Method for removing aflatoxin B1 in rice protein

Abstract

A method for removing aflatoxin B1 in rice protein comprises the following steps: primary treatment of a raw material: treating the rice protein raw material into pH alkaline semi-dry powder, preparing a conjugate into a conjugate system by adopting an entrainment regulator, and treating enzyme into an enzyme system by adopting an entrainer; fully and uniformly mixing the semi-dry powder, a conjugate system and an enzyme system to obtain a mixed material; and (2) putting the mixed material obtained in the step (2) into a high-pressure reaction kettle, and under the conditions of medium temperature and high pressure, filling a regulator to enable the material to fully react in the reaction kettle to degrade the AFB1. Under the conditions of high pressure, medium temperature and a regulator, the efficiency of enzyme catalysis substrate reaction, conjugate addition, substitution and other reactions is improved by adjusting the pressure and the temperature, so that the degradation efficiency of AFB1 under the condition of low ppb can be improved, the AFB1 is degraded to be lower than 2ppb and even is not detected, meanwhile, brown stain and charring of rice protein are avoided, and the quality of AFB1 is improved. The nutrition of the rice protein is reduced or not changed.

Description

Technical Field

[0001] The invention relates to the technical field of rice protein processing, in particular to a method for removing aflatoxin B1 in rice protein. Background Art

[0002] Aflatoxin is a highly toxic substance. Its basic structure contains difuran ring and oxaquinone (also known as coumarin). The derivatives that have been separated and identified include aflatoxin B1, B2, G1, G2, M1, M2, P1, Q1, H1, etc. Aflatoxin B1 (AFB1) is one of the most toxic and harmful biological toxins known so far. AFB1 is a colorless or light yellow crystal with a molecular formula of C 17 H 12 O6, molecular weight is 312.27.

[0003] According to the different sites of AFB1 degradation, its degradation mainly occurs in four positions: (1) the lactone ring part in the coumarin structure, which generates phenolic compounds through hydrolysis, converting AFB1 into less toxic compounds; (2) the furan ring double bond, which is the binding site with nucleic acids, proteins, etc. The furan ring double bond has a great relationship with carcinogenicity, teratogenicity and mutagenicity. This double bond reacts with H2O, hydrogen atoms, etc. to reduce the toxicity of AFB1; (3) the cyclopentenone ring, which affects the toxicity of AFB1 through addition and substitution reactions; (4) the methoxy site on the benzene ring, which reacts with hydroxyl, hydrogen atoms, and aldehyde groups to reduce its toxicity. At the same time, some degradation products of AFB1 can be converted into each other through substitution reactions.

[0004] AFB1 is the most toxic biotoxin in rice protein. The national standard currently requires that it be controlled at less than 10ppb. High-end rice protein products, infants and special medical foods require even higher levels, and less than 2ppb or no detection. AFB1 can only be degraded under ultra-high temperature (greater than 200°C), but this processing condition also causes great damage to rice protein. In addition, although ultraviolet radiation and ozone reaction have a destructive effect on AFB1, ultraviolet rays cannot penetrate solid powders, and ozone is not suitable for rice protein processing due to its poor penetration and specificity. Microbial methods are limited by controllability and the current status of commercial implementation of scaled-up production, and are not suitable for the actual production of rice protein. Enzymatic hydrolysis is a relatively effective way to degrade AFB1, but most of them are aimed at a limit of 10ppb, and it is difficult to achieve the demand for AFB1 less than 5ppb or even lower. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for removing aflatoxin B1 from rice protein.

[0006] The technical solution adopted by the present invention to solve the above technical problems is:

[0007] A method for removing aflatoxin B1 from rice protein comprises the following steps:

[0008] (1) initial treatment of raw materials: the rice protein raw material is treated into a semi-dry powder with alkaline pH, the conjugate is prepared into a conjugate system by using an entrainment regulator, and the enzyme is treated into an enzyme system by using an entrainment agent;

[0009] (2) fully mixing the semi-dry powder, the conjugate system and the enzyme system to obtain a mixed material;

[0010] (3) The mixed material obtained in step (2) is placed in a high-pressure reactor, and a regulator is added under medium temperature and high pressure conditions to allow the material to fully react in the reactor to degrade AFB1.

[0011] Further, in step (1), the rice protein raw material processing method is as follows: take rice protein powder and spray it with alkaline water to adjust the pH value to 8-9, the moisture content to 15-28%, and the rice protein raw material is rice protein to be processed.

[0012] Furthermore, the preparation method of the conjugate system is as follows: valeric aldehyde, shikonin and ocimene are used as conjugates, the mass of the conjugates is 0.15-0.3% of the rice protein raw material, 60-95% ethanol is used as an entrainment regulator, and the conjugates are placed in the entrainment regulator and fully stirred to form the conjugate system; the preparation method of the enzyme system is as follows: at least one of laccase, catalase and superoxide dismutase that catalyzes redox reactions is used, the mass of which is 0.05-0.2% of the rice protein raw material, a small amount of water is used as an entrainment agent, and the enzyme is placed in the entrainment agent and fully stirred to form the enzyme system.

[0013] Furthermore, the medium temperature and high pressure conditions in step (3) are 8-50 MPa, 35-50° C., and 1-6 h.

[0014] Furthermore, the regulator is a single or mixed agent of carbon dioxide, nitrogen and oxygen under medium temperature and high pressure conditions.

[0015] Furthermore, in step (3), the high-pressure reactor is continuously stirred or rotated to allow the mixed material to flow continuously, and the solvent is continuously countercurrently flowed in and out to allow the mixed material to fully react in the reactor.

[0016] The present application adopts valeric aldehyde, shikonin, and ocimene as the basis of the conjugate system, wherein valeric aldehyde has conjugated double bonds, epoxy conjugated double bonds, and double bonds, shikonin contains a naphthoquinone structure of p-phenol ketone conjugated double bonds, and ocimene contains a conjugated double bond structure. Valeric aldehyde, shikonin, and ocimene all have conjugated structures, but there are differences in their structures, and the double bond arrangement is different from the structure of oxygen, the redox potential is different, and the degree of reaction with AFB1 is also different. The combination can effectively improve the overall reaction efficiency and the degree of degradation of AFB1.

[0017] The high-pressure and medium-temperature reaction conditions are adopted, and the regulator is placed in a critical state by adjusting the pressure and temperature, so that the solubility of the conjugated system substance in the regulator fluid can be changed, and the efficiency of the enzyme-catalyzed substrate reaction can be greatly improved. Water, ethanol, etc. are used as entrainment agents or entrainment regulators, and the polarity and non-polarity of the entire solvent system can be adjusted, which is beneficial for the direct contact between the substrate and the reactant in the reaction. Under the high permeability of the solvent, the conjugate is efficiently contacted with aflatoxin B1 for addition, substitution and other reactions and enzymatic reactions, so as to solve the degradation efficiency of AFB1 under the situation of low ppb (<2ppb, <0.5ppb, <0.1ppb), and at the same time, the rice protein is prevented from being obviously browned and burnt, and the edibility of the rice protein is not changed, and the influence on the high-quality nutrition of the rice protein is reduced.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Under the conditions of high pressure, medium temperature and a regulator, the present invention improves the efficiency of enzyme-catalyzed substrate reaction and conjugate addition, substitution and other reactions by adjusting pressure and temperature, so that the degradation efficiency of AFB1 under low ppb conditions can be solved, and AFB1 can be degraded to less than 2 ppb or even undetectable, while avoiding browning and burning of rice protein, and reducing or not changing the nutritional value of rice protein. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. In the following embodiments, the same batch of raw materials are used for processing and testing.

[0021] Example 1

[0022] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8, and moisture 15% obtains rice protein semi-dry powder, adopts rice protein powder quality 4% ethanol (volume fraction 60%) to carry out solubilizing conjugate system (valeric aldehyde 0.05%+shikonin 0.05%+ocimene 0.05%), and uses the laccase of 0.1% of water dissolution for rice protein powder quality 1%, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, reload autoclave, at 8.5mpa, under 38 ℃, be charged with the mixture of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make the mixture countercurrent flow inflow out, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 1.7pbb from 9.9ppb.

[0023] Example 2

[0024] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8.5, and 21% of moisture obtains rice protein semi-dry powder, adopts 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (valerian aldehyde 0.08%+shikonin 0.08%+ocimene 0.08%), and dissolves 0.05% laccase+0.05% catalase with 1% water, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, then load into autoclave, at 30mpa, under 60 ℃, charge with the mixture of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 1.2pbb from 9.9ppb.

[0025] Example 3

[0026] Rice protein raw material is sprayed with alkaline water and is adjusted to pH9, and 28% of moisture obtains rice protein semi-dry powder, adopts 4% ethanol (volume fraction 90%) to carry out solubilizing conjugate system (valeric aldehyde 0.1%+shikonin 0.1%+ocimene 0.1%), and dissolves 0.1% laccase+0.1% catalase with 1% water, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, then load into autoclave, at 50mpa, under 80 ℃, charge with the mixed solvent of carbon dioxide 95%, nitrogen 2%, oxygen 3%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 0.45pbb from 9.9ppb.

[0027] Comparative Example 1

[0028] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8.5, and 21% of moisture obtains rice protein semi-dry powder, adopts 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (shikonin 0.08%+ocimene 0.08%), and dissolves 0.05% laccase+0.05% catalase with 1% water, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, then load into autoclave, at 30mpa, under 60 ℃, charge with the mixed solvent of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 2.5pbb from 9.9ppb.

[0029] Comparative Example 2

[0030] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8.5, and 21% of moisture obtains rice protein semi-dry powder, adopts 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (valerian aldehyde 0.08%+ocimene 0.08%), and dissolves 0.05% laccase+0.05% catalase with 1% water, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, then load into autoclave, at 30mpa, under 60 ℃, charge with the mixed solvent of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 2.8pbb from 9.9ppb.

[0031] Comparative Example 3

[0032] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8.5, and 21% of moisture obtains rice protein semi-dry powder, adopts 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (valerian aldehyde 0.08%+shikonin 0.08%), and dissolves 0.05% laccase+0.05% catalase with 1% water, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, then load into autoclave, at 30mpa, under 60 ℃, charge with the mixed solvent of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 3.5pbb from 9.9ppb.

[0033] Comparative Example 4

[0034] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8.5, and 21% of moisture obtains rice protein semi-dry powder, adopts 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (valerian aldehyde 0.08%+shikonin 0.08%+ocimene 0.08%), and dissolves 0.05% laccase+0.05% catalase with 1% water, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, then load into autoclave, at normal pressure, under 60 ℃, charge with the mixed solvent of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to high-efficiency contact and enzymatic reaction, aflatoxin B1 is reduced to 8.8pbb from 9.9ppb.

[0035] Comparative Example 5

[0036] Rice protein raw material alkaline water spray is adjusted to pH8.5, moisture 21% obtains rice protein semi-dry powder, adopt 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (valerian aldehyde 0.08%+shikonin 0.08%+ocimene 0.08%), and use 1% water dissolution 0.05% laccase+0.05% catalase, and conjugate system solution, enzyme system solution and rice protein semi-dry powder are mixed, pack into autoclave again, at 30mpa, under 140 ℃, be charged with the mixed solvent of carbon dioxide 90%, nitrogen 5%, oxygen 5%, keep pressure to react 4h.But because temperature of charge is too high, during discharging, rice protein can't keep original edible state, so the detection of B1 is also meaningless.

[0037] Comparative Example 6

[0038] Rice protein raw material is sprayed with alkaline water and is adjusted to pH8.5, and moisture 21% obtains rice protein semi-dry powder, adopts 3% ethanol (volume fraction 75%) to carry out solubilizing conjugate system (valeric aldehyde 0.08%+shikonin 0.08%+ocimene 0.08%), conjugate system solution and rice protein semi-dry powder are mixed, then load into autoclave, at 30mpa, under 60 ℃, charge with the mixed solvent of carbon dioxide 90%, nitrogen 5%, oxygen 5%, and stir or rotate reactor, constantly make solvent countercurrent flow inflow and outflow, make material react 4h in reactor.Under the high permeability effect of solvent, conjugate and aflatoxin B1 are carried out to contact and reaction efficiently, aflatoxin B1 is reduced to 7.1pbb from 9.9ppb.

[0039] The present application adopts valeric aldehyde, shikonin, and ocimene as the basis of the conjugate system, wherein valeric aldehyde has conjugated double bonds, epoxy conjugated double bonds and double bonds, shikonin contains a conjugated double bond of a naphthoquinone structure to a phenol ketone, and ocimene contains a conjugated double bond structure. Valeric aldehyde, shikonin, and ocimene all have conjugated structures, but there are differences in their structures, and the double bond arrangement is also different from the structure of oxygen, the redox potential is different, and the degree of reaction with AFB1 is also different. Its combination can effectively improve the overall reaction efficiency and the degree of degradation of AFB1. In comparative examples 1-3, the lack of any one of the conjugates leads to the loss of effective reaction substrate groups in the conjugate system, reducing their mutual synergistic and complementary effects, so reducing the degradation efficiency of AFB1.

[0040] By adopting high pressure and medium temperature reaction conditions, the solubility of the conjugated system substance in the regulator fluid can be changed by adjusting the pressure and temperature, greatly improving the efficiency of the enzyme-catalyzed substrate reaction, and then supplemented with water, ethanol, etc. as entrainers or entrainment regulators, the polarity and non-polarity of the entire solvent system can be adjusted, which is conducive to the direct contact between the substrate and the reactant in the reaction. Under the high permeability of the solvent, the conjugate is efficiently contacted with aflatoxin B1 for addition, substitution and other reactions and enzymatic reactions to solve the degradation efficiency of AFB1 under low ppb (<2ppb, <0.5ppb, <0.1ppb). In Comparative Example 4, under normal pressure reaction conditions, the state of the regulator is only gaseous, the density of the regulator is small, the viscosity is low, and the polar interaction is weak, resulting in its low solubility for the reaction substrate, entrainer and enzyme, etc., and low mass transfer efficiency, thereby greatly reducing the reaction speed, so that the degradation efficiency of AFB1 is greatly discounted.

[0041] In Comparative Example 5, under high temperature conditions, the material undergoes obvious denaturation, the semi-dry powder forms hard lumps, and the color undergoes obvious browning, which is no longer acceptable for use as food.

[0042] As a catalyst, the enzyme can significantly reduce the activation energy required for the reaction, thereby greatly improving the enzymatic efficiency. In Comparative Example 6, in the absence of the enzyme, the reaction efficiency was greatly reduced, so the degradation efficiency of AFB1 was significantly reduced.

[0043] It should be understood that the present invention is not limited to the forms disclosed herein, nor should it be regarded as excluding other embodiments. Modifications and changes made by technicians in this field without departing from the spirit and scope of the present invention should be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for removing aflatoxin B1 from rice protein, characterized in that, The following steps are involved: (1) initial treatment of raw materials: the rice protein raw material is treated into a semi-dry powder with alkaline pH, the conjugate is prepared into a conjugate system by using an entrainment regulator, and the enzyme is treated into an enzyme system by using an entrainment agent; (2) fully mixing the semi-dry powder, the conjugate system and the enzyme system to obtain a mixed material; (3) The mixed material obtained in step (2) is placed in a high-pressure reactor, and a regulator is added under medium temperature and high pressure conditions to allow the material to fully react in the reactor to degrade AFB1.

2. The method for removing aflatoxin B1 in rice protein according to claim 1, wherein: In step (1), the rice protein raw material processing method is as follows: take rice protein powder and spray it with alkaline water to adjust the pH value to 8-9, with a moisture content of 15-28%.

3. the method for removing aflatoxin B1 in rice protein according to claim 1, is characterized in that: The medium temperature and high pressure conditions described in step (3) are 8-50 MPa, 35-50° C., and 1-6 h.

4. The method for removing aflatoxin B1 in rice protein according to claim 1, wherein: The regulator is a single or mixed agent of carbon dioxide, nitrogen and oxygen under medium temperature and high pressure conditions.

5. The method for removing aflatoxin B1 in rice protein according to claim 1, wherein: In step (3), the high-pressure reactor is continuously stirred or rotated to allow the mixed material to flow continuously, and the solvent is continuously counter-currently flowed in and out to allow the mixed material to fully react in the reactor.