CN115428887A - Biological degradation method for mycotoxin in grains or byproducts thereof - Google Patents
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- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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Abstract
The invention provides a biological degradation method of mycotoxin in grains or byproducts thereof, which relates to the technical field of toxin degradation and comprises the following steps: the method comprises the following steps: collecting grain or its byproduct polluted by fungi, pulverizing, sieving to remove impurities to obtain grain pieces; step two: adding mitochondrial cytochrome P450, flavin-containing monooxygenase, prostaglandin synthetase, amino oxidase and alcohol dehydrogenase into crushed grains for oxidation reaction; step three: after oxidation reaction, adding epoxy hydrolase and aldehyde reductase into the crushed grains for reduction reaction; step four: after the reduction reaction, adding coupling enzyme and microsomal glucuronic acid transferase into the grain fragments; the invention simulates the temperature environment of internal organs of mammals, takes the elements such as enzyme in biological tissue body fluid as main degradation raw materials to carry out oxidation reaction, reduction reaction and conjugation reaction, reduces the toxicity of mycotoxin, increases the solubility of mycotoxin in water, and is convenient for dry and wet separation to discharge toxin.
Description
Technical Field
The invention relates to the technical field of toxin degradation, in particular to a biodegradation method of mycotoxin in grains or byproducts thereof.
Background
Mycotoxins are secondary metabolites produced by toxin-producing fungi in a hazard process, and mainly comprise aflatoxin, deoxynivalenol (also called vomitoxin, DON), zearalenone (ZEN), fumonisin (FUM), ochratoxin and the like, in recent years, due to frequent occurrence of extreme climate, the prevalence of gibberellic disease mainly damaging wheat and corn is caused, the pollution of DON and ZEN produced by fungi is increased, and the wheat and corn with overproof pollution is increased continuously, so that research on safe utilization of grains with overproof toxins is urgently carried out, not only is the grain benefit of farmers protected, but also the situation that the grains flow into grain markets is prevented, and the food quality safety of grain countries is maintained;
for the removal of mycotoxin, the current common methods mainly comprise physical removal, adsorption, chemical treatment and the like, the adsorbent adsorbs mycotoxin and simultaneously adsorbs a large amount of micronutrients in feed and food, and the toxin adsorbed by clay cannot be decomposed and causes secondary pollution; in the prior art, for example, application No. 201210335523.2 discloses "a biodegradation method for mycotoxins in grains and/or byproducts thereof", and specifically discloses: the method is characterized in that grains and/or byproducts thereof polluted by mycotoxin are used as raw materials, engineering bacteria containing mycotoxin degrading enzyme genes are utilized to ferment and produce ethanol, and the byproducts in the ethanol production process are further prepared into vinasse protein.
Disclosure of Invention
In view of the above problems, the present invention provides a method for biodegrading mycotoxins in grains or by-products thereof, which reduces the toxicity of mycotoxins and increases their solubility in water, thereby facilitating the removal of mycotoxins by wet-dry separation.
In order to realize the purpose of the invention, the invention is realized by the following technical scheme: a method for biodegrading mycotoxin in grains or byproducts thereof comprises the following steps:
the method comprises the following steps: collecting grain or its byproduct polluted by fungi, pulverizing, sieving to remove impurities to obtain grain pieces;
step two: adding micro-granular cytochrome P450, flavin-containing monooxygenase, prostaglandin synthetase, amino oxidase and alcohol dehydrogenase into the crushed grains for oxidation reaction;
step three: after oxidation reaction, adding epoxy hydrolase and aldehyde reductase into the crushed grains for reduction reaction;
step four: after the reduction reaction, adding coupling enzyme, microsomal glucuronic acid transferase, cytoplasmic sulfur transferase, methyltransferase, aminoacyl transferase, s-glutathione transferase and n-acetyltransferase into the grain fragments to carry out conjugation reaction;
step five: drying and wet separating the processed crushed grain, introducing a culture solution, collecting bacillus in natto, and inputting the bacillus into the culture solution to process the crushed grain;
step six: collecting white-rot fungi in oyster mushrooms, recombining laccase of the white-rot fungi into aspergillus niger, and inputting the aspergillus niger and ozone into a culture solution to crush grains for treatment;
step seven: separating CK1 type bacillus licheniformis from soil, inputting the bacillus licheniformis into a culture solution to process grain crushing;
step eight: and crushing and air-drying the treated grains, performing high-density compaction and bundling, then filling the grains into plastic bags, fastening bag openings, sealing and stacking, thereby creating an anaerobic fermentation environment, and finally completing the lactic acid fermentation process to obtain finished products.
The further improvement is that: in the first step, the grains comprise wheat, corn and rice barley, and the grain byproducts comprise starch, wheat bran and corn steep liquor.
The further improvement is that: in the second step, before the oxidation reaction, the temperature environment of the internal organs of the mammals is simulated by using the reaction chamber, water is introduced, the temperature is controlled to be 35-40 ℃, and the environment is maintained until the fourth step.
The further improvement is that: and in the third step, collecting nonspecific esterase and amidase in tissues and body fluids of mammals, and introducing the nonspecific esterase and the amidase into the grain fragments in the process of reduction reaction.
The further improvement is that: in the second step, the third step and the fourth step, bile acid is introduced in the processes of oxidation reaction, reduction reaction and conjugation reaction, so that the three reactions are carried out in the environment of bile acid.
The further improvement lies in that: and in the fifth step, collecting bacillus in natto, culturing the bacillus in a broth agar culture medium for 48 hours, and then inputting the bacillus into the culture medium to process the grain fragments, wherein the broth agar culture medium comprises 10-12 parts of peptone, 13-16 parts of beef extract, 14-16 parts of NACL, 800-1000 parts of distilled water and 18-20 parts of agar.
The further improvement is that: and in the sixth step, recombining the laccase of the white rot fungi into the Aspergillus niger to obtain the expressed recombinant laccase, and inputting the expressed recombinant laccase into the culture solution to process the crushed grains.
The further improvement lies in that: and seventhly, separating and obtaining CK1 type bacillus licheniformis from soil, culturing the bacillus licheniformis in an LB culture medium for 36 hours, inputting the culture medium into a culture solution to crush the grains, wherein the LB culture medium comprises 4-6 parts of yeast extract, 9-12 parts of peptone, 5-7 parts of NaCl, 15-20 parts of agar and 800-1000 parts of water.
The further improvement lies in that: in the fifth step, the sixth step and the seventh step, in the process of crushing the grains, the nutrient solution of the amino acid is selected as the culture solution, and the temperature is controlled to be 20-30 ℃.
The further improvement is that: and step eight, performing dry-wet separation on the processed grain pieces, air-drying, and introducing a lactic acid fermentation agent for anaerobic fermentation for 30-40 days.
The invention has the beneficial effects that:
1. the invention simulates the temperature environment of internal organs of mammals, takes the elements such as enzyme in biological tissue body fluid as main degradation raw materials to carry out oxidation reaction, reduction reaction and conjugation reaction, reduces the toxicity of mycotoxin, increases the solubility of mycotoxin in water, and is convenient for dry and wet separation to discharge toxin.
2. According to the invention, bacillus, recombinant laccase, CK1 type bacillus licheniformis and ozone are subjected to detoxification degradation treatment, the detoxification rate of ochratoxin reaches 88%, the detoxification rate of aflatoxin reaches 80%, the detoxification rate of zearalenone and fumonisin reaches 95.8%, the detoxification rate of deoxynivalenol reaches 93.6%, and multiple reactions in the previous stage are matched, so that the grain breakage reaches the edible standard.
3. The invention obtains finished products by lactic acid fermentation of grain fragments, and is convenient to be applied to various fields such as feed and the like, thereby utilizing wastes and avoiding secondary pollution.
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FIG. 1 is a flow chart of the present invention.
Detailed Description
In order to further understand the present invention, the following detailed description will be made with reference to the following examples, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example one
According to fig. 1, the present embodiment provides a method for biodegrading mycotoxin in food or its byproducts, comprising the following steps:
the method comprises the following steps: collecting grain or its byproduct polluted by fungi, pulverizing, sieving to remove impurities to obtain grain pieces;
step two: adding micro-granular cytochrome P450, flavin-containing monooxygenase, prostaglandin synthetase, amino oxidase and alcohol dehydrogenase into the crushed grains for oxidation reaction;
step three: after oxidation reaction, adding epoxy hydrolase and aldehyde reductase into the crushed grains for reduction reaction;
step four: after the reduction reaction, adding coupling enzyme, microsomal glucuronic acid transferase, cytoplasmic sulfur transferase, methyltransferase, aminoacyl transferase, s-glutathione transferase and n-acetyltransferase into the grain fragments to carry out conjugation reaction;
step five: drying and wet separating the processed crushed grain, introducing a culture solution, collecting bacillus in natto, and inputting the bacillus into the culture solution to process the crushed grain;
step six: collecting white-rot fungi in oyster mushrooms, recombining laccase of the white-rot fungi into aspergillus niger, and inputting the aspergillus niger and ozone into a culture solution in a matching manner to treat crushed grains;
step seven: separating CK1 type bacillus licheniformis from soil, inputting into culture solution, and processing crushed grains;
step eight: and crushing and air-drying the treated grains, performing high-density compaction and bundling, then filling the grains into plastic bags, fastening bag openings, sealing and stacking, thereby creating an anaerobic fermentation environment, and finally completing the lactic acid fermentation process to obtain finished products.
The invention fully considers the digestion degradation performance of the animal system, simulates the temperature environment of internal organs of mammals, takes elements such as enzyme in biological tissue body fluid as main degradation raw materials, carries out oxidation reaction, reduction reaction and conjugation reaction, reduces the toxicity of mycotoxins, increases the solubility of mycotoxins in water, and is convenient for dry-wet separation and toxin discharge. The grain scraps after toxin separation are subjected to secondary treatment, and are subjected to detoxification degradation treatment through bacillus, recombinant laccase, CK1 type bacillus licheniformis and ozone, and multiple reactions in the previous stage are matched, so that the grain scraps reach the edible standard.
Example two
According to fig. 1, the present embodiment provides a method for biodegrading mycotoxin in food or its byproducts, comprising the following steps:
collecting grain or its byproduct polluted by fungi, pulverizing, sieving to remove impurities to obtain grain pieces; the grain comprises wheat, corn and rice barley, and the grain by-products comprise starch, wheat bran and corn steep liquor;
simulating the temperature environment of internal organs of mammals by using a reaction chamber, introducing water, controlling the temperature to be 35-40 ℃, and then adding mitochondrial cytochrome P450, flavin-containing monooxygenase, prostaglandin synthetase, amino oxidase and alcohol dehydrogenase into the crushed grains for oxidation reaction; after the oxidation reaction, adding epoxy hydrolase and aldehyde reductase into the grain fragments to carry out reduction reaction, collecting nonspecific esterase and amidase in tissues and body fluid of mammals, and introducing nonspecific esterase and amidase into the grain fragments in the process of the reduction reaction; after the reduction reaction, adding coupling enzyme, microsomal glucuronidase, cytoplasmic thiotransferase, methyltransferase, aminoacyltransferase, s-glutathione transferase and n-acetyltransferase into the grain fragments to carry out conjugation reaction; in the process of the three reactions, bile acid is introduced, so that the three reactions are carried out in the environment of the bile acid; in the reaction process, the digestion degradation performance of the animal system is fully considered, the temperature environment of internal organs of mammals is simulated, elements such as enzyme in biological tissue body fluid are used as main degradation raw materials, oxidation reaction, reduction reaction and conjugation reaction are carried out, the toxicity of mycotoxins is reduced, the solubility of mycotoxins in water is increased, and the mycotoxins are conveniently separated and discharged in a dry mode and a wet mode. The process is carried out in the environment of bile acid, the unique hydrophilic and lipophilic amphoteric properties of the bile acid mix mycotoxin and various enzymes together to promote oxidation, reduction, hydrolysis and conjugation reactions.
Crushing the processed grain, performing dry-wet separation and drying, introducing a culture solution, collecting bacillus in natto, culturing in a broth agar culture medium for 48 hours, and inputting the broth agar culture medium into the culture solution to process the grain crush, wherein the broth agar culture medium comprises 10 parts of peptone, 15 parts of beef extract, 15 parts of NACL, 1000 parts of distilled water and 19 parts of agar; collecting white-rot fungi in oyster mushrooms, recombining laccase of the white-rot fungi into aspergillus niger to obtain expressed recombinant laccase (118U/L), and inputting the expressed recombinant laccase into a culture solution in cooperation with ozone to carry out treatment on crushed grains; separating and obtaining CK1 type bacillus licheniformis from soil, culturing in an LB culture medium for 36 hours, inputting the culture medium into a culture solution to process crushed grains, wherein the LB culture medium comprises 5 parts of yeast extract, 10 parts of peptone, 5 parts of NaCl, 18 parts of agar and 1000 parts of water; in the process of crushing grain, the nutrient solution of amino acid is selected as the culture solution, and the temperature is controlled to be 20-30 ℃; in the reaction process, a broth agar culture medium is used for fully culturing bacillus, an LB culture medium is used for fully culturing CK1 type bacillus licheniformis, the detoxification rate of bacillus on ochratoxin reaches 88%, the detoxification rate of recombinant laccase on aflatoxin reaches 80%, the detoxification rate of CK1 type bacillus licheniformis on zearalenone and fumonisin reaches 95.8%, the detoxification rate of ozone on deoxynivalenol reaches 93.6%, and multiple reactions in the previous stage are matched, so that the crushed grains reach the edible standard;
and (3) carrying out dry-wet separation on the treated grain fragments, air-drying, carrying out high-density compaction and bundling, then loading into a plastic bag, fastening the opening of the bag, sealing and stacking, introducing a lactic acid fermentation agent, thereby creating an anaerobic fermentation environment, carrying out anaerobic fermentation for 30-40 days, and finally completing the lactic acid fermentation process to obtain a finished product. After detoxification reaction, the finished product is obtained by crushing the grains and performing lactic acid fermentation, and the method is convenient to be applied to various fields such as feed and the like, so that waste is utilized, and secondary pollution is avoided.
The invention fully considers the digestion degradation performance of the animal system, simulates the temperature environment of internal organs of mammals, takes elements such as enzyme in biological tissue body fluid as main degradation raw materials, carries out oxidation reaction, reduction reaction and conjugation reaction, reduces the toxicity of mycotoxins, increases the solubility of mycotoxins in water, and is convenient for dry-wet separation and toxin discharge. The invention is carried out in the environment of bile acid, and the unique hydrophilic and lipophilic amphoteric characteristics of the bile acid mix mycotoxins with various enzymes to promote oxidation, reduction, hydrolysis and conjugation reactions. Meanwhile, the grain crumbs subjected to toxin separation are subjected to secondary treatment, detoxification degradation treatment is performed through bacillus, recombinant laccase, CK1 type bacillus licheniformis and ozone, the detoxification rate of ochratoxin reaches 88%, the detoxification rate of aflatoxin reaches 80%, the detoxification rate of zearalenone and fumonisin reaches 95.8%, the detoxification rate of deoxynivalenol reaches 93.6%, and the grain crumbs reach the edible standard by matching with multiple reactions in the previous stage. In addition, the finished product is obtained by performing lactic acid fermentation on the crushed grains, and is convenient to be applied to various fields such as feeds, so that the waste is utilized, and the secondary pollution is avoided.
The foregoing shows and describes the general principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A method for biodegrading mycotoxin in grains or byproducts thereof is characterized by comprising the following steps:
the method comprises the following steps: collecting grain or its byproduct polluted by fungi, pulverizing, sieving to remove impurities to obtain grain pieces;
step two: adding micro-granular cytochrome P450, flavin-containing monooxygenase, prostaglandin synthetase, amino oxidase and alcohol dehydrogenase into the crushed grains for oxidation reaction;
step three: after oxidation reaction, adding epoxy hydrolase and aldehyde reductase into the crushed grains for reduction reaction;
step four: after the reduction reaction, adding coupling enzyme, microsomal glucuronidase, cytoplasmic thiotransferase, methyltransferase, aminoacyltransferase, s-glutathione transferase and n-acetyltransferase into the grain fragments to carry out conjugation reaction;
step five: drying and wet separating the processed crushed grain, introducing a culture solution, collecting bacillus in natto, and inputting the bacillus into the culture solution to process the crushed grain;
step six: collecting white-rot fungi in oyster mushrooms, recombining laccase of the white-rot fungi into aspergillus niger, and inputting the aspergillus niger and ozone into a culture solution to crush grains for treatment;
step seven: separating CK1 type bacillus licheniformis from soil, inputting the bacillus licheniformis into a culture solution to process grain crushing;
step eight: after being crushed and dried, the processed grains are compacted in high density and bundled, then are put into plastic bags, bag openings are tied, and the stacking is sealed, so that an anaerobic fermentation environment is created, and finally, the lactic acid fermentation process is completed to obtain finished products.
2. The method of claim 1 for biodegrading mycotoxins in foodstuffs or by-products thereof, wherein the method comprises: in the first step, the grains comprise wheat, corn and rice barley, and the grain byproducts comprise starch, wheat bran and corn steep liquor.
3. The method of claim 2, wherein the step of biodegrading the mycotoxins in the food or by-product comprises: in the second step, before the oxidation reaction, the temperature environment of the internal organs of the mammals is simulated by using the reaction chamber, water is introduced, the temperature is controlled to be 35-40 ℃, and the environment is maintained until the fourth step.
4. The method of claim 3, wherein the step of biodegrading the mycotoxins in the grain or by-product comprises: and in the third step, collecting nonspecific esterase and amidase in tissues and body fluids of mammals, and introducing the nonspecific esterase and the amidase into the grain fragments in the process of reduction reaction.
5. The method of claim 4 for biodegrading mycotoxins in foodstuffs or by-products thereof, wherein the method comprises: in the second step, the third step and the fourth step, bile acid is introduced in the processes of oxidation reaction, reduction reaction and conjugation reaction, so that the three reactions are carried out in the environment of bile acid.
6. The method of claim 1 for biodegrading mycotoxins in foodstuffs or by-products thereof, wherein the method comprises: and in the fifth step, collecting bacillus in the natto, culturing the bacillus in a broth agar culture medium for 48 hours, and then inputting the bacillus into the culture medium to crush the grain, wherein the broth agar culture medium comprises 10-12 parts of peptone, 13-16 parts of beef extract, 14-16 parts of NACL, 800-1000 parts of distilled water and 18-20 parts of agar.
7. The method of claim 6, wherein the step of biodegrading the mycotoxins in the food or by-product comprises: in the sixth step, the laccase of the white rot fungi is recombined into the aspergillus niger to obtain the expressed recombined laccase, and the expressed recombined laccase is input into the culture solution to process the grain fragments.
8. The method of claim 7 for biodegrading mycotoxins in foodstuffs or by-products thereof, wherein the method comprises: and seventhly, separating and obtaining CK1 type bacillus licheniformis from the soil, culturing the bacillus licheniformis in an LB culture medium for 36 hours, and inputting the bacillus licheniformis into a culture solution to process the crushed grain, wherein the LB culture medium comprises 4-6 parts of yeast extract, 9-12 parts of peptone, 5-7 parts of NaCl, 15-20 parts of agar and 800-1000 parts of water.
9. The method of claim 8, wherein the step of biodegrading the mycotoxins in the food or by-product comprises: in the fifth step, the sixth step and the seventh step, in the process of crushing the grains, the nutrient solution of the amino acid is selected as the culture solution, and the temperature is controlled to be 20-30 ℃.
10. The method of claim 1 for biodegrading mycotoxins in foodstuffs or by-products thereof, wherein the method comprises: and step eight, performing dry-wet separation on the processed grain fragments, air-drying, and introducing a lactic acid fermentation agent for anaerobic fermentation for 30-40 days.
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