculated in and carries out aerobic fermentation in the aseptic culture medium containing carbon source, nitrogenous source, inorganic salt and trace element and make fermented liquid;
Fermention medium is made up of the component of following mass percent:
Bagasse 1.9%-2.3%, wheat bran 0.1%-0.12%, soybean cake powder 0.1%-0.13%, glucose 0.5%-0.7%, fructose 0.5%-0.7%, ammonium sulfate 0.08%-0.11%, potassium primary phosphate 0.15%-0.2%, magnesium sulfate 0.04%-0.06%, vitamins B
10.00001%-0.00003%, methyl catechol 0.00013%, the volume ratio that trace element solution accounts for aseptic culture medium is 0.07%-0.12%, and surplus is sterilized water, pH=4.5;
The processing condition of aerobic fermentation are as follows:
Inoculum size is volume ratio=6-8: 100, temperature 29 DEG C-31 DEG C, air flow 1: 0.7-1.2vvm, and pressure is 0.03Mpa-0.05Mpa, and culture cycle is for being not less than 220 hours.
2. the preparation method of Trametes versicolor fermented liquid according to claim 1, is characterized in that described Trametes versicolor bacterial classification before access aseptic culture medium through enlarged culturing.
3. the preparation method of Trametes versicolor fermented liquid according to claim 2, is characterized in that described enlarged culturing comprises the preparation of the preparation of slant strains, the preparation of first order seed and secondary seed.
4. the preparation method of Trametes versicolor fermented liquid according to claim 3, it is characterized in that in the preparation of described slant strains, substratum adopts the one in potato culture or dextrose culture-medium, temperature is 29 DEG C-31 DEG C, culture cycle 4-5 days.
5. the preparation method of Trametes versicolor fermented liquid according to claim 3, is characterized in that the preparation of described first order seed comprises following processing step:
Slant strains being inoculated into by the inoculum size that every inclined-plane connects 3 bottles is equipped with in primary-seed medium triangular flask, under 30 DEG C of conditions, shaking culture is not less than 90 hours and makes first order seed, loading amount is that the triangular flask of 1000ml loads 200ml primary-seed medium, have a certain amount of mycelium suspended to estimate in primary-seed medium, uniformity sturdy with microscopy mycelial growth cultivates terminal for first order seed.
6. the preparation method of Trametes versicolor fermented liquid according to claim 3, is characterized in that the preparation of described secondary seed comprises following processing step:
Volume ratio=10 according to first order seed and secondary seed medium: the ratio of 100, be equipped with after first order seed being accessed sterilizing in the fermentor tank of secondary seed medium, loading amount is volume ratio=60-70:100, it is 29 DEG C-31 DEG C in temperature, air flow is 1: 0.5-0.7vvm, pressure is that the aerated culture carrying out being not less than 72 hours under the condition of 0.03Mpa-0.05Mpa makes secondary seed, have a large amount of mycelium suspended to estimate in liquid nutrient medium, uniformity sturdy with microscopy mycelial growth cultivates terminal for secondary seed.
7. the preparation method of Trametes versicolor fermented liquid according to claim 3, is characterized in that described primary-seed medium and secondary seed medium adopt the raw material of following mass fraction to form:
Glucose 0.9%-1.1%, yeast extract paste 0.1%-0.12%, ammonium sulfate 0.08%-0.11%, potassium primary phosphate 0.15%-0.2%, magnesium sulfate 0.04%-0.06%, urea 0.012%-0.015%, the volume ratio that trace element solution accounts for seed culture medium is 0.07%-0.12%, surplus is water, pH=4.5.
8. the preparation method of the Trametes versicolor fermented liquid according to claim 1 or 7, is characterized in that described trace element solution is adopted and prepares with the following method: calcium chloride 100mg, ferrous sulfate 100mg, zinc sulfate 20mg, copper sulfate 25mg, is settled to 1000ml.
9. the Trametes versicolor fermented liquid prepared of method according to claim 1 is at degraded aflatoxin degradation B
1in application.
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| CN108034690A (en) * | 2017-06-28 | 2018-05-15 | 山东省花生研究所 | A kind of method that aflatoxin degrades and synchronously prepares peanut protein peptide in peanut meal |
| CN108703313A (en) * | 2018-05-29 | 2018-10-26 | 广东日可威富硒食品有限公司 | One kind nourishes the liver to improve visual acuity compound bacteria vermicelli and preparation method thereof |
| CN108967833A (en) * | 2018-08-02 | 2018-12-11 | 合肥工业大学 | A kind of complex enzyme formulation and preparation method thereof for removing aflatoxin |
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