CN110604175B - Method for inhibiting growth of aspergillus flavus and toxin generation in grains - Google Patents

Method for inhibiting growth of aspergillus flavus and toxin generation in grains Download PDF

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CN110604175B
CN110604175B CN201911031450.6A CN201911031450A CN110604175B CN 110604175 B CN110604175 B CN 110604175B CN 201911031450 A CN201911031450 A CN 201911031450A CN 110604175 B CN110604175 B CN 110604175B
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film
grain
aspergillus flavus
growth
ethyl alcohol
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CN110604175A (en
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邢福国
靳婧
任瑶瑶
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Institute of Food Science and Technology of CAAS
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/18—Vapour or smoke emitting compositions with delayed or sustained release
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/02—Acyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/144—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of gases, e.g. fumigation; Compositions or apparatus therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B9/00—Preservation of edible seeds, e.g. cereals
    • A23B9/16—Preserving with chemicals
    • A23B9/18—Preserving with chemicals in the form of gases, e.g. fumigation; Compositions or apparatus therefor
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
    • C08G73/10—Polyimides; Polyester-imides; Polyamide-imides; Polyamide acids or similar polyimide precursors
    • C08G73/1067—Wholly aromatic polyimides, i.e. having both tetracarboxylic and diamino moieties aromatically bound
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2379/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2361/00 - C08J2377/00
    • C08J2379/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors
    • C08J2379/08—Polyimides; Polyester-imides; Polyamide-imides; Polyamide acids or similar polyimide precursors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2479/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2461/00 - C08J2477/00
    • C08J2479/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors
    • C08J2479/08—Polyimides; Polyester-imides; Polyamide-imides; Polyamide acids or similar polyimide precursors

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  • Dentistry (AREA)
  • Plant Pathology (AREA)
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Abstract

The invention discloses a method for inhibiting growth of aspergillus flavus and toxin generation in grains, which comprises the steps of taking absolute ethyl alcohol as a fumigant, burying and doping the absolute ethyl alcohol in the grains, wherein the buried doping volume of the absolute ethyl alcohol is 4-10% of the volume of the grains, rapidly sealing a grain pile by using a film after doping is finished, and continuously fumigating the grain pile for 1-10 days at the temperature of 20-45 ℃. The invention takes the absolute ethyl alcohol as the active component of the aspergillus flavus growth inhibitor, the used absolute ethyl alcohol has mature preparation process, wide source of preparation resources, volatility, good safety to people and livestock, no pollution to the environment, effective inhibition of the growth of the aspergillus flavus in grains, good effects of inhibiting the growth and producing toxin of the aspergillus flavus, safety, no toxic or side effect, convenient use and easy industrial production, thereby having extremely wide application prospect.

Description

Method for inhibiting growth of aspergillus flavus and toxin generation in grains
Technical Field
The invention relates to the technical field of grain preservation. More particularly, the invention relates to a method for inhibiting the growth of aspergillus flavus and the production of toxin in grains.
Background
The aspergillus flavus is an aerobic saprophytic fungus widely distributed in nature, can parasitize in grains, foods and feeds to grow and breed, and causes the foods and the feeds to go mouldy and deteriorate, and the aflatoxin generated by secondary metabolism is a highly toxic substance with strong carcinogenicity, has a destructive effect on liver tissues of people and animals, and can cause liver cancer and even death in severe cases. At present, a method of fumigating grains is usually adopted to solve the mildew of grains, and a commonly used fumigant is phosphine. Researches find that phosphine has a relatively remarkable inhibition effect on the respiration intensity of mould cells, but the inhibition rate of phosphine is the best when the phosphine is used for preparing the whole strain of aspergillus, the inhibition effect in the mycelium stage is not obvious, the prevention and control effect cannot be achieved, and the phosphine belongs to a highly toxic and flammable substance, is easy to leak if not operated properly during grain storage fumigation, harms the safety of operators, and causes toxic residues on the fumigated grains.
Therefore, the fumigant capable of efficiently inhibiting the growth of aspergillus flavus in grains and the generation of toxin is screened and developed, is used for preventing and controlling the grains from being polluted by the aflatoxin, improves the safety level of foods, and has very important economic value and social significance.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for inhibiting the growth of aspergillus flavus and the generation of toxin in grains, which utilizes absolute ethyl alcohol as a fumigant and is quickly sealed by the gas separation composite membrane prepared by the invention after the application of the fumigant, wherein the absolute ethyl alcohol has mature preparation process, wide preparation resource source, volatility, good safety to people and livestock, no pollution to the environment, effective inhibition of the growth of aspergillus flavus in grains, good effects of inhibiting the growth of aspergillus flavus and generating toxin, safety and no toxic or side effect.
In order to achieve the objects and other advantages according to the present invention, a method for inhibiting growth of aspergillus flavus and production of toxins in grains is provided, wherein absolute ethyl alcohol is used as a fumigant, absolute ethyl alcohol is buried and doped in grains, the buried and doped volume of the absolute ethyl alcohol is 4-10% of the volume of the grains, the grain pile is rapidly sealed with a film after doping is finished, and continuous fumigation is performed for 1-10 days at 20-45 ℃.
Preferably, the fumigating temperature is 28 ℃, the fumigating time is 7d, and the embedding doping volume of the absolute ethyl alcohol is 4-6% of the grain volume.
Preferably, the grain is at least one of wheat, barley, rice, corn, oat, rye, buckwheat, soybean, broad bean, potato, sweet potato and peanut.
Preferably, the water activity of the grain is 0.92.
Preferably, the membrane is a gas separation composite membrane, and the preparation method mainly comprises the following steps:
dissolving 4,4 '-diaminodiphenylmethane in an N-methylpyrrolidone solvent, mechanically stirring in an ice-water bath and under a nitrogen atmosphere, and slowly adding pyromellitic dianhydride in batches, wherein the molar ratio of the 4,4' -diaminodiphenylmethane to the pyromellitic dianhydride to the N-methylpyrrolidone is 1 (1-1.1) to (20-30), and the reaction time is 4-8 h, so as to prepare a PMDA-ODA type polyamic acid casting solution;
step two, uniformly blade-coating the PMDA-ODA type polyamide acid casting solution prepared in the step one on a clean glass plate, soaking the glass plate in deionized water for 45-80 min, separating the film on the glass plate, freeze-drying the film, placing the film in a vacuum drying oven, carrying out temperature programming to 300 ℃, and carrying out heat preservation for 3-5 h to prepare a polyimide base film, wherein the thickness of the polyimide base film is 100-200 mu m;
adding hexafluoro dianhydride and 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt into a dimethyl sulfoxide solvent, wherein the molar ratio of the hexafluoro dianhydride to the 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt is 1:1, reacting at room temperature for 3-5 h to obtain 6FDA type polyamic acid, adding acetic anhydride and triethylamine into the system, wherein the molar ratio of the acetic anhydride, the triethylamine and the hexafluoro dianhydride is 1.5:1.5:1, heating to 100 ℃ under the protection of nitrogen, reacting for 5-8 h, pouring a reaction product into acetone, settling a polymer, filtering, washing and drying to obtain 6FDA type polyimide powder;
step four, dissolving the 6FDA type polyimide powder prepared in the step three in an N-methyl pyrrolidone solvent to prepare a 6FDA type polyimide film casting solution with the concentration of 15-20%, blade-coating the 6FDA type polyimide film casting solution on the polyimide base film prepared in the step two, heating to 60-80 ℃, preserving heat for 1-2 h, placing the film in a vacuum drying oven, and drying at 130 ℃ for 12h to finally prepare the gas separation composite film, wherein the thickness of the gas separation composite film is 150-300 mu m.
Preferably, the molar ratio of the 4,4' -diaminodiphenylmethane to the pyromellitic dianhydride to the N-methylpyrrolidone is 1:1.02:24, and the reaction time is 6 hours.
Preferably, the soaking time in the second step is 60min, and the thickness of the polyimide-based film is 150 μm.
Preferably, the temperature programming process of the vacuum drying oven in the second step is as follows: heating to 200 ℃ at the heating rate of 5 ℃/min, keeping the temperature for 2h, then heating to 250 ℃ and keeping the temperature for 1.5h, and heating to 300 ℃ again and keeping the temperature for 1 h.
Preferably, the concentration of the 6FDA type polyimide casting solution in the fourth step is 18%, and the thickness of the gas separation composite membrane is 230 μm.
The invention at least comprises the following beneficial effects:
firstly, the invention adopts the absolute ethyl alcohol as the fumigant, so that the effect of inhibiting aflatoxin pollution is good, the preparation process of the absolute ethyl alcohol is mature, the absolute ethyl alcohol is safe and harmless to people and livestock, has no pollution to the environment, is safe and has no toxic or side effect, and has very important economic value and social significance for improving the safety level of food;
secondly, the gas separation composite membrane provided by the invention takes the PMDA-ODA type polyimide membrane as a base membrane, and the surface of the base membrane is coated with a layer of 6FDA type polyimide membrane, so that the finally prepared gas separation composite membrane has good solvent resistance and good membrane selectivity, can effectively prevent ethanol gas from seeping under the condition of allowing oxygen and carbon dioxide to permeate, prolongs the circulation of ethanol in the membrane, and prolongs the sterilization period and sterilization effect of medicines.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples to enable those skilled in the art to practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example 1>
Grain variety: wheat (Triticum aestivum L.).
Fumigant: anhydrous ethanol.
The fumigation method comprises the following steps: adding absolute ethyl alcohol into grain sample, the adding and embedding volume of the absolute ethyl alcohol is 6% of grain volume, after the doping is finished, quickly sealing grain pile with commercial PVC film, and continuously fumigating at 28 deg.C for 7 d.
< example 2>
Grain variety: corn.
Fumigant: anhydrous ethanol.
The fumigation method comprises the following steps: adding absolute ethyl alcohol into grain sample, the adding and embedding volume of the absolute ethyl alcohol is 4% of grain volume, after the doping is finished, quickly sealing grain pile with commercial PVC film, and continuously fumigating at 45 deg.C for 10 d.
< example 3>
Grain variety: and (4) soybeans.
Fumigant: anhydrous ethanol.
The fumigation method comprises the following steps: adding absolute ethyl alcohol into grain sample, the adding and embedding volume of the absolute ethyl alcohol is 10% of grain volume, after the doping is finished, quickly sealing grain pile with commercial PVC film, and continuously fumigating for 5d at 20 deg.C.
< example 4>
Grain variety: wheat (Triticum aestivum L.).
Fumigant: anhydrous ethanol.
The fumigation method comprises the following steps: adding and burying the doped absolute ethyl alcohol in the grain sample, wherein the adding and burying doping volume of the absolute ethyl alcohol is 6% of the grain volume, rapidly sealing the grain pile by using a gas separation composite membrane after doping is finished, and continuously fumigating for 7d at 28 ℃.
The preparation method of the gas separation composite membrane mainly comprises the following steps:
dissolving 4,4 '-diaminodiphenylmethane in an N-methylpyrrolidone solvent, mechanically stirring in an ice-water bath and in a nitrogen atmosphere, slowly adding pyromellitic dianhydride in batches, wherein the molar ratio of the 4,4' -diaminodiphenylmethane to the pyromellitic dianhydride to the N-methylpyrrolidone is 1:1.02:24, and the reaction time is 6 hours to prepare a PMDA-ODA type polyamic acid casting solution;
step two, uniformly blade-coating the PMDA-ODA type polyamide acid casting solution prepared in the step one on a clean glass plate, soaking the glass plate in deionized water for 60min, removing a film on the glass plate, freeze-drying the film, placing the film in a vacuum drying oven, heating to 200 ℃ at the heating rate of 5 ℃/min, keeping the temperature for 2h, then heating to 250 ℃ and keeping the temperature for 1.5h, heating to 300 ℃ again and keeping the temperature for 1h to prepare a polyimide base film, wherein the thickness of the polyimide base film is 150 microns;
adding hexafluoro dianhydride and 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt into a dimethyl sulfoxide solvent, wherein the molar ratio of the hexafluoro dianhydride to the 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt is 1:1, reacting for 4 hours at room temperature to prepare 6FDA type polyamic acid, adding acetic anhydride and triethylamine into the system, wherein the molar ratio of the acetic anhydride, the triethylamine and the hexafluoro dianhydride is 1.5:1, heating to 100 ℃ under the protection of nitrogen, reacting for 7 hours, pouring a reaction product into acetone, settling a polymer, filtering, washing and drying to prepare 6FDA type polyimide powder;
step four, 6FDA type polyimide powder prepared in the step three is dissolved in N-methyl pyrrolidone solvent to prepare 6FDA type polyimide casting solution with the concentration of 18%, the 6FDA type polyimide casting solution is blade-coated on the polyimide base film prepared in the step two, the polyimide base film is heated to 70 ℃ and is kept warm for 1.5h, the film is placed in a vacuum drying oven and is dried for 12h at the temperature of 130 ℃, and finally the gas separation composite film is prepared, wherein the thickness of the gas separation composite film is 230 microns.
< comparative example 1>
Grain variety: wheat (Triticum aestivum L.).
Fumigant: anhydrous ethanol.
The fumigation method comprises the following steps: adding absolute ethyl alcohol into grain sample, the adding and embedding volume of the absolute ethyl alcohol is 2% of grain volume, after the doping is finished, quickly sealing grain pile with commercial PVC film, and continuously fumigating at 28 deg.C for 7 d.
< comparative example 2>
Grain variety: wheat (Triticum aestivum L.).
Fumigant: and (3) phosphine.
The fumigation method comprises the following steps: adding doped hydrogen phosphide into grain sample, the doping volume of the hydrogen phosphide is 4% of the grain volume, after doping, rapidly sealing the grain pile with commercial PVC film, and continuously fumigating at 28 deg.C for 7 d.
< comparative example 3>
The grain variety, fumigant, and fumigation method were the same as in example 4, except that a PMDA-ODA type polyimide base film was used as a gas separation membrane to seal the grain pile. The preparation method of the PMDA-ODA type polyimide base film mainly comprises the following steps:
dissolving 4,4 '-diaminodiphenylmethane in an N-methylpyrrolidone solvent, mechanically stirring in an ice-water bath and in a nitrogen atmosphere, slowly adding pyromellitic dianhydride in batches, wherein the molar ratio of the 4,4' -diaminodiphenylmethane to the pyromellitic dianhydride to the N-methylpyrrolidone is 1:1.02:24, and the reaction time is 6 hours to prepare a PMDA-ODA type polyamic acid casting solution;
step two, uniformly blade-coating the PMDA-ODA type polyamide acid casting solution prepared in the step one on a clean glass plate, soaking the glass plate in deionized water for 60min, removing a film on the glass plate, freeze-drying the film, placing the film in a vacuum drying oven, heating to 200 ℃ at the heating rate of 5 ℃/min, keeping the temperature for 2h, then heating to 250 ℃ and keeping the temperature for 1.5h, heating to 300 ℃ again and keeping the temperature for 1h to prepare the PMDA-ODA type polyimide base film, wherein the thickness of the PMDA-ODA type polyimide base film is 150 microns.
< comparative example 4>
The grain variety, fumigant, and fumigation method were the same as in example 4, except that the 6FDA type polyimide film was used as the gas separation membrane to seal the grain pile. The preparation method of the 6FDA type polyimide film mainly comprises the following steps:
adding hexafluoro dianhydride and 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt into a dimethyl sulfoxide solvent, wherein the molar ratio of the hexafluoro dianhydride to the 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt is 1:1, reacting for 4 hours at room temperature to prepare 6FDA type polyamic acid, adding acetic anhydride and triethylamine into a system, wherein the molar ratio of the acetic anhydride, the triethylamine and the hexafluoro dianhydride is 1.5:1, heating to 100 ℃ under the protection of nitrogen, reacting for 7 hours, pouring a reaction product into acetone, settling a polymer, filtering, washing and drying to prepare 6FDA type polyimide powder;
and step two, dissolving the 6FDA type polyimide powder prepared in the step one in an N-methyl pyrrolidone solvent to prepare a 6FDA type polyimide casting solution with the concentration of 18%, heating to 70 ℃, preserving the temperature for 1.5h, placing the membrane in a vacuum drying oven, and drying at 130 ℃ for 12h to finally prepare the gas separation membrane, wherein the thickness of the gas separation composite membrane is 150 microns.
< test for determining inhibitory Effect of Anhydrous ethanol liquid culture >
PDA culture medium: boiling 200g of peeled potato for 30min, collecting the filtrate, adding 20g of glucose and 16g of agar, diluting to 1000mL with distilled water, adding distilled water to make up 1L, autoclaving at 115 deg.C for 30min, cooling to about 55 deg.C, and pouring into flat plate (20 mL per flat plate).
YES medium: 2% yeast extract, 15% sucrose, 0.5% MgSO4·7H2And O, adding deionized water to complement 1L, subpackaging into 250mL triangular bottles with each bottle being 100mL, and autoclaving at 121 ℃ for 20 min.
Activating strains: inoculating Aspergillus flavus to PDA culture medium, growing at 28 deg.C for 7 days, and storing at 4 deg.C.
Preparing an aspergillus flavus spore suspension: on a flat plate culture medium with a sterile flat toothpickDissolving Aspergillus flavus spore in 0.1% Tween 80, and diluting the initial spore solution with sterile water to concentration of 1 × 107cfu/mL。
Respectively dropwise adding anhydrous ethanol in YES, wherein the dropwise adding amount of the anhydrous ethanol accounts for 1-6% of the volume of the YES culture medium, uniformly mixing, and dropwise adding 100 mu L (1 multiplied by 10) of aspergillus flavus suspension into each bottle7cfu/mL), shaking uniformly, sealing the bottle mouth, placing in a shaking table for dark culture for 7d, setting the temperature at 28 ℃ and the rotation speed at 180 rpm. After 7 days, the cultured Aspergillus flavus flask was taken out of the shaker, the mycelia were filtered in a fume hood, and the mycelia were dried in an oven at 60 ℃ to constant weight, and the dry weight (g) of the mycelia was weighed. The culture solution is taken to extract aflatoxin, the content of aflatoxin is measured by HPLC, and the final inhibition rate result on the generation of aflatoxin is shown in Table 1:
TABLE 1
Figure BDA0002250275020000061
Test results show that the absolute ethyl alcohol can play a certain role in inhibiting the generation of aflatoxin, the inhibiting effect is enhanced along with the increase of the addition amount of the absolute ethyl alcohol, and when the addition amount of the absolute ethyl alcohol accounts for more than 4% of the volume of the YES culture medium, the inhibiting rate of the absolute ethyl alcohol on the generation of aflatoxin can reach 100%.
<Anhydrous ethanol fumigation for inhibiting aspergillus flavus in peanuts (water activity a)w0.92) growth and toxicity test>
The peanut sample is cleaned and disinfected by 1% sodium hypochlorite solution, washed by sterile water, and the surface water is absorbed by sterile filter paper. Adding quantitative sterile water according to the difference value of the water activity and the preset target water activity, standing at 4 ℃ for 24h to balance the water content, and obtaining the water activity (a) required by the experimentw0.92) peanut. The peanuts were divided into 200g units in 1L Erlenmeyer flasks, and 1mL of spore suspension (concentration 1X 10) was added7one/mL), gently shaking the bottle to evenly cover the spores onto the peanuts. Dropping anhydrous ethanol on sterile filter paper, placing into conical flask, and making the anhydrous ethanol content account for 0, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 5% and the spore suspension volume in the flask6 percent, sealing the bottle mouth, placing the bottle in a constant temperature and humidity box, culturing the bottle for 7d at the temperature of 28 ℃, and measuring the aflatoxin pollution rate and the aflatoxin content of the peanuts, wherein the experimental results are shown in table 2.
The test result shows that the absolute ethyl alcohol has a remarkable effect of reducing the aflatoxin pollution rate in the peanuts, a certain inhibiting effect on the aflatoxin production in the peanuts can be achieved, the aflatoxin pollution rate is gradually reduced along with the increase of the addition amount of the absolute ethyl alcohol, the inhibiting effect on the aflatoxin production is gradually enhanced, when the addition amount of the absolute ethyl alcohol accounts for more than 4% of the volume of the spore suspension, the aflatoxin pollution rate in the peanuts is reduced to 0%, and the inhibiting rate on the aflatoxin production in the peanuts reaches 100%.
TABLE 2
Figure BDA0002250275020000071
< test for gas permeability of Membrane >
The gas permeation coefficient and the separation performance of the gas separation composite membrane prepared in example 4 and the gas separation membranes prepared in comparative examples 3 to 4 were measured by the following measurement methods:
the gas permeability coefficient is 7cm2Each membrane of (a) was placed in a stainless steel tank and measured by a differential pressure method. Specifically, carbon dioxide (CO) was used at a temperature of 23 deg.C2) Oxygen (O)2) And ethanol gas (C)2H5OH) as a test gas, the permeability coefficient and separation performance (ratio of permeability coefficients of respective gases) of each gas were measured. The measurement results of the gas permeability coefficients are shown in table 3, and the separation performance results are shown in table 4.
Experiment results show that the gas separation composite membrane prepared by the preparation method in example 4 has good permeability to oxygen and carbon dioxide and poor permeability to ethanol gas, and can effectively prevent the ethanol gas from permeating under the condition of ensuring the normal permeation of oxygen and carbon dioxide; while comparative example 3 used only a PMDA-ODA type polyimide-based film as a sealing film, comparative example 46FDA type polyimide film is used as a sealing film, the air tightness of the two films is relatively poor, and the sealing film is resistant to CO2/C2H5OH，O2/C2H5The OH separating effect is also poor, and the exudation of the ethanol gas cannot be prevented well.
TABLE 3
Transmittance (Barrer) CO2 O2 C2H5OH
Example 4 78 79 1.3
Comparative example 3 110 106 98
Comparative example 4 98 89 63
TABLE 4
Separation Performance CO2/C2H5OH O2/C2H5OH
Example 4 60 61
Comparative example 3 1.1 1.1
Comparative example 4 1.6 1.4
< determination of bacteriostatic Effect in grain bulk >
The grain samples fumigated in examples 1 to 4 and comparative examples 1 to 4 were subjected to mold detection with a microscope at regular intervals in a storage environment, and the detection results are shown in table 5. In addition, the color of the grain sample treated by the fumigation of each example is observed by a color difference meter without obvious change.
Detection results show that in examples 1-3, the fumigation method disclosed by the invention is adopted, and absolute ethyl alcohol is used as a fumigant, so that the growth of mold is effectively inhibited, but due to the fact that the sealing performance of a commercial PVC film is poor, the ethyl alcohol gas gradually leaks in the fumigation process, and the sterilization period of the medicine is short; the gas separation composite membrane used in example 4 has good selectivity, can effectively prevent the ethanol gas from seeping out under the condition of allowing oxygen and carbon dioxide to permeate, and prolongs the circulation of ethanol in the membrane, thereby prolonging the sterilization period and sterilization effect of the medicine. Compared with the comparative example 1, the anhydrous ethanol is less, and the sterilization period is shorter than that of the examples 1-3; in the comparative example 2, phosphine is used as a fumigant, and although the sterilizing effect is the same as that of the examples 1-3, the phosphine is extremely toxic and is easy to cause toxic residues on fumigated grains and cause potential safety hazards of foods; in the comparative examples 3 and 4, the PMDA-ODA type polyimide base film and the 6FDA type polyimide film are respectively adopted as sealing films, the sealing effect is poor, the seepage of ethanol gas cannot be effectively prevented, the ethanol content is gradually reduced in the fumigation process, the inhibition effect on the growth of mould is weakened, the sterilization period of the medicament is short, and finally the mould is generated.
TABLE 5
Fumigating time (d) 1 2 4 6 10 30
Example 1 Without mould Without mould Without mould Without mould Without mould With mould
Example 2 Without mould Without mould Without mould Without mould Without mould With mould
Example 3 Without mould Without mould Without mould Without mould Without mould With mould
Example 4 Without mould Without mould Without mould Without mould Without mould Without mould
Comparative example 1 Without mould Without mould Without mould Without mould With mould With mould
Comparative example 2 Without mould Without mould Without mould Without mould Without mould With mould
Comparative example 3 Without mould Without mould Without mould With mould With mould With mould
Comparative example 4 Without mould Without mould Without mould With mould With mould With mould
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the examples shown and described without departing from the generic concept as defined by the claims and their equivalents.

Claims (8)

1. The method for inhibiting the growth of aspergillus flavus and the generation of toxin in grain is characterized in that absolute ethyl alcohol is taken as a fumigant, the absolute ethyl alcohol is buried and doped in the grain, the buried doping volume of the absolute ethyl alcohol is 4-10% of the volume of the grain, after doping is finished, a grain pile is rapidly sealed by a membrane, continuous fumigation is carried out for 1-10 days at the temperature of 20-45 ℃, the membrane is a gas separation composite membrane, and the preparation method mainly comprises the following steps:
dissolving 4,4 '-diaminodiphenylmethane in an N-methylpyrrolidone solvent, mechanically stirring in an ice-water bath and under a nitrogen atmosphere, and slowly adding pyromellitic dianhydride in batches, wherein the molar ratio of the 4,4' -diaminodiphenylmethane to the pyromellitic dianhydride to the N-methylpyrrolidone is 1 (1-1.1) to (20-30), and the reaction time is 4-8 h, so as to prepare a PMDA-ODA type polyamic acid casting solution;
step two, uniformly blade-coating the PMDA-ODA type polyamide acid casting solution prepared in the step one on a clean glass plate, soaking the glass plate in deionized water for 45-80 min, separating the film on the glass plate, freeze-drying the film, placing the film in a vacuum drying oven, carrying out temperature programming to 300 ℃, and carrying out heat preservation for 3-5 h to prepare a polyimide base film, wherein the thickness of the polyimide base film is 100-200 mu m;
adding hexafluoro dianhydride and 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt into a dimethyl sulfoxide solvent, wherein the molar ratio of the hexafluoro dianhydride to the 3, 5-diamino-2, 4, 6-trimethylbenzene sulfonic acid lithium salt is 1:1, reacting at room temperature for 3-5 h to obtain 6FDA type polyamic acid, adding acetic anhydride and triethylamine into the system, wherein the molar ratio of the acetic anhydride, the triethylamine and the hexafluoro dianhydride is 1.5:1.5:1, heating to 100 ℃ under the protection of nitrogen, reacting for 5-8 h, pouring a reaction product into acetone, settling a polymer, filtering, washing and drying to obtain 6FDA type polyimide powder;
step four, dissolving the 6FDA type polyimide powder prepared in the step three in an N-methyl pyrrolidone solvent to prepare a 6FDA type polyimide film casting solution with the concentration of 15-20%, blade-coating the 6FDA type polyimide film casting solution on the polyimide base film prepared in the step two, heating to 60-80 ℃, preserving heat for 1-2 h, placing the film in a vacuum drying oven, and drying at 130 ℃ for 12h to finally prepare the gas separation composite film, wherein the thickness of the gas separation composite film is 150-300 mu m.
2. The method for inhibiting the growth and toxin production of aspergillus flavus in grain according to claim 1, wherein the fumigating temperature is 28 ℃, the fumigating time is 7d, and the embedding doping volume of the absolute ethyl alcohol is 4-6% of the grain volume.
3. The method of inhibiting aspergillus flavus growth and toxin production in grain according to claim 1, wherein said grain is at least one of wheat, barley, rice, corn, oat, rye, buckwheat, soybean, broad bean, potato, sweet potato, peanut.
4. The method of inhibiting aspergillus flavus growth and toxin production in foodstuff according to claim 1, wherein said foodstuff has a water activity of 0.92.
5. The method for inhibiting the growth and toxin production of aspergillus flavus in grain according to claim 1, wherein the molar ratio of the 4,4' -diaminodiphenylmethane, the pyromellitic dianhydride and the N-methyl pyrrolidone is 1:1.02:24, and the reaction time is 6 h.
6. The method for inhibiting the growth and toxin production of aspergillus flavus in grain according to claim 1, wherein the soaking time in the second step is 60min, and the thickness of the polyimide-based film is 150 μm.
7. The method for inhibiting the growth and toxin production of aspergillus flavus in grain according to claim 1, wherein the temperature programming process of the vacuum drying oven in the second step is as follows: heating to 200 ℃ at the heating rate of 5 ℃/min, keeping the temperature for 2h, then heating to 250 ℃ and keeping the temperature for 1.5h, and heating to 300 ℃ again and keeping the temperature for 1 h.
8. The method for inhibiting the growth of aspergillus flavus and the generation of toxins in food grains according to claim 1, wherein the concentration of the 6FDA type polyimide casting solution in the fourth step is 18%, and the thickness of the gas separation composite membrane is 230 μm.
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