阅读说明：本技术 一种大豆脱毒的方法及脱毒的大豆 (Method for detoxifying soybeans and detoxified soybeans ) 是由 郑小娟 付夏娜 余佳 王翠霞 唐丹 王灿 张瑞华 于 2020-12-18 设计创作，主要内容包括：本发明提出了一种大豆脱毒的方法,将大豆处理后浸泡于复合微生物菌液中,微波处理,发酵,再加入钝化剂处理后,有机溶液蒸汽处理,干燥,得到脱毒大豆。本发明采用微波辅助,钝化剂参与反应,从而钝化抗营养因子,复合微生物菌液进行发酵脱毒,进一步改善大豆口感和营养,化学溶剂+物理蒸汽脱除残余抗营养因子,方法简单,能够对大豆起到有效脱毒,且不会重新引发毒性,可以实现长效脱毒的效果。(The invention provides a method for detoxifying soybeans, which comprises the steps of soaking the processed soybeans in a compound microbial liquid, performing microwave treatment, fermenting, adding a passivator for treatment, performing steam treatment on an organic solution, and drying to obtain detoxified soybeans. The invention adopts microwave assistance, the passivator participates in the reaction, thereby passivating the anti-nutritional factors, the compound microbial liquid is fermented and detoxified, the taste and nutrition of the soybeans are further improved, the residual anti-nutritional factors are removed by chemical solvent and physical steam, the method is simple, the soybeans can be effectively detoxified, the toxicity is not caused again, and the effect of long-acting detoxification can be realized.)

1. A method for detoxifying soybeans is characterized in that soybeans are treated and then soaked in a compound microbial liquid, subjected to microwave treatment and fermentation, then added with a passivating agent for treatment, subjected to steam treatment by using an organic solution, and dried to obtain detoxified soybeans.

2. The method for detoxifying soybeans according to claim 1, comprising the steps of:

s1, cleaning and drying soybeans, and then polishing the surfaces of the soybeans;

s2, dissolving a carbon source, a nitrogen source, vitamins and inorganic salts with sterile water, uniformly mixing, adjusting the pH value of a culture medium with a PBS (phosphate buffer solution), and sterilizing with ultraviolet rays for later use to obtain a nutrient solution;

s3, respectively inoculating bifidobacterium, lactobacillus bulgaricus, candida utilis and bacillus licheniformis into a Gao's culture medium for streaking, carrying out anaerobic culture, and then respectively culturing into strain seed solutions;

s4, respectively inoculating the strain seed liquid prepared in the step S3 into the nutrient solution obtained in the step S2, carrying out anaerobic culture, mixing and diluting to obtain a compound microorganism bacterium liquid;

s5, adding the soybeans processed in the step S1 into the compound microbial liquid in the step S4, completely soaking the processed soybeans by the compound microbial liquid, performing microwave treatment, controlling the temperature of a bacterial liquid, and performing fermentation treatment for a first time period to obtain a first detoxified soybean system;

s6, adding cysteine and ficin into the first detoxified soybean system in the step S5, keeping the original treatment conditions, continuing to treat for a second time period, and filtering to obtain second detoxified soybeans;

s7, dissolving glycerol, citric acid and ethylenediamine in water, and uniformly mixing to obtain a mixed solution;

s8, filling the mixed liquid obtained in the step S7 into a container, placing the second detoxified soybeans obtained in the step S6 into an upper-layer sieve net, covering the upper-layer sieve net with a cover, installing a condenser pipe, heating, and performing steam treatment for a third time period to obtain third detoxified soybeans;

s9, performing microwave drying on the third detoxified soybean;

s10, performing ultraviolet sterilization on the soybeans after microwave drying, weighing and packaging.

3. The method for detoxifying soybean according to claim 2, wherein the carbon source is selected from one or more of glucose, maltose, lactose, sucrose, fructose, starch, molasses; the nitrogen source is selected from ammonia water, urea, ammonium salt, nitrate and amino acid; the vitamins are selected from one or more of vitamin C, vitamin B1, vitamin B2, vitamin A, vitamin K, vitamin B12, vitamin D and vitamin E; the inorganic salt is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride.

4. The method of soybean detoxification according to claim 3, wherein the molasses comprises cane molasses, beet molasses, in a mass ratio of 1: (1-5); the amino acid is selected from one or more of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine and phenylalanine.

5. The method for detoxifying soybean according to claim 4, wherein the mass ratio of said carbon source, nitrogen source, vitamins and inorganic salts in step S2 is (12-20): (3-7): (0.5-1): (0.1-0.5).

6. The method for detoxification of soybean according to claim 2, wherein the pH is adjusted to 6.5-7.5 in step S2; in step S3, the bifidobacteria, Lactobacillus bulgaricus and Candida utilis are addedThe mass ratio of mother bacteria to bacillus licheniformis is 10: (2-5): (1-3): (2-7); the anaerobic culture condition is 27-35 ℃, the humidity is 75-85%, and the culture lasts for 1-2 days; the strain seed liquid has a bacteria content of 10⁸-10⁹cfu/mL。

7. The method for detoxifying soybeans according to claim 2, wherein the inoculation amounts of the bifidobacterium, the lactobacillus bulgaricus, the candida utilis and the bacillus licheniformis in step S4 are 2-5% (v/v), 1-3% (v/v), 1-4% (v/v) and 2-4% (v/v), respectively, the anaerobic culture conditions are 27-35 ℃, the humidity is 75-85%, the culture is carried out for 2-4 days, and the dilution factor is 1000 times that of 100-.

8. The method for detoxifying soybean as claimed in claim 2, wherein the microwave treatment power in step S5 is 1000-2000W, the temperature of the bacterial liquid is controlled between 27-37 ℃, and the first time period is 2-3 h; in step S6, the addition amounts of cysteine and ficin are 5-12 wt% and 10-17 wt%, respectively, and the second time period is 1-2 h.

9. The method for detoxifying soybean according to claim 2, wherein the mass ratio of glycerol, citric acid, ethylenediamine and water in step S7 is (2-5): (1-7): (2-4): 100, respectively; the third time period in the step S8 is 10-20min, and the power of the microwave drying in the step S9 is 200-.

10. A detoxified soybean produced by the method of soybean detoxification as claimed in any of claims 1 to 9.

Technical Field

The invention relates to the technical field of agricultural product processing, in particular to a soybean detoxification method and detoxified soybeans.

Background

Soybeans and soybean products are one of the important food for Chinese people, and play an extremely important role in the reproduction and the rest of Chinese nationality. Since the beginning of the 50 s of the last century, in order to make up for the shortage of protein nutrition in food, soybean protein nutrition has been vigorously developed as a donor of new proteins and widely used and studied in many countries.

According to the book of the Chinese light industry publishing company, namely Soybean product technology: about 7-10 kinds of tryptatins are contained in soybean, and two kinds of tryptase have been purified by scientists so far, and the rest of the tryptatins cannot be extracted so far. The heat stability of the trypsin inhibin is very high, the residual activity of the trypsin inhibin is still more than 89% when the soybeans are heated at 100 ℃ for 60 minutes, and the activity of the trypsin inhibin cannot be obviously reduced by increasing the heat treatment time. This is the most interesting problem in the soybean production process. Soybean is food which can not be eaten by human beings, and human beings need to take protein nutrition from the soybean by heat processing, because of the existence of trypsin inhibin in the soybean, once the soybean is heated to 55-65 ℃, the trypsin inhibin generates heat denaturation by chemical and biological actions such as high temperature, high acid, high alkali and the like through catalysis of the soybean protein, thereby greatly destroying the natural structure of the soybean protein and leading part or most of the protein to be denatured and disappear. As long as trypsin inhibin remains in soybeans and bean products, when people eat whole soybeans, the following phenomena occur: the digestibility is less than 35 percent, most of the rest bean slice components are discharged out of the body from excrement, so that dyspepsia is caused, and the soybean and bean products are also difficult to eat by part of people; however, when people eat soybeans and bean products into intestines and stomach, the trypsin inhibitor not only prevents the digestion and absorption of protein nutrition of the soybeans, but also destroys and prevents the digestion and absorption of other protein nutrition in the body, and also generates toxicity which is harmful to human health and can cause slight food poisoning of eaters, wherein the poisoning symptoms mainly comprise abdominal pain, nausea, stomach distension, diarrhea, dizziness and malnutrition and cause various diseases.

CN102511746B discloses a detoxification curing technology for whole soybeans, which comprises polishing whole soybeans, putting the polished soybeans into a distillation retort under the condition of no hydrophilicity, connecting the distillation retort with a steam source, and adding ethanol into water of the steam source to perform detoxification distillation under normal pressure; adding acetic acid into water of a steam source, and distilling; then, ethanol was further added to the steam source, followed by distillation. And packaging to obtain the product. Although the method can effectively reduce trypsin inhibitors and purine substances in the soybeans, the effect is poor, and substances dissolved in acetic acid solution or ethanol solution in the soybeans are dissolved along with the distillation process, so that the nutritional ingredients in the soybeans are reduced.

CN 107183543 a discloses a quantum detoxification curing method for whole soybeans, which comprises the following steps: the method comprises the steps of material selection, quantum permeation, quantum resonance entanglement, quantum rapid hydrophilic treatment, rapid drying, ethanol detoxification distillation and acetic acid distillation. The method can reduce anti-nutritional factors and harmful components in the soybean, but can not effectively improve the beany flavor of the soybean and improve the solubility of the soybean protein, thereby improving the mouthfeel and nutrition of the soybean.

Therefore, the development of a method which can remove most of toxins in soybeans by a simple and efficient method and can keep the high nutritional value of the soybeans so that the soybeans which can be eaten by people safely have wide application prospect.

Disclosure of Invention

The invention aims to provide a soybean detoxification method and detoxified soybeans, wherein microwave assistance is adopted, a passivating agent participates in reaction, so that anti-nutritional factors are passivated, the compound microbial liquid is fermented and detoxified, the taste and nutrition of the soybeans are further improved, residual anti-nutritional factors are removed by using a chemical solvent and physical steam, the method is simple, the soybeans can be effectively detoxified, toxicity is not caused again, and the effect of long-acting detoxification can be realized.

The technical scheme of the invention is realized as follows:

the invention provides a method for detoxifying soybeans, which comprises the steps of treating soybeans, soaking the treated soybeans in a compound microbial liquid, performing microwave treatment, fermenting, adding a passivating agent for treatment, performing steam treatment on an organic solution, and drying to obtain detoxified soybeans.

As a further improvement of the invention, the method specifically comprises the following steps:

s1, cleaning and drying soybeans, and then polishing the surfaces of the soybeans;

s2, dissolving a carbon source, a nitrogen source, vitamins and inorganic salts with sterile water, uniformly mixing, adjusting the pH value of a culture medium with a PBS (phosphate buffer solution), and sterilizing with ultraviolet rays for later use to obtain a nutrient solution;

s3, respectively inoculating bifidobacterium, lactobacillus bulgaricus, candida utilis and bacillus licheniformis into a Gao's culture medium for streaking, carrying out anaerobic culture, and then respectively culturing into strain seed solutions;

s4, respectively inoculating the strain seed liquid prepared in the step S3 into the nutrient solution obtained in the step S2, carrying out anaerobic culture, mixing and diluting to obtain a compound microorganism bacterium liquid;

s5, adding the soybeans processed in the step S1 into the compound microbial liquid in the step S4, completely soaking the processed soybeans by the compound microbial liquid, performing microwave treatment, controlling the temperature of a bacterial liquid, and performing fermentation treatment for a first time period to obtain a first detoxified soybean system;

s6, adding cysteine and ficin into the first detoxified soybean system in the step S5, keeping the original treatment conditions, continuing to treat for a second time period, and filtering to obtain second detoxified soybeans;

s7, dissolving glycerol, citric acid and ethylenediamine in water, and uniformly mixing to obtain a mixed solution;

s8, filling the mixed liquid obtained in the step S7 into a container, placing the second detoxified soybeans obtained in the step S6 into an upper-layer sieve net, covering the upper-layer sieve net with a cover, installing a condenser pipe, heating, and performing steam treatment for a third time period to obtain third detoxified soybeans;

s9, performing microwave drying on the third detoxified soybean;

s10, performing ultraviolet sterilization on the soybeans after microwave drying, weighing and packaging.

As a further improvement of the invention, the carbon source is selected from one or a mixture of more of glucose, maltose, lactose, sucrose, fructose, starch and molasses; the nitrogen source is selected from ammonia water, urea, ammonium salt, nitrate and amino acid; the vitamins are selected from one or more of vitamin C, vitamin B1, vitamin B2, vitamin A, vitamin K, vitamin B12, vitamin D and vitamin E; the inorganic salt is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride.

As a further improvement of the invention, the molasses comprises cane molasses and beet molasses, and the mass ratio of the cane molasses to the beet molasses is 1: (1-5); the amino acid is selected from one or more of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine and phenylalanine.

As a further improvement of the invention, the mass ratio of the carbon source, the nitrogen source, the vitamins and the inorganic salt is (12-20): (3-7): (0.5-1): (0.1-0.5).

As a further improvement of the invention, the pH value is adjusted to 6.5-7.5 in step S2; in the step S3, the mass ratio of the bifidobacteria to the lactobacillus bulgaricus to the candida utilis to the bacillus licheniformis is 10: (2-5): (1-3): (2-7); the anaerobic culture condition is 27-35 ℃, the humidity is 75-85%, and the culture lasts for 1-2 days; the strain seed liquid has a bacteria content of 10⁸-10⁹cfu/mL。

As a further improvement of the invention, the inoculation amounts of the bifidobacterium, the lactobacillus bulgaricus, the candida utilis and the bacillus licheniformis in the step S4 are respectively 2-5% (v/v), 1-3% (v/v), 1-4% (v/v) and 2-4% (v/v), the anaerobic culture condition is 27-35 ℃, the humidity is 75-85%, the culture is carried out for 2-4 days, and the dilution factor is 1000 times of 100.

As a further improvement of the invention, the microwave treatment power in step S5 is 1000-2000W, the temperature of the bacterial liquid is controlled between 27-37 ℃, and the first time period is 2-3 h; in step S6, the addition amounts of cysteine and ficin are 5-12 wt% and 10-17 wt%, respectively, and the second time period is 1-2 h.

In a further improvement of the present invention, in step S7, the mass ratio of glycerol, citric acid, ethylenediamine, and water is (2-5): (1-7): (2-4): 100, respectively; the third time period in the step S8 is 10-20min, and the power of the microwave drying in the step S9 is 200-.

The invention further protects a detoxified soybean prepared by the method for detoxifying soybeans.

The invention has the following beneficial effects: the trypsin inhibin in soybean is mainly divided into 2 types, namely Kunitz (Kunitz) trypsin inhibin (KTI) and Bowman-Birk inhibin (BBI), wherein the BBI is a molecule formed by crosslinking 7 disulfide bonds, and the 7 disulfide bonds endow the stability of the molecular structure, therefore, the BBI is extremely stable to heat, acid, alkali and the like, the invention adopts a mixed microbial preparation to detoxify whole soybean, can efficiently decompose complex and variable disulfide bonds and intermediate products thereof due to the complicated enzyme system of the microbe, and finally can thoroughly decompose the trypsin inhibin in the soybean, and can decompose other anti-nutritional factors, such as phytic acid, tannin, cellulose and the like, thereby greatly improving the taste of vegetable soybean, improving the utilization rate of nutritional ingredients in the soybean, and finally, the microbe utilizes the self-metabolism, can generate flavor substance or flavor precursor substance to improve flavor of detoxified semen glycines.

According to the trypsin inhibitor, the passivating agent cysteine and the ficin are added, the ficin is thiol protease, the trypsin inhibitor can be effectively passivated by the synergy of the cysteine and the ficin, and the disulfide bond is cracked due to the exchange reaction of thiol and disulfide, so that the trypsin inhibitor is effectively decomposed and removed; under the microwave treatment condition, the invention can passivate anti-nutritional factors in the soybeans, such as purine, trypsin inhibin and the like, thereby assisting in reducing the toxicity of the soybeans; the invention also adopts a chemical solvent and physical steam combined method for further detoxification treatment, trypsin inhibin and other anti-nutritional factors are dissolved in high-temperature steam with a chemical solvent at high temperature, part of the high-temperature decomposition generates volatile substances to be dissipated along with the steam, and part of the high-temperature decomposition reacts with organic substances, such as amine can react with a sulfur bond, thereby achieving the effects of effectively detoxifying and improving the taste.

The invention adopts microwave assistance, the passivator participates in the reaction, thereby passivating the anti-nutritional factors, the compound microbial liquid is fermented and detoxified, the taste and nutrition of the soybeans are further improved, the residual anti-nutritional factors are removed by chemical solvent and physical steam, the method is simple, the soybeans can be effectively detoxified, the toxicity is not caused again, and the effect of long-acting detoxification can be realized.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1A method for detoxification of soybeans

The method specifically comprises the following steps:

s1, cleaning and drying soybeans, and then polishing the surfaces of the soybeans;

s2, dissolving 12g of sucrose, 3g of alanine, 0.5g of vitamin E and 0.1g of zinc sulfate in 100mL of sterile water, uniformly mixing, adjusting the pH value of a culture medium to 6.5 by using a PBS (phosphate buffer solution), and sterilizing by using ultraviolet rays for later use to obtain a nutrient solution;

s3, respectively inoculating 10g of bifidobacterium, 2g of lactobacillus bulgaricus, 1g of candida utilis and 2g of bacillus licheniformis into a Gao's medium for streaking, carrying out anaerobic culture under the conditions of 27 ℃ and 75% of humidity for 1 day, and respectively culturing to obtain strain seed solutions with the strain content of 10⁸cfu/mL；

S4, respectively inoculating the strain seed solutions prepared in the step S3 into the nutrient solution obtained in the step S2, wherein the inoculation amounts of bifidobacterium, lactobacillus bulgaricus, candida utilis and bacillus licheniformis are respectively 2% (v/v), 1% (v/v) and 2% (v/v), carrying out anaerobic culture under the anaerobic culture condition of 27 ℃ and the humidity of 75%, culturing for 2 days, mixing and diluting by 100 times to obtain a composite microbial strain solution;

s5, adding the soybeans processed in the step S1 into the compound microbial liquid in the step S4, completely soaking the processed soybeans by the compound microbial liquid, performing microwave treatment with the power of 1000W, controlling the temperature of a bacterial liquid at 27 ℃, and performing fermentation treatment for 2 hours to obtain a first detoxified soybean system;

s6, adding cysteine and ficin into the first detoxified soybean system in the step S5, wherein the addition amounts are 5% and 10% of the total mass of the system respectively, keeping the original treatment conditions for continuous treatment for 1h, and filtering to obtain second detoxified soybeans;

s7, dissolving 2-5g of glycerol, 1g of citric acid and 2g of ethylenediamine in 100g of water, and uniformly mixing to obtain a mixed solution;

s8, filling the mixed liquid obtained in the step S7 into a container, placing the second detoxified soybeans obtained in the step S6 into an upper-layer sieve mesh, covering the upper-layer sieve mesh with a cover, installing a condensing tube, heating, and performing steam treatment for 10min to obtain third detoxified soybeans;

s9, performing microwave drying on the third detoxified soybean at the power of 200W;

s10, performing ultraviolet sterilization on the soybeans after microwave drying, weighing and packaging.

Example 2A method for detoxification of soybeans

The method specifically comprises the following steps:

s1, cleaning and drying soybeans, and then polishing the surfaces of the soybeans;

s2, dissolving 20g of maltose, 3g of threonine, 4g of tryptophan, 1g of vitamin B12 and 0.5g of calcium chloride in 100mL of sterile water, uniformly mixing, adjusting the pH value of a culture medium to 7.5 by using a PBS solution, and sterilizing by ultraviolet rays for later use to obtain a nutrient solution;

s3, respectively inoculating 10g of bifidobacterium, 5g of lactobacillus bulgaricus, 3g of candida utilis and 7g of bacillus licheniformis into a Gao's medium for streaking, carrying out anaerobic culture under the conditions of 35 ℃ and 85% humidity for 2 days,then respectively culturing to obtain strain seed liquid with a strain content of 10⁹cfu/mL；

S4, respectively inoculating the strain seed solutions prepared in the step S3 into the nutrient solution obtained in the step S2, wherein the inoculation amounts of bifidobacterium, lactobacillus bulgaricus, candida utilis and bacillus licheniformis are respectively 5% (v/v), 3% (v/v), 4% (v/v) and 4% (v/v), carrying out anaerobic culture under the anaerobic culture condition of 35 ℃ and 85% of humidity for 4 days, mixing and diluting by 1000 times to obtain a composite microbial strain solution;

s5, adding the soybeans processed in the step S1 into the compound microbial liquid in the step S4, completely soaking the processed soybeans by the compound microbial liquid, performing microwave treatment with the power of 2000W, controlling the temperature of a bacterial liquid at 37 ℃, and performing fermentation treatment for 3 hours to obtain a first detoxified soybean system;

s6, adding cysteine and ficin into the first detoxified soybean system in the step S5, wherein the addition amounts are 12% and 17% of the total mass of the system respectively, keeping the original treatment conditions for continuous treatment for 2h, and filtering to obtain second detoxified soybean;

s7, dissolving 5g of glycerol, 7g of citric acid and 4g of ethylenediamine in 100g of water, and uniformly mixing to obtain a mixed solution;

s8, filling the mixed liquid obtained in the step S7 into a container, placing the second detoxified soybeans obtained in the step S6 into an upper-layer sieve mesh, covering the upper-layer sieve mesh with a cover, installing a condensing tube, heating, and performing steam treatment for 20min to obtain third detoxified soybeans;

s9, performing microwave drying on the third detoxified soybean with the power of 400W;

s10, performing ultraviolet sterilization on the soybeans after microwave drying, weighing and packaging.

Example 3A method for detoxification of soybeans

The method specifically comprises the following steps:

s1, cleaning and drying soybeans, and then polishing the surfaces of the soybeans;

s2, dissolving 16g of molasses (the molasses is cane molasses and beet molasses in a mass ratio of 1: 3), 5g of urea, 0.3g of vitamin A, 0.4g of vitamin B1 and 0.3g of magnesium sulfate in 100mL of sterile water, uniformly mixing, adjusting the pH value of a culture medium to 7 by using a PBS solution, and performing ultraviolet sterilization for later use to obtain a nutrient solution;

s3, respectively inoculating 10g of bifidobacterium, 3.5g of lactobacillus bulgaricus, 2g of candida utilis and 5g of bacillus licheniformis into a Gao's medium for streaking, carrying out anaerobic culture under the conditions of 30 ℃ and 80% of humidity for 1.5 days, and respectively culturing to obtain strain seed solutions with the bacterium content of 10⁸cfu/mL；

S4, respectively inoculating the strain seed solutions prepared in the step S3 into the nutrient solution obtained in the step S2, wherein the inoculation amounts of bifidobacterium, lactobacillus bulgaricus, candida utilis and bacillus licheniformis are respectively 3.5% (v/v), 2% (v/v), 2.5% (v/v) and 3% (v/v), anaerobic culture is carried out for 3 days under the anaerobic culture condition of 30 ℃ and the humidity of 80%, and the composite microbial strain solution is obtained after mixing and diluting by 500 times;

s5, adding the soybeans processed in the step S1 into the compound microbial liquid in the step S4, completely soaking the processed soybeans by the compound microbial liquid, performing microwave treatment with the power of 1500W, controlling the temperature of the liquid at 32 ℃, and performing fermentation treatment for 2.5 hours to obtain a first detoxified soybean system;

s6, adding cysteine and ficin into the first detoxified soybean system in the step S5, wherein the addition amounts are 8% and 14% of the total mass of the system respectively, keeping the original treatment conditions for continuous treatment for 1.5h, and filtering to obtain second detoxified soybean;

s7, dissolving 3.5g of glycerol, 5g of citric acid and 3g of ethylenediamine in 100g of water, and uniformly mixing to obtain a mixed solution;

s8, filling the mixed liquid obtained in the step S7 into a container, placing the second detoxified soybeans obtained in the step S6 into an upper-layer sieve mesh, covering the upper-layer sieve mesh with a cover, installing a condensing tube, heating, and performing steam treatment for 15min to obtain third detoxified soybeans;

s9, performing microwave drying on the third detoxified soybean, wherein the power is 300W;

s10, performing ultraviolet sterilization on the soybeans after microwave drying, weighing and packaging.

Comparative example 1

In step S1, the polishing treatment was not performed, and other conditions were not changed, as compared with example 3.

S1, cleaning and drying soybeans.

Comparative example 2

Compared with the example 3, no bacillus licheniformis is added in the step S3, and other conditions are not changed.

S3, respectively inoculating 15g of bifidobacterium, 3.5g of lactobacillus bulgaricus and 2g of candida utilis into a Gao's medium for streaking, carrying out anaerobic culture under the conditions of 30 ℃ and 80% of humidity for 1.5 days, and respectively culturing to obtain strain seed solutions with the strain content of 10⁸cfu/mL。

S4, respectively inoculating the strain seed liquid prepared in the step S3 into the nutrient solution obtained in the step S2, wherein the inoculation amounts of bifidobacterium, lactobacillus bulgaricus and candida utilis are respectively 6.5% (v/v), 2% (v/v), 2.5% (v/v) and 3% (v/v), carrying out anaerobic culture under the anaerobic culture condition of 30 ℃ and the humidity of 80% for 3 days, mixing and diluting by 500 times to obtain a composite microbial strain liquid;

comparative example 3

In step S3, no bifidobacterium was added as compared with example 3, and the other conditions were not changed.

S3, respectively inoculating 3.5g of lactobacillus bulgaricus, 2g of candida utilis and 5g of bacillus licheniformis into a Gao's medium for streaking, carrying out anaerobic culture under the conditions of 30 ℃ and 80% of humidity for 1.5 days, and respectively culturing to obtain strain seed solutions with the strain content of 10⁸cfu/mL。

S4, respectively inoculating the strain seed solutions prepared in the step S3 into the nutrient solution obtained in the step S2, wherein the inoculation amounts of lactobacillus bulgaricus, candida utilis and bacillus licheniformis are respectively 2% (v/v), 2.5% (v/v) and 6.5% (v/v), carrying out anaerobic culture under the conditions of 30 ℃ and 80% of humidity for 3 days, and mixing and diluting by 500 times to obtain a composite microbial strain solution;

comparative example 4

In step S5, the microwave treatment was not performed, and other conditions were not changed, as compared with example 3.

S5, adding the soybeans processed in the step S1 into the compound microbial liquid in the step S4, completely soaking the processed soybeans by the compound microbial liquid, controlling the temperature of the liquid to be 32 ℃, and fermenting for 2.5 hours to obtain a first detoxified soybean system.

Comparative example 5

In comparison with example 3, no cysteine was added in step S6, and the other conditions were not changed.

S6, adding the ficin into the first detoxified soybean system in the step S5, wherein the adding amount is 22% of the total mass of the system, keeping the original treatment condition for continuous treatment for 1.5h, and filtering to obtain the second detoxified soybean.

Comparative example 6

In step S6, ficin was not added, and other conditions were not changed, as compared with example 3.

S6, adding cysteine into the first detoxified soybean system in the step S5, wherein the adding amount is 22% of the total mass of the system, keeping the original treatment condition, continuing to treat for 1.5h, and filtering to obtain second detoxified soybeans.

Comparative example 7

In step S7, ethylene diamine was not added, and the other conditions were not changed, as compared with example 3.

S7, dissolving 6.5g of glycerol and 5g of citric acid in 100g of water, and uniformly mixing to obtain a mixed solution.

Comparative example 8

In step S7, glycerin was not added, and other conditions were not changed, as compared with example 3.

S7, dissolving 5g of citric acid and 6.5g of ethylenediamine in 100g of water, and uniformly mixing to obtain a mixed solution.

Comparative example 9

Compared with the embodiment 3, the microwave drying is not carried out in the step S9, the drying is carried out by adopting a common oven, and other conditions are not changed.

S9, drying the third detoxified soybean at 45 ℃.

Test example 1 detection of anti-nutrient substance

The test object is: detoxified soybeans prepared in examples 1 to 3 and comparative examples 1 to 9 and commercially available normal soybeans.

And (4) checking items: hypoxanthine nucleotide, guanine nucleotide, adenine nucleotide, trypsin inhibitor activity.

The detection method comprises the following steps:

trypsin inhibitor activity: GB/T21498-2008;

hypoxanthine nucleotide, guanine nucleotide, and adenine nucleotide, HPLC (determination of active ingredients in functional food, chemical industry Press, chapter ninth and twenty third paragraphs of 2005).

And (4) testing results: see table 1.

TABLE 1

As can be seen from the table above, the detoxified soybeans prepared by the embodiment of the invention can obviously reduce the anti-nutritional factors in the soybeans, the purine content of the detoxified soybeans is reduced to 3.82-4.45mg/100g, the trypsin inhibitor content of the detoxified soybeans is reduced to less than 0.2mg/g, and compared with common soybeans, most of the anti-nutritional factors are removed, so that the standard of qualified detoxified soybeans is reached.

Test example 2 detection of ingredients of beany flavor

The test object is: detoxified soybeans prepared in examples 1 to 3 and comparative examples 1 to 9 and commercially available normal soybeans.

And (4) checking items: a beany flavor component.

The detection method comprises the following steps:

and determining the beany flavor components by adopting HS-SPME-GC-MS. Crushing a soybean sample, and sieving the crushed soybean sample with a 40-mesh sieve to obtain the soybean powder. Placing 2g soybean powder and 3mL deionized water in a headspace bottle, adding 10 μ L p-dichlorobenzene-methanol solution (internal standard substance) with mass concentration of 0.12mg/mL, mixing, water bathing at 50 deg.C for 30min, adsorbing with extraction head for 40min, and desorbing at 250 deg.C for 5 min. GC conditions were as follows: SH-Rxi-5 SiLMS chromatographic column; the temperature raising program is that the temperature is kept for 1min at 50 ℃, is raised to 150 ℃ at 10 ℃/min, is kept for 3min, is raised to 290 ℃ at 8 ℃/min, and is kept for 2 min; the temperature of a sample inlet is 250 ℃; helium is taken as carrier gas, and the flow rate is 1.0 mL/min; adopts a non-shunting sample introduction mode. MS conditions: bombarding an ion source with electrons; an ionization mode EI; ionization voltage 70 eV; the temperature of the electron source is 200 ℃; filament current 150 μ A; scanning a mass range (m/z) of 45-500; the scanning mode is Scan. The results of the experiments were compared with the database of NIST14/NIST14s, and compounds having a degree of similarity of more than 80% were identified as the beany flavor component of the soybean sample, and the content thereof was tested.

And (4) testing results: see table 2.

TABLE 2 Soy bean odor component and content (unit. mu.g/kg)

Note that: in the table, "/" indicates that the content thereof was not detected.

The beany flavor is a special flavor generated in the soybean processing process and is composed of alcohol, aldehyde, ketone and other substances generated by the oxidative degradation of unsaturated fatty acid, wherein the content of hexanal determines the beany flavor degree of the soybean product. As can be seen from table 2, the soybean odor components of the detoxified soybeans obtained in the examples of the present invention were significantly reduced, and some of the components were not detected, so that the soybean odor was significantly improved by treating the soybeans according to the present invention.

Test example 3 lipoxygenase Activity assay

The test object is: detoxified soybeans prepared in examples 1 to 3 and comparative examples 1 to 9 and commercially available normal soybeans.

And (4) checking items: lipoxygenase activity.

The detection method comprises the following steps:

preparing a crude enzyme solution: grinding 20g of soybean and 200mL of deionized water in ice water bath, filtering with 100-mesh double-layer filter cloth, centrifuging at 4 deg.C and 8000r/min for 30min, and collecting the supernatant as crude enzyme solution.

Preparing a substrate solution: 0.1mL of linoleic acid and 0.1mL of Tween 20 were added to 0.2mol/L of a boric acid buffer solution (pH9.0) to make a volume of 100 mL.

Lipoxygenase activity assay: the substrate solution was diluted 5-fold with 0.2mol/L boric acid buffer solution (pH 9.0). 2.0mL of the substrate dilution, 0.9mL of 0.2mol/L boric acid buffer (pH9.0), and 0.1mL of the crude enzyme solution diluted by an appropriate amount were mixed, and the absorbance at 234nm was measured at intervals of 10 seconds (A234). An increase of 0.001 in A234 within 1min is defined as one enzyme activity unit (U). The relative activity of lipoxygenase was calculated as follows.

Relative activity ═ treated soybean lipoxygenase activity/untreated soybean lipoxygenase activity × 100%

And (3) detection results: see table 3.

TABLE 3

Group of	Relative Activity of Soybean lipoxygenase (%)
		Commercially available common soybeans	100
Example 1	1.9
		Example 2	1.7
Example 3	1.4
		Comparative example 1	4.5
Comparative example 2	32.5
		Comparative example 3	30.3
Comparative example 4	2.7
		Comparative example 5	2.2
Comparative example 6	2.4
		Comparative example 7	24.8
Comparative example 8	26.4
		Comparative example 9	3.1

As can be seen from Table 3, the relative activity of the lipoxygenase in the detoxified soybeans prepared by the embodiment of the invention is remarkably reduced by 98.1-98.6%, and the soybeans subjected to steam treatment possibly change the conformation of the active center of the enzyme or the dissociation state of related groups, so that the enzyme activity is effectively reduced.

Test example 4 protein solubility assay

The test object is: detoxified soybeans prepared in examples 1 to 3 and comparative examples 1 to 9 and commercially available normal soybeans.

And (4) checking items: protein solubility.

The detection method comprises the following steps: the soluble protein content of the soybean sample was measured by the Coomassie Brilliant blue method with reference to SN/T3926-2014. The total protein content of the soybean sample was measured by Kjeldahl method in GB 5009.5-2016 (protein conversion factor: 5.71). The protein solubility of the soybean sample was calculated as follows.

Protein solubility ═ soluble protein content in soybean/total protein content in soybean x 100%

And (3) detection results: see table 4.

TABLE 4

Group of	Protein solubility (%)
		Commercially available common soybeans	32.7
Example 1	46.5
		Example 2	47.2
Example 3	48.4
		Comparative example 1	42.3
Comparative example 2	37.2
		Comparative example 3	38.4
Comparative example 4	44.7
		Comparative example 5	45.2
Comparative example 6	44.9
		Comparative example 7	42.4
Comparative example 8	41.7
		Comparative example 9	43.9

As can be seen from Table 4, the solubility of protein in the detoxified soybean obtained in the examples of the present invention is slightly increased, and the soluble protein is increased, probably because part of insoluble protein is decomposed into small molecule soluble protein after the microbial fermentation treatment, thereby increasing the nutrition of soybean.

In comparative example 1, the soybeans were not polished, and the protective film on the surface of the soybeans was not removed, so that the effect of detoxifying the soybeans was slightly reduced. Compared with the comparative example 2 and the comparative example 3, the bacillus licheniformis or the bifidobacterium is not added, the removal of antinutritional factors in the soybean is obviously reduced, most of trypsin inhibitor is remained, the content of fishy smell components of the soybean is increased, and meanwhile, the protein solubility is reduced, because the mixed microbial preparation is adopted to detoxify the whole soybean, the complex and changeable disulfide bonds and intermediate products thereof can be efficiently decomposed due to the complex enzyme system of the microbe, the trypsin inhibitor in the soybean can be completely decomposed finally, meanwhile, the taste of the vegetable soybean is greatly improved, the utilization rate of the nutrient components in the soybean is improved, finally, the microbe utilizes the self metabolism, can generate precursor substances of flavor substances or flavor, thereby improving the flavor of the soybean after detoxification, and part of insoluble protein is decomposed into small-molecule soluble protein after microbial fermentation, therefore, the nutrition of the soybean is increased, and the addition of the bacillus licheniformis and the bifidobacterium has a synergistic effect. In comparative example 4, the detoxification effect is slightly reduced as a whole without microwave treatment, and thus, the microwave treatment can effectively promote the decomposition of soybean anti-nutritional factors such as purine and trypsin inhibin. Cysteine or ficin is not added in comparative example 5 and comparative example 6, the content of trypsin inhibin is obviously increased, passivating agent cysteine and ficin are added, the ficin is thiol protease, the thiol protease and the ficin can effectively passivate the trypsin inhibin in a synergistic manner, and the exchange reaction of thiol and disulfide causes the cleavage of disulfide bonds, so that the trypsin inhibin is effectively decomposed and removed, and the thiol protease and the ficin have synergistic effect. In comparative example 7 and comparative example 8, ethylenediamine or glycerol was not added, and the lipoxygenase activity, the content of soybean odor components, and the trypsin inhibitor were increased, probably because trypsin inhibin and other antinutritional factors were dissolved in high temperature steam with chemical solvent at high temperature, and partially decomposed at high temperature to generate volatile substances and lost with steam, and partially reacted with organic substances, for example, ethylenediamine could react with sulfur bond, and glycerol could dissolve soybean odor substances such as 1-octen-3-ol, n-hexanol, and n-pentanol, thereby achieving effective detoxification and taste improvement effects. In comparative example 9, microwave drying was not employed, but a common oven was employed for drying, and the microwave treatment was performed to decompose and remove a small part of the remaining anti-nutritional factors in the soybeans again.

Compared with the prior art, the trypsin inhibin in the soybean is mainly divided into 2 types, namely Kunitz (Kunitz) trypsin inhibin (KTI) and Bowman-Belck (Bowman-Birk) inhibin (BBI), wherein the BBI is a molecule formed by crosslinking 7 disulfide bonds, and the 7 disulfide bonds endow the molecular structure with stability, so that the BBI is extremely stable to heat, acid, alkali and the like. Can generate flavor substance or flavor precursor substance to improve flavor of detoxified semen glycines.

According to the trypsin inhibitor, the passivating agent cysteine and the ficin are added, the ficin is thiol protease, the trypsin inhibitor can be effectively passivated by the synergy of the cysteine and the ficin, and the disulfide bond is cracked due to the exchange reaction of thiol and disulfide, so that the trypsin inhibitor is effectively decomposed and removed; under the microwave treatment condition, the invention can passivate anti-nutritional factors in the soybeans, such as purine, trypsin inhibin and the like, thereby assisting in reducing the toxicity of the soybeans; the invention also adopts a chemical solvent and physical steam combined method for further detoxification treatment, trypsin inhibin and other anti-nutritional factors are dissolved in high-temperature steam with a chemical solvent at high temperature, part of the high-temperature decomposition generates volatile substances to be dissipated along with the steam, and part of the high-temperature decomposition reacts with organic substances, such as amine can react with a sulfur bond, thereby achieving the effects of effectively detoxifying and improving the taste.

The invention adopts microwave assistance, the passivator participates in the reaction, thereby passivating the anti-nutritional factors, the compound microbial liquid is fermented and detoxified, the taste and nutrition of the soybeans are further improved, the residual anti-nutritional factors are removed by chemical solvent and physical steam, the method is simple, the soybeans can be effectively detoxified, the toxicity is not caused again, and the effect of long-acting detoxification can be realized.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.