CN108094712B - Degradation method of aflatoxin B1 in distiller's grains - Google Patents

Degradation method of aflatoxin B1 in distiller's grains Download PDF

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CN108094712B
CN108094712B CN201711472042.5A CN201711472042A CN108094712B CN 108094712 B CN108094712 B CN 108094712B CN 201711472042 A CN201711472042 A CN 201711472042A CN 108094712 B CN108094712 B CN 108094712B
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culture solution
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CN108094712A (en
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常敬华
何志明
栾雨婷
刘聪
项莹
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Liaoning Baijiali feed Sales Co.,Ltd.
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract

Aflatoxin B in distiller's grains1Belonging to the technical field of feed safety, the degradation method comprises the following steps: culturing the straw mushroom strain by a solid-liquid culture medium to form a straw mushroom culture solution, culturing the straw mushroom culture solution in white spirit vinasse, and screening a target object meeting laccase activity for later use; culturing Aspergillus flavus culture solution, adding the Aspergillus flavus culture solution into the distiller's grains according to the proportion for culturing, and completing inoculation; adding culture components into inoculated distiller's grains, mixing, inoculating and screening straw mushroom culture solution, mixing, culturing under controlled culture condition to obtain aflatoxin B1Measured, aflatoxin B1The degradation rate reaches above 86.02 percent. Aflatoxins B in distillers' grains of the invention1The degradation method can obviously reduce the aflatoxin B in the distiller's grains1The content of the compound can be used for reducing aflatoxin B of livestock and poultry1The intake of the feed improves the product titer, and has important significance in reducing economic loss and health risks of people and animals.

Description

Aflatoxin B in distiller's grains1By a degradation method
The technical field is as follows:
the invention belongs to the technical field of feed safety, and particularly relates to aflatoxin B in distiller's grains1The degradation method of (1).
Background art:
the aflatoxin is a compound with very similar chemical structure generated by aspergillus flavus, aspergillus parasiticus and other moulds, and is the most polluted grain and oil crops, food and feed in China and worldThe main mycotoxins have strong toxicity and carcinogenicity, and the most important of the mycotoxins is aflatoxin B1(AFB1) The compound has been classified as a class I carcinogen by the world health organization, and seriously harms human and animal health. And its secondary metabolite residues in animal food can potentially pose a hazard to human health through the food chain. For example aflatoxin MlIs aflatoxin B1Major metabolites in the mammalian body. It is present in the milk, liver, eggs and other edible parts of animals, and is commonly found in milk, and it has strong toxicity and carcinogenicity. In actual production, the AFB in the feed is strictly controlled1The content of the (D) has direct practical significance for guaranteeing the safety of people and livestock and reducing economic loss.
The distiller's grains are residues of alcohol extracted from sorghum, wheat, corn, etc. by fermentation and distillation, and the residual raw materials contain most of protein, fat, calcium, phosphorus, etc. The fodder produced by the distiller's grains can save more than 350 ten thousand tons of fodder each year. However, the fresh vinasse has large water content and is rich in abundant nutrient elements, so that the mold, especially aflatoxin, is easy to breed. In the brewing process, 1 time of vinasse by-products are generated from 3 times of cereal raw materials, and if the raw materials are polluted, aflatoxin in the white spirit vinasse is concentrated to 3 times of the raw materials, which seriously harms the safety of livestock and poultry. However, aflatoxins are stable in nature and cannot be removed by common processing methods. Therefore, the aflatoxin in the white spirit vinasse is effectively controlled and eliminated, and a safe, efficient and environment-friendly detoxification method is urgently needed.
The invention content is as follows:
the invention aims to overcome the defects in the prior art and provide aflatoxin B in distiller's grains1The method provided by the invention can be used for degrading the aflatoxin B in the distiller's grains1Obviously reduces the biological value of the vinasse and increases the biological value of the vinasse.
In order to achieve the purpose, the invention adopts the following technical scheme:
aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium for culture to finish culture, wherein: the culture conditions are as follows: the culture temperature is 25-40 ℃, and the culture time is 3-8 d;
(2) liquid medium culture:
cooling the PD liquid culture medium to room temperature, inoculating the straw mushroom strains cultured by the solid culture medium into the PD liquid culture medium, and culturing to form straw mushroom culture solution for later use; wherein: the culture conditions are as follows: the culture temperature is 25-40 ℃, the culture time is 3-8 d, and the spore concentration of the straw mushroom culture solution is 105～107cfu/mL；
In the step 1(2), the preparation process of the PD liquid culture medium comprises the following steps: 250g of peeled potato, 20g of glucose and 1000ml of distilled water are mixed, the pH is adjusted to 6-7, and a PD liquid culture medium is prepared.
In the step 1(2), the PD liquid culture medium is sterilized in advance, and the sterilization process comprises the following steps: subpackaging PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volume, sealing the tube opening, and sterilizing with high pressure steam sterilization pot at 121 deg.C for 30 min.
In the step 1(2), the PD liquid culture medium is placed in a super clean workbench platform for cooling, the super clean workbench is sterilized in advance, and the treatment process comprises the following steps: wiping the clean bench surface with alcohol cotton soaked in 75% alcohol, and sterilizing with ultraviolet for 30 min.
In the step 1(2), the liquid culture medium is cultured in a constant-temperature shaking incubator.
(3) Screening a white spirit vinasse Chinese herbal mushroom culture solution:
adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture, uniformly mixing, adding distilled water into the cultured mixture, uniformly mixing to form a diluted mixture, measuring the laccase activity of the supernatant after centrifugation, screening the diluted mixture with the laccase activity higher than 1800U/L, and further screening the straw mushroom culture solution meeting the activity requirement of the cultured diluted laccase for later use;
in the step 1 and the step 3, the ratio of the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is (10-50): 1 in g/mL.
In the step 1(3), the addition amount of the distilled water is as follows according to the mixture ratio, the mass of the cultured mixture solid is as follows: distilled water volume 1: (1-4) in g/mL.
In the step 1(3), the centrifugation process is as follows: placing the cultured mixture into a 50mL centrifuge tube, adding distilled water according to the proportion to dissolve, uniformly mixing by using a homogenizer, and centrifuging.
In the step 1(3), the centrifugation conditions are as follows: rotation speed 4000r min-1And centrifuging for 10 min.
In the step 1 and the step 3, the laccase activity test method is an ABTS method.
In the step 1(3), one laccase unit (U) is the amount of laccase required for 1min to convert 1 mu mol of ABTS.
In the step 1(3), the culture conditions after the straw mushroom culture solution is added with the distiller's grains are as follows: the culture temperature is 25-35 ℃, and the culture time is 3-7 d.
In the step 1(3), the distiller's grains are sterilized before inoculation of the straw mushroom culture solution, the sterilization operation is performed in a high-pressure steam sterilization pot, the sterilization temperature is 121 ℃, and the sterilization time is 60 min.
Step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
preparing Aspergillus flavus suspension from strain producing toxin Aspergillus flavus, adding Aspergillus flavus suspension into PD culture solution, and culturing until spore concentration is 105～107cfu/mL to obtain an aspergillus flavus culture solution;
in the step 2(1), the preparation process of the aspergillus flavus suspension comprises the following steps: inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3-7 d at 25-35 ℃, activating, washing with sterile water, and preparing the aspergillus flavus suspension.
In the step 2(1), the culture process of the aspergillus flavus culture solution is carried out in a constant-temperature shaking incubator, and the culture conditions are as follows: the culture temperature was 30 ℃ and the culture time was 7d, and the number of shaking revolutions was 200 rpm.
(2) Inoculating distiller's grains with aspergillus flavus for producing toxin:
adding an aspergillus flavus culture solution into the distiller's grains of distillers for culture, and completing inoculation through sterilization treatment, wherein: the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added according to the proportion of (5-20): 1, unit g: ml;
in the step 2(2), the culture conditions after the distiller's grains are added into the aspergillus flavus culture solution are as follows: 7d at 28 ℃; the sterilization conditions are as follows: 121 ℃ and 60 min.
In the step 2(2), the distiller's grains are sterilized before the inoculation of the aspergillus flavus for producing the toxin, the sterilization operation is carried out in a high-pressure steam sterilization pot, the sterilization temperature is 121 ℃, and the sterilization time is 60 min.
Step 3, aflatoxin B in distiller's grains1Degradation of
Adding culture components into inoculated distiller's grains, mixing, inoculating and screening straw mushroom culture solution, mixing, and culturing to obtain aflatoxin B in distiller's grains1Degradation of (2); the culture medium comprises carbon sources, nitrogen sources, inorganic salts and trace elements, wherein the solid mass of the distiller's grains after inoculation and the liquid volume ratio of the screened straw mushroom culture solution are (10-50): 1 in g/mL, said culture conditions being: the temperature is 25-35 ℃ and the time is 8-20 d.
In the step 3, the inoculated distiller's grains are sterilized in advance.
In the step 3, the culture conditions are preferably: the temperature was 28 ℃ for 14 d.
And in the step 3, the inoculated white spirit vinasse is subjected to pH and water content adjustment treatment, wherein the pH is adjusted to 4.5-7, and the water content is adjusted to 55-70%.
In the step 3, the pH value of the inoculated white spirit vinasse is adjusted to be 6, and the water content is adjusted to be 65%.
In the step 3, the carbon source is one of glucose, soluble starch, sucrose or maltose, and the adding mass of the carbon source is 1-4% of the mass of the distiller's grains after inoculation;
the nitrogen source is peptone, beef extract, KNO3Yeast powder, NH4NO3The adding mass of the nitrogen source is 1-2.5% of the mass of the white spirit vinasse after inoculation;
the inorganic salt is KH2PO4，CaCl2Or MgSO 24The inorganic salt is added in an amount which is 0.05-0.35% of the mass of the distiller's grains after inoculation;
the microelement is CuSO4，ZnSO4，MnSO4The addition mass of the trace elements is 0.1-0.4 mg/kg of the mass of the white spirit vinasse after inoculation.
In the step 3, the addition quality of the culture medium is preferably as follows:
the adding mass of the carbon source is 3 percent of the mass of the white spirit vinasse after inoculation;
the adding mass of the nitrogen source is 1.5 percent of the mass of the white spirit vinasse after inoculation;
the adding mass of the inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation;
the adding mass of the trace elements is 0.2mg/kg of the mass of the distiller's grains after inoculation.
The straw mushroom is used for degrading aflatoxin B in distiller's grains of white spirit1Application in the field of application.
The invention has the beneficial effects that:
the aflatoxin B in the distiller's grains of the invention1The degradation method can obviously reduce the aflatoxin B in the distiller's grains1The content of the compound can be used for reducing aflatoxin B of livestock and poultry1The intake of the feed improves the product titer, and has important significance in reducing economic loss and health risks of people and animals.
Description of the drawings:
FIG. 1 is a 50. mu.g/L AFB1HPLC (high performance liquid chromatography) spectrum of the standard substance;
FIG. 2 shows 20. mu.g/L AFB1HPLC (high performance liquid chromatography) spectrum of the standard substance;
FIG. 3 is 10. mu.g/L AFB1Standard articleHPLC profile;
FIG. 4 is 1. mu.g/L AFB1HPLC (high performance liquid chromatography) spectrum of the standard substance;
FIG. 5 is 0.5. mu.g/L AFB1HPLC (high performance liquid chromatography) spectrum of the standard substance;
FIG. 6 shows aflatoxin B in distillers grains of example 21In the degradation method of (1), AFB in the inoculated distiller's grains1HPLC profile of (a);
FIG. 7 is aflatoxin B in distillers grains of example 21In the degradation method, AFB in the degraded spirit lees1HPLC profile of (a).
The specific implementation mode is as follows:
the present invention will be described in further detail with reference to examples.
The experimental methods used in the previous experiments and examples described below are conventional methods unless otherwise specified.
The materials, reagents and the like used in the previous experiments and examples described below are commercially available unless otherwise specified.
Aflatoxin B used in the previous experiments and examples described below1(AFB1) And (3) standard substance: purchased from SIGMA, 50. mu.g/L AFB1The HPLC chromatogram of the standard is shown in FIG. 1, 20 μ g/L AFB1The HPLC chromatogram of the standard is shown in FIG. 2, 10 μ g/L AFB1The HPLC chromatogram of the standard is shown in FIG. 3, 1 μ g/L AFB1The HPLC chromatogram of the standard is shown in FIG. 4, and the HPLC chromatogram is 0.5 μ g/L AFB1The HPLC chromatogram of the standard is shown in FIG. 5;
the following earlier experiments and examples were bought from the Fuxin Sangou winery;
the following prophase experiments and AFB in examples 1-81The content determination method comprises the following steps:
drying distiller's grains inoculated with Aspergillus flavus and distiller's grains subjected to aflatoxin degradation at 60 deg.C for 12 hr, pulverizing about 20g of dried distiller's grains with pulverizer, and measuring water content and AFB of distiller's grains1The content and toxin extraction treatment method refers to GB/T30955-.
Chromatographic conditions are as follows: the chromatographic column is Venusil MP C18(5 μm, 4.6 mm. times.250 mm); the column temperature was 40 ℃; the mobile phase is methanol and water (V: V)53: 47); the flow rate is 0.8 mL/min; post-column photochemical derivatization: 254nm for photochemical derivitizer; detecting with fluorescence detector, exciting wavelength is 360nm, emission wavelength is 450nm, and sample amount is 20 μ L, and calculating aflatoxin B1And (4) content.
Earlier stage experiment 1, the optimal conditions for culturing the straw mushroom strains are determined:
inoculating the straw mushroom strain into a PDA plate culture medium for culturing, wherein the temperature is respectively set to be 25 ℃, 28 ℃ and 40 ℃, and the time is respectively set to be 3d, 6d, 7d and 8 d; mixing 250g of peeled potatoes, 20g of glucose and 1000ml of distilled water, adjusting the pH value to 6-7, preparing a PD liquid culture medium, subpackaging the PD liquid culture medium into a triangular flask to 2/3 of the volume of a bottle body, sealing a pipe orifice, sterilizing by using a high-pressure steam sterilization pot at the sterilization temperature of 121 ℃ for 30min, wiping the surface of an ultra-clean workbench by using alcohol cotton soaked by 75% alcohol, sterilizing by using ultraviolet rays for 30min, placing the sterilized PD liquid culture medium into the ultra-clean workbench, cooling to room temperature, inoculating straw mushroom strains cultured by using a solid culture medium into the PD liquid culture medium, placing the PD liquid culture medium into a constant-temperature shaking incubator, and culturing at 25 ℃, 28 ℃ and 40 ℃ for 3d, 6d, 7d and 8d respectively to form a straw mushroom culture solution;
taking distiller's grains, adjusting the pH to be 4.5, 6 and 7 respectively, adjusting the water content to be 55%, 65% and 70% respectively, inoculating the straw mushroom culture solution, culturing for 8d, 14d and 20d at 25 ℃, 28 ℃ and 35 ℃ respectively, finishing the culture, and observing the growth condition of hypha in the straw mushroom culture solution shows that the optimal culture condition of the straw mushroom strain is as follows: the solid culture temperature in the PDA plate culture medium is 28 ℃, the culture time is 6d, the liquid culture temperature in the PD liquid culture medium is 28 ℃, the culture time is 6d, the pH value of the distilled spirit grains is adjusted to be 6, the water content is adjusted to be 65%, the culture time of the straw mushroom culture solution in the distilled spirit grains is 14d, and the hypha in the straw mushroom strain grows most vigorously when the culture temperature is 28 ℃;
in the earlier stage experiment 2, the optimal carbon source, the optimal nitrogen source, the optimal trace elements and the corresponding addition quality are determined:
(1) inoculating Aspergillus flavus strain to PDA slant test tube culture medium, and culturing at 28 deg.CCulturing for 3 days, activating, washing with sterile water to obtain Aspergillus flavus suspension, adding Aspergillus flavus suspension into PD culture solution, culturing at 30 deg.C under 200rpm in a constant temperature shaking incubator for 7 days until spore concentration is 105cfu/mL to obtain an aspergillus flavus culture solution;
(2) taking distiller's grains, sterilizing, and adding aspergillus flavus culture solution, wherein the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are 10: 1, unit g: ml, culturing distiller's grains inoculated with Aspergillus flavus at 28 deg.C for 7d, sterilizing, and determining AFB1Content (c);
(3) adjusting the water content of the sterilized distiller's grains to 65 percent, adjusting the pH value to 6.0, respectively adding glucose, soluble starch, sucrose and maltose as unique external carbon sources, and setting 4 adding amounts which are 1 percent, 2 percent, 3 percent and 4 percent of the mass of the distiller's grains after determining the optimal carbon source; selecting yeast powder, peptone, beef extract, KNO3、NH4NO3As the only additional nitrogen source, after determining the optimal nitrogen source, setting 4 addition amounts which are respectively 1%, 1.5%, 2% and 2.5% of the mass of the vinasse; adding CaCl respectively2、MgSO4、 KH2PO4After determining the optimal inorganic salt, setting 4 addition amounts of three inorganic salts, which are respectively 0.05%, 0.15%, 0.25% and 0.35% of the mass of the vinasse; selection of zinc (ZnSO)4) Manganese (MnSO)4) Copper (CuSO)4) Determining the optimal addition of three additional trace elements, setting 4 addition amounts of 0.1mg/kg, 0.2mg/kg, 0.3mg/kg and 0.4mg/kg of distiller's grains, respectively, mixing, inoculating straw mushroom culture solution, mixing, culturing at 28 deg.C for 14 days, sterilizing, and determining AFB1Calculating the degradation rate, and determining the optimal carbon source, nitrogen source, inorganic salt and trace elements and the corresponding optimal addition amount.
The experimental results are as follows:
measuring aflatoxin B of straw mushroom under different culture conditions1The degradation rate of the straw mushroom shows that the optimal carbon source is glucose, so that the straw mushroom can be subjected to AFB1The degradation rate of (A) is 82.07%; the optimal nitrogen source is peptone, and the degradation rate is 80.94%; the optimum inorganic salt is KH2PO4The degradation rate is 79.35 percent; the optimum trace element is copper (CuSO)4) The degradation rate is 77.16%, and the specific experimental data are shown in the following tables 1-4;
TABLE 1 carbon Source pairing AFB1Influence of degradation Rate
Figure BDA0001532108650000061
TABLE 2 Nitrogen Source for AFB1Influence of degradation Rate
Figure BDA0001532108650000071
TABLE 3 inorganic salt vs AFB1Influence of degradation Rate
Figure BDA0001532108650000072
TABLE 4 Trace element vs AFB1Influence of degradation Rate
Figure BDA0001532108650000081
The optimal addition mass of the glucose obtained by optimization is 3 percent of the mass of the white spirit vinasse after inoculation, and the straw mushroom pairs AFB1The average degradation rate of the composite material can reach 82.06 percent; the best adding mass of the peptone is 1.5 percent of the mass of the distiller's grains after inoculation, and the degradation rate is 85.74 percent; KH (Perkin Elmer)2PO4The best adding quality of the strain is 0.15 percent of the quality of the white spirit vinasse after inoculation, and the degradation rate is 80.08 percent; adding CuSO with the mass of 0.2mg/kg4Is most beneficial to the straw mushroom pair AFB1The degradation rate is 77.05%, and the final optimal conditions are as follows: 3% glucose, 1.5% peptone, 0.15% KH2PO40.2mg/kg of CuSO4The straw mushroom can be cultured in distiller's grains of 53.52 μ g/kg at 28 deg.C, pH 6, and culture temperature of 28 deg.C for 14 days-1AFB1Reduced to 7.48. mu.g.kg-1The degradation rate is 86.02%, and the specific experimental data are shown in the following table 5-8;
table 5 addition of glucose to AFB of different masses1Influence of degradation Rate
Figure BDA0001532108650000082
Table 6 addition of peptone to AFB in different masses1Influence of degradation Rate
Figure BDA0001532108650000091
Table 7 addition of KH in different masses2PO4For AFB1Influence of degradation Rate
Figure BDA0001532108650000092
Table 8 addition of different masses of CuSO4For AFB1Influence of degradation Rate
Figure BDA0001532108650000101
The following examples 1 to 8 were carried out on the basis of this preliminary experiment:
example 1
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 25 ℃ for 8d to finish the culture;
(2) liquid medium culture:
mixing peeled rhizoma Solani Tuber osi 250g, glucose 20g, and distilled water 1000ml, adjusting pH to 6 to obtain PD liquid culture medium, subpackaging the PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volume, sealingStopping the pipe orifice, sterilizing with high pressure steam sterilization pot at 121 deg.C for 30min, wiping clean the surface of the clean workbench with alcohol cotton soaked in 75% alcohol, sterilizing with ultraviolet for 30min, placing sterilized PD liquid culture medium in the clean workbench, cooling to room temperature, inoculating Volvariella volvacea strain cultured with solid culture medium in PD liquid culture medium, placing in constant temperature shaking incubator at 25 deg.C, culturing for 8d to obtain Volvariella volvacea culture solution with spore concentration of 106cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, wherein the ratio of the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 10: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at the temperature of 25 ℃ for 7 days, uniformly mixing, taking 2.5g of cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 2.5mL of distilled water for dissolving, uniformly mixing by using a homogenizer to form diluted mixture, placing the diluted mixture in a 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 7d at 25 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 106cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, adding the aspergillus flavus culture solution into the distiller's grains, wherein the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added in a ratio of 5: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 91.03 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 4.5 and the water content to 70%, adding glucose as a carbon source, wherein the adding mass is 1% of the mass of the inoculated distiller's grains; peptone is used as a nitrogen source, and the adding mass is 1 percent of the mass of the white spirit vinasse after inoculation; KH (Perkin Elmer)2PO4The added mass of the inorganic salt is 0.05 percent of the mass of the white spirit vinasse after inoculation; CuSO4As trace elements, the mass of the trace elements added is 0.1mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the volume ratio of the liquid of the screened straw mushroom culture solution are 10: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 20 days at the temperature of 25 ℃, so that the aflatoxin B in the white spirit vinasse is completed1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content of the extract is 46.32 mug/kg-1Aflatoxins B1The degradation rate was 49.12%.
Example 2
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 28 ℃ for 6d to finish the culture;
(2) liquid medium culture:
mixing peeled rhizoma Solani Tuber osi 250g, glucose 20g, and distilled water 1000ml, adjusting pH to 6 to obtain PD liquid culture medium, subpackaging the PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volume, sealing the tube opening, and steaming with high pressure steamSterilizing in a sterilizing pot at 121 deg.C for 30min, wiping clean the surface of the clean bench with alcohol cotton soaked in 75% alcohol, sterilizing with ultraviolet rays for 30min, placing the sterilized PD liquid culture medium in the clean bench, cooling to room temperature, inoculating the straw mushroom strain cultured with solid culture medium in the PD liquid culture medium, culturing at 28 deg.C in a constant temperature shaking incubator for 6d to obtain straw mushroom culture solution with spore concentration of 105cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 30: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at 28 ℃ for 7 days, mixing uniformly, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 10mL of distilled water for dissolving, mixing uniformly by a homogenizer to form a diluted mixture, placing the diluted mixture in 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3d at 28 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 106cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
high pressure of distiller's grainsAnd (3) in a steam sterilization pot, after sterilizing for 60min at the temperature of 121 ℃, adding the aspergillus flavus culture solution into the distiller's grains with the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution in a ratio of 10: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 53.52 mug kg-1AFB in inoculated distillers grains1The HPLC profile of (A) is shown in FIG. 6;
step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 6 and the water content to 65%, adding glucose as a carbon source, wherein the adding mass is 3% of the mass of the inoculated distiller's grains; peptone is used as a nitrogen source, and the adding mass is 1.5 percent of the mass of the white spirit vinasse after inoculation; KH (Perkin Elmer)2PO4The added mass of the inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation; CuSO4As trace elements, the mass of the trace elements added is 0.2mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the liquid volume ratio of the screened straw mushroom culture solution are 30: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 14 days at the temperature of 28 ℃, so that the aflatoxin B in the distiller's grains is finished1Degradation of AFB in distiller's grains after degradation1The HPLC chromatogram of (A) is shown in FIG. 7, and AFB in the degraded distiller's grains is determined1The content of the extract is 7.48 mug/kg-1Aflatoxins B1The degradation rate was 86.02%.
Example 3
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 28 ℃ for 5d to finish the culture;
(2) liquid medium culture:
mixing peeled potato 250g, glucose 20g and distilled water 1000ml, adjusting pH to 6, preparing into PD liquid culture medium,subpackaging PD liquid culture medium into triangular flasks to 2/3 of the bottle volume, sealing the tube mouth, sterilizing with a high-pressure steam sterilization pot at 121 ℃ for 30min, wiping the clean bench surface with alcohol cotton soaked in 75% alcohol, sterilizing with ultraviolet rays for 30min, placing the sterilized PD liquid culture medium in a clean bench, cooling to room temperature, inoculating straw mushroom strains cultured by a solid culture medium into the PD liquid culture medium, placing in a constant-temperature shaking culture box, culturing at 28 ℃ for 7d to form straw mushroom culture solution, wherein the spore concentration of the straw mushroom culture solution is 105cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 30: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at 28 ℃ for 7 days, mixing uniformly, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 10mL of distilled water for dissolving, mixing uniformly by a homogenizer to form a diluted mixture, placing the diluted mixture in 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3d at 28 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 105cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, adding the aspergillus flavus culture solution into the distiller's grains, wherein the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added in a ratio of 10: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 53.52 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 6 and the water content to 65%, adding glucose as a carbon source, wherein the adding mass is 2% of the mass of the inoculated distiller's grains; peptone is used as a nitrogen source, and the adding mass is 2% of the mass of the white spirit vinasse after inoculation; KH (Perkin Elmer)2PO4The added mass of the inorganic salt is 0.25 percent of the mass of the white spirit vinasse after inoculation; CuSO4As trace elements, the mass of the trace elements added is 0.3mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the liquid volume ratio of the screened straw mushroom culture solution are 30: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 14 days at the temperature of 28 ℃, so that the aflatoxin B in the distiller's grains is finished1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content of the extract is 10.07 mug/kg-1Aflatoxins B1The degradation rate was 81.18%.
Example 4
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 28 ℃ for 6d to finish the culture;
(2) liquid medium culture:
mixing peeled rhizoma Solani Tuber osi 250g, glucose 20g, and distilled water 1000ml, adjusting pH to 6 to obtain PD liquid culture medium, and packaging PD liquid culture medium into three bagsSealing the opening of the tube at 2/3 of the bottle body in the angle flask, sterilizing with a high-pressure steam sterilization pot at 121 deg.C for 30min, wiping the clean bench surface with alcohol cotton soaked in 75% alcohol, sterilizing with ultraviolet rays for 30min, placing the sterilized PD liquid culture medium in the clean bench, cooling to room temperature, inoculating straw mushroom strain cultured with solid culture medium in the PD liquid culture medium, and culturing in a constant-temperature shaking incubator at 28 deg.C for 6d to obtain straw mushroom culture solution with spore concentration of 105cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 30: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at 28 ℃ for 7 days, mixing uniformly, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 10mL of distilled water for dissolving, mixing uniformly by a homogenizer to form a diluted mixture, placing the diluted mixture in 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3d at 28 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 105cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, adding the aspergillus flavus culture solution into the distiller's grains, wherein the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added in a ratio of 10: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 53.52 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 6 and the water content to 65%, adding glucose as a carbon source, wherein the adding mass is 4% of the mass of the inoculated distiller's grains; peptone is used as a nitrogen source, and the adding mass is 2.5 percent of the mass of the white spirit vinasse after inoculation; KH (Perkin Elmer)2PO4The added mass of the inorganic salt is 0.35 percent of the mass of the white spirit vinasse after inoculation; CuSO4As trace elements, the mass of the trace elements added is 0.4mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the liquid volume ratio of the screened straw mushroom culture solution are 30: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 14 days at the temperature of 28 ℃, so that the aflatoxin B in the distiller's grains is finished1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content of the extract is 10.55 mug/kg-1Aspergillus toxin B1The degradation rate was 80.29%.
Example 5
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 28 ℃ for 6d to finish the culture;
(2) liquid medium culture:
mixing peeled rhizoma Solani Tuber osi 250g, glucose 20g, and distilled water 1000ml, adjusting pH to 7 to obtain PD liquid culture medium, and subpackaging the PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volumeSealing the pipe orifice, sterilizing by adopting a high-pressure steam sterilization pot at 121 ℃ for 30min, wiping the surface of a super-clean workbench by using alcohol cotton soaked in 75% alcohol, sterilizing by ultraviolet rays for 30min, placing the sterilized PD liquid culture medium in the super-clean workbench, cooling to room temperature, inoculating straw mushroom strains cultured by a solid culture medium in the PD liquid culture medium, placing in a constant-temperature shaking incubator, culturing for 6d at 28 ℃ to form straw mushroom culture solution, wherein the spore concentration of the straw mushroom culture solution is 105cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 30: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at 28 ℃ for 7 days, mixing uniformly, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 5mL of distilled water for dissolving, mixing uniformly by using a homogenizer to form a diluted mixture, placing the diluted mixture in 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3d at 28 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 105cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, adding the aspergillus flavus culture solution into the distiller's grains, wherein the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added in a ratio of 10: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 53.52 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 6 and the water content to 65%, adding maltose serving as a carbon source into the distiller's grains, wherein the adding mass is 3% of the mass of the inoculated distiller's grains; KNO3As a nitrogen source, the adding mass is 1.5 percent of the mass of the white spirit vinasse after inoculation; MgSO (MgSO)4·7H2The adding mass of O as inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation; ZnSO4As trace elements, the mass of the trace elements added is 0.2mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the liquid volume ratio of the screened straw mushroom culture solution are 30: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 14 days at the temperature of 28 ℃, so that the aflatoxin B in the distiller's grains is finished1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content of the extract is 29.22 mug/kg-1Aflatoxins B1The degradation rate was 45.40%.
Example 6
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 28 ℃ for 6d to finish the culture;
(2) liquid medium culture:
mixing peeled rhizoma Solani Tuber osi 250g, glucose 20g, and distilled water 1000ml, adjusting pH to 7 to obtain PD liquid culture medium, subpackaging PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volume, sealing the tube opening, and collectingSterilizing with high pressure steam sterilizer at 121 deg.C for 30min, wiping clean the surface of the work bench with alcohol cotton soaked in 75% alcohol, sterilizing with ultraviolet for 30min, placing sterilized PD liquid culture medium in the work bench, cooling to room temperature, inoculating Volvariella volvacea strain cultured with solid culture medium in PD liquid culture medium, placing in constant temperature shaking incubator, culturing at 28 deg.C for 6d to obtain Volvariella volvacea culture solution with spore concentration of 105cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 30: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture for 7d at the temperature of 28 ℃, uniformly mixing, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 7.5mL of distilled water for dissolving, uniformly mixing by using a homogenizer to form a diluted mixture, placing the diluted mixture in a 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3d at 28 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 105cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
white spiritSterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, adding an aspergillus flavus culture solution into the distiller's grains with the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution in a ratio of 10: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 53.52 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 6 and the water content to 65%, adding sucrose serving as a carbon source into the distiller's grains, wherein the adding mass is 3% of the mass of the inoculated distiller's grains; beef extract is used as nitrogen source, and the adding mass is 1.5% of the mass of the distiller's grains after inoculation; CaCl2The added mass of the inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation; MnSO4As trace elements, the mass of the trace elements added is 0.2mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the liquid volume ratio of the screened straw mushroom culture solution are 30: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 14 days at the temperature of 28 ℃, so that the aflatoxin B in the distiller's grains is finished1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content is 24.28 mug/kg-1Aflatoxins B1The degradation rate was 54.63%.
Example 7
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 28 ℃ for 6d to finish the culture;
(2) liquid medium culture:
mixing peeled potato 250g, glucose 20g and distilled water 1000ml, adjusting pH to 7 to obtain PD liquid culture medium, subpackaging the PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volume, sealing tube orifice, sterilizing with high pressure steam sterilization pot, sterilizing at room temperatureSterilizing at 121 deg.C for 30min, wiping clean the surface of the clean bench with alcohol cotton soaked in 75% alcohol, sterilizing with ultraviolet rays for 30min, placing the sterilized PD liquid culture medium in the clean bench, cooling to room temperature, inoculating Volvariella volvacea strain cultured with solid culture medium in the PD liquid culture medium, culturing at 28 deg.C in a constant temperature shaking incubator for 6d to obtain Volvariella volvacea culture solution with spore concentration of 105cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 30: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at 28 ℃ for 7 days, mixing uniformly, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 10mL of distilled water for dissolving, mixing uniformly by a homogenizer to form a diluted mixture, placing the diluted mixture in 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 3d at 28 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 105cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
sterilizing distiller's grains in high pressure steam sterilizing pot at 121 deg.CAfter the strain is cultured for 60min, adding an aspergillus flavus culture solution into the distiller's grains of distillers, wherein the addition ratio of the solid mass of the distiller's grains to the liquid volume of the aspergillus flavus culture solution is 10: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 53.52 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 6 and the water content to 65%, adding soluble starch serving as a carbon source into the distiller's grains, wherein the adding mass is 3% of the mass of the inoculated distiller's grains; yeast powder is used as a nitrogen source, and the adding mass is 1.5 percent of the mass of the white spirit vinasse after inoculation; KH (Perkin Elmer)2PO4The added mass of the inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation; CuSO4As trace elements, the mass of the trace elements added is 0.2mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the liquid volume ratio of the screened straw mushroom culture solution are 30: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 14 days at the temperature of 28 ℃, so that the aflatoxin B in the distiller's grains is finished1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content of the extract is 15.63 mug/kg-1Aflatoxins B1The degradation rate was 70.79%.
Example 8
Aflatoxin B in distiller's grains1The degradation method comprises the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium, and culturing at 40 ℃ for 3d to finish the culture;
(2) liquid medium culture:
mixing peeled potato 250g, glucose 20g and distilled water 1000ml, adjusting pH to 7 to obtain PD liquid culture medium, subpackaging the PD liquid culture medium into Erlenmeyer flask to 2/3 of bottle volume, sealing tube orifice, sterilizing with high pressure steam sterilization pot at 121 deg.CWiping the clean workbench surface with alcohol cotton soaked with 75% alcohol for 30min, sterilizing with ultraviolet rays for 30min, placing the sterilized PD liquid culture medium in the clean workbench, cooling to room temperature, inoculating Volvariella volvacea strain cultured with solid culture medium in the PD liquid culture medium, and culturing at 40 deg.C in constant temperature shaking incubator for 3d to obtain Volvariella volvacea culture solution with spore concentration of 107cfu/mL for standby;
(3) screening a white spirit vinasse Chinese herbal mushroom culture solution:
sterilizing the distiller's grains in a high-pressure steam sterilization pot at 121 ℃ for 60min, and mixing the distiller's grains in proportion that the solid mass of the distiller's grains to the liquid volume of the straw mushroom culture solution is 50: 1, taking the unit as g/mL, adding straw mushroom culture solution into white spirit vinasse for solid state fermentation culture at 35 ℃ for 3 days, mixing uniformly, taking 2.5g of the cultured mixture, placing the mixture into a 50mL centrifuge tube, adding 10mL of distilled water for dissolving, mixing uniformly by a homogenizer to form a diluted mixture, placing the diluted mixture in 4000 r.min-1Centrifuging for 10min under the condition, measuring the laccase activity of the supernatant by adopting an ABTS method, screening a diluted mixture with the laccase activity higher than 1800U/L, and further screening a volvariella volvacea culture solution meeting the laccase activity requirement after dilution for later use, wherein one laccase unit (U) is the amount of laccase required for 1 mu mol ABTS conversion in 1 min;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
inoculating the strain of the aspergillus flavus producing the toxin on a PDA slant test tube culture medium, culturing for 7d at 25 ℃, activating, adding 4mL of sterile water on the slant test tube culture medium, washing to prepare aspergillus flavus suspension, adding 50mL of PD culture solution into a 150mL triangular flask, adding the aspergillus flavus suspension into the PD culture solution, and placing in a constant-temperature shaking incubator, wherein the culture conditions are as follows: culturing at 30 deg.C for 7d with shaking rotation number of 200rpm until spore concentration is 107cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
sterilizing distiller's grains in autoclave at 121 deg.C for 60min, adding Chinese liquorAdding an aspergillus flavus culture solution into the grains, wherein the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added in a ratio of 20: 1, unit g: ml, culturing at 28 deg.C for 7 days, sterilizing at 121 deg.C for 60min to complete inoculation, and determining AFB in distiller's grains after inoculation1The content of 46.59 mug kg-1；
Step 3, aflatoxin B in distiller's grains1Degradation of
Taking the inoculated distiller's grains, sterilizing, adjusting the pH to 7 and the water content to 55%, adding glucose as a carbon source, wherein the adding mass is 3% of the mass of the inoculated distiller's grains; NH (NH)4NO3As a nitrogen source, the adding mass is 1.5 percent of the mass of the white spirit vinasse after inoculation; KH (Perkin Elmer)2PO4The added mass of the inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation; CuSO4As trace elements, the mass of the trace elements added is 0.2mg/kg of the mass of the inoculated white spirit vinasse, and the solid mass of the inoculated white spirit vinasse and the volume ratio of the liquid of the screened straw mushroom culture solution are 50: 1, the unit is g/mL, the screened straw mushroom culture solution is inoculated and mixed evenly, and the mixture is cultured for 8 days at the temperature of 35 ℃ to finish the aflatoxin B in the white spirit vinasse1The degradation is determined, and the AFB in the white spirit vinasse after the degradation is1The content is 16.88 mug/kg-1Aflatoxins B1The degradation rate was 63.76%.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible, such as the degradation of white spirit vinasse and other vinasse by other varieties of straw mushrooms. All the above variants in the field also belong to the scope of protection of the present invention.

Claims (7)

1. Aflatoxin B in distiller's grains1The degradation method is characterized by comprising the following steps:
step 1, straw mushroom strain culture
(1) Solid medium culture: inoculating the straw mushroom strain into a PDA plate culture medium for culture to finish culture, wherein: the culture conditions are as follows: the culture temperature is 25-40 ℃, and the culture time is 3-8 d;
(2) liquid medium culture:
cooling the PD liquid culture medium to room temperature, inoculating the straw mushroom strains cultured by the solid culture medium into the PD liquid culture medium, and culturing to form straw mushroom culture solution for later use; wherein: the culture conditions are as follows: the culture temperature is 25-40 ℃, the culture time is 3-8 d, and the spore concentration of the straw mushroom culture solution is 105~107 cfu/mL；
(3) Screening a white spirit vinasse Chinese herbal mushroom culture solution:
taking straw mushroom culture solution, adding the straw mushroom culture solution into white spirit vinasse for solid state fermentation culture, uniformly mixing, taking the cultured mixture, adding distilled water for uniformly mixing to form a diluted mixture, measuring the laccase activity of supernatant after centrifugation, screening the diluted mixture with the laccase activity higher than 1800U/L, and further screening the straw mushroom culture solution meeting the activity requirement of the diluted laccase after culture for later use, wherein the solid mass of the white spirit vinasse and the liquid volume ratio of the straw mushroom culture solution are (10-50): 1, the unit is g/mL, and the culture conditions after the straw mushroom culture solution is added with the white spirit vinasse are as follows: the culture temperature is 25-35 ℃, and the culture time is 3-7 d;
step 2, inoculating distiller's grains to produce aspergillus flavus
(1) Activating and culturing the strain of the toxigenic aspergillus flavus:
preparing Aspergillus flavus suspension from strain producing toxin Aspergillus flavus, adding Aspergillus flavus suspension into PD culture solution, and culturing until spore concentration is 105~107cfu/mL to obtain an aspergillus flavus culture solution;
(2) inoculating distiller's grains with aspergillus flavus for producing toxin:
adding an aspergillus flavus culture solution into the distiller's grains of distillers for culture, and completing inoculation through sterilization treatment, wherein: the solid mass of the distiller's grains and the liquid volume of the aspergillus flavus culture solution are added according to the proportion of (5-20): 1, unit g: ml;
step 3, aflatoxin B in distiller's grains1Degradation of
Collecting inoculated distiller's grains, adding culture medium components, mixing, inoculating screened straw mushroom, and culturingMixing the solutions, and culturing to obtain aflatoxin B in distiller's grains1Degradation of (2); wherein, the components of the culture medium comprise a carbon source, a nitrogen source, inorganic salts and trace elements;
the carbon source is glucose, and the adding mass of the carbon source is 2-4% of the mass of the white spirit vinasse after inoculation;
the nitrogen source is peptone, and the adding mass of the nitrogen source is 1.5-2.5% of the mass of the white spirit vinasse after inoculation;
the inorganic salt is KH2PO4The adding mass of the inorganic salt is 0.15-0.35% of the mass of the white spirit vinasse after inoculation;
the microelement is CuSO4The adding mass of the trace elements is 0.2-0.4 mg/kg of the mass of the white spirit vinasse after inoculation;
the volume ratio of the inoculated white spirit vinasse solid mass to the screened straw mushroom culture solution liquid is (10-50): 1 in g/mL, said culture conditions being: the temperature is 25-28 ℃ and the time is 8-20 d.
2. Aflatoxin B in distillers' grains according to claim 11The degradation method is characterized in that in the step 2(1), the culture process of the aspergillus flavus culture solution is carried out in a constant-temperature shaking incubator, and the culture conditions are as follows: the culture temperature was 30 ℃ and the culture time was 7d, and the number of shaking revolutions was 200 rpm.
3. Aflatoxin B in distillers' grains according to claim 11The degradation method is characterized in that in the step 2(2), the culture conditions of the distiller's grains after being added into the aspergillus flavus culture solution are as follows: 28 ℃ and 7 d.
4. Aflatoxin B in distillers' grains according to claim 11The degradation method of (3), wherein the culture conditions in step (3) are: the temperature was 28 ℃ for 14 d.
5. The aflatoxin in distillers' grains of claim 1B1The degradation method is characterized in that in the step 3, the inoculated white spirit vinasse is subjected to pH and water content adjustment treatment, the pH is adjusted to 4.5-7, and the water content is adjusted to 55-70%.
6. Aflatoxin B in distillers' grains according to claim 51The degradation method of (3), wherein the pH of the inoculated distiller's grains is adjusted to 6 and the water content is adjusted to 65%.
7. Aflatoxin B in distillers' grains according to claim 11The degradation method is characterized in that in the step 3, the adding mass of the culture medium is as follows:
the adding mass of the carbon source is 3 percent of the mass of the white spirit vinasse after inoculation;
the adding mass of the nitrogen source is 1.5 percent of the mass of the white spirit vinasse after inoculation;
the adding mass of the inorganic salt is 0.15 percent of the mass of the white spirit vinasse after inoculation;
the adding mass of the trace elements is 0.2mg/kg of the mass of the distiller's grains after inoculation.
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