CN112205512A - Preparation method of hypoallergenic plant protein meat - Google Patents
Preparation method of hypoallergenic plant protein meat Download PDFInfo
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Abstract
The invention discloses a preparation method of hypoallergenic plant protein meat, and belongs to the technical field of food processing. The invention reduces soybean tissue protein, peanut tissue protein, wheat tissue protein, pea tissue protein or a mixture thereof, or beta-conglycinin, soybean hydrophobin, soybean hull protein, soybean arrestin or a mixture thereof by adding bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, aspergillus oryzae or a mixture thereof and/or adding alkaline protease, neutral protease, acid protease, pepsin, chymotrypsin, amylase, cellulase, trypsin or a mixture thereof in the preparation process of the vegetable protein meat.
Description
Technical Field
The invention relates to a preparation method of hypoallergenic plant protein meat, and belongs to the technical field of food processing.
Background
Anaphylaxis is a common autoimmune disease of human beings, and is a great problem in the medical field for a long time because symptoms are complex, various and wide in harm and difficult to cure radically. There are many types of allergens in nature, pollen proteins and food proteins being the two most common allergens among plant allergens. About 90% of the food allergies currently known are caused by peanuts, soybeans, eggs, milk, nuts, fish, crustaceans and wheat. Different food processing modes in the food processing process inevitably influence the structure of protein in food. Thus, pre-processing of food that is susceptible to sensitization may result in the elimination of the allergen. At present, the research on low-sensitization soybean food is relatively more abroad, and the researched desensitization method mainly comprises heat treatment, extrusion and puffing, ultrahigh pressure treatment, radiation, pulsed ultraviolet and the like, but has the defects of environmental unfriendliness, energy consumption, low efficiency and the like. Therefore, development of a more green and efficient biological method for desensitization and reduction of sensitization of plant proteins are of great significance.
Disclosure of Invention
The first purpose of the invention is to provide a method for degrading allergen in plant tissue protein, which takes plant protein as substrate, adds microorganism and/or enzyme to degrade, the microorganism is bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, aspergillus oryzae or mixture thereof, the enzyme is alkaline protease, neutral protease, acid protease, pepsin, chymotrypsin, amylase, cellulase, trypsin or mixture thereof, when adding enzyme, the degradation reaction condition is: the addition amount is 10-100U/g substrate, the reaction temperature is 25-37 ℃, and the reaction time is 24-120 h; when the microorganism is added, the degradation reaction conditions are as follows: the addition amount is 20-200CFU/g substrate, the reaction temperature is 25-37 ℃, the rotation speed is 150-; when the enzyme and the microorganism are added simultaneously, the reaction conditions are as follows: adding microbial biomass 10-100CFU/g substrate, adding enzyme 10-50U/g substrate, reacting at 25-37 deg.C for 24-120 h.
In one embodiment of the invention, when single protease is added, the addition amount is 10-100U/g, the reaction condition is 25-37 ℃, and the reaction time is 24-48 h.
In one embodiment of the invention, when the complex enzyme is added, the adding amount is 10-100U/g substrate, including alkaline protease, neutral protease, acid protease, amylase and cellulase, the mass ratio of the alkaline protease, the neutral protease, the acid protease, the amylase and the cellulase is 3: 1: 1: 10: 2, the reaction condition is 25-37 ℃, and the reaction time is 24-36 h.
In one embodiment of the invention, when the mixed microorganism is added, the addition amount is 20-200CFU/g substrate, including Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus oryzae, and the volume ratio of the added Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus oryzae is 3: 1: 2: 10, the reaction condition is 25-37 ℃, and the reaction time is 24-120 h.
In one embodiment of the invention, when a single Aspergillus oryzae is added, the addition amount is 40-300CFU/g substrate, the reaction conditions are 28-30 ℃ and the reaction time is 24-120 h.
In one embodiment of the invention, the vegetable protein includes, but is not limited to, soy tissue protein, peanut tissue protein, wheat tissue protein, pea tissue protein, or mixtures thereof.
In one embodiment of the present invention, the vegetable protein includes, but is not limited to, including but not limited to, beta-conglycinin, soy hydrophobin, soy hull protein, soy arrestin or mixtures thereof.
In one embodiment of the present invention, the added microorganism is lactobacillus plantarum, and the fermentation conditions for degrading the total allergenicity of soybean by liquid fermented soybean meal are as follows: the initial pH of the culture medium is 5.0-5.5, the concentration of the soybean meal is 10% -15%, and the inoculation concentration is 1 multiplied by 108-5×108Inoculating lactobacillus plantarum suspension with volume ratio of 1-3% in a shaker at 37 deg.C, and fermenting at 100rpm for 24-120 h.
In one embodiment of the invention, the added microorganism is aspergillus oryzae, and the fermentation conditions for degrading the total soybean allergen by solid state fermentation of soybean meal are as follows: ratio of material to water 11.0-1.5, fermentation temperature 25-37 deg.C, initial pH of culture medium 6.0-6.5, and adding 0.05-0.5mL/g total protein of soybean with concentration of 1 × 108-5×108And fermenting the Aspergillus oryzae spore suspension for 100-120 h.
In one embodiment of the present invention, the added microorganism is lactobacillus plantarum, and the fermentation conditions for degrading the total allergenicity of soybean by liquid fermented soybean meal are as follows: the fermentation system is that 50mL fermentation liquor is placed in a 250mL triangular flask, the initial pH of the culture medium is 5.0, the concentration of the soybean meal is 15%, and 1mL fermentation liquor with the concentration of 1 × 10 is inoculated8The lactobacillus plantarum suspension is fermented for 60 hours in a shaker at 25-37 ℃ at 100-.
In one embodiment of the invention, the added microorganism is aspergillus oryzae, and the fermentation conditions for degrading the total soybean allergen by solid state fermentation of soybean meal are as follows: the fermentation system is that 20g of soybean total protein is placed in a 250mL triangular flask, the ratio of material to water is 1:1.5, the fermentation temperature is 30 ℃, the initial pH of the culture medium is 6.0, and 1mL of the culture medium with the concentration of 2 multiplied by 10 is inoculated8And fermenting the Aspergillus oryzae spore suspension for 120 h.
The second purpose of the invention is to provide the application of the method for degrading the allergen in the plant tissue protein in the preparation of hypoallergenic plant protein meat.
In one embodiment of the invention, the components of the vegetable protein meat include normal vegetable tissue protein and highly fibrillated trabecular protein.
The invention has the beneficial effects that:
plant allergens (allergenic proteins) are a special class of proteins in plants that may cause different allergic symptoms to the consumer. The invention provides a biological enzyme method for degrading allergens in plant tissue proteins, which can realize efficient and specific degradation of different allergens. In addition, the invention also provides a corresponding biological enzyme preparation method, which comprises the steps of microbial protein expression and biological enzyme purification and is used for producing artificial meat. The method can effectively reduce the problems of vegetable protein allergen and the like, improve the quality of the vegetable protein and apply the vegetable protein to the preparation of vegetable protein meat.
Detailed Description
(I) the related strains and culture media
Strain: bacillus subtilis (Bacillus subtilis); lactobacillus plantarum (Lactobacillus plantarum); saccharomyces cerevisiae (Saccharomyces cerevisiae); aspergillus oryzae (Aspergillus oryzae).
LB medium (bacillus subtilis medium): 950mL of distilled water; 10g of tryptone; 10g of NaCl; 5g of yeast extract, adjusting pH to 7.0 with 1mol/mL NaOH (about 1mL), adding distilled water to a total volume of 1L, and performing moist heat sterilization at 121 deg.C for 30 min.
MRS broth medium (lactobacillus plantarum medium): 10g of peptone; 10g of beef extract; 5g of yeast extract powder; 20g of glucose; 2g of dipotassium phosphate; 2g of diammonium hydrogen citrate; 5g of sodium acetate; magnesium sulfate 0.5 g; 0.25g of manganese sulfate tetrahydrate; 0.5g of cysteine; adjusting pH to 6.0 of Tween 801 g, adding distilled water to total volume of 1L, and sterilizing at 121 deg.C for 15 min.
YPD medium (saccharomyces cerevisiae medium): 10g of yeast extract; dissolving peptone 20g in 900mL water (20 g agar powder should be added when preparing flat plate or inclined plane), and autoclaving at 121 deg.C for 20 min; after sterilization, 100mL of a 20% glucose solution was added.
PDA medium (aspergillus oryzae medium): 200g of potatoes; 20g of cane sugar; 1000mL of water; boiling in beaker for 30min, filtering with double-layer gauze, collecting filtrate, adding agar 15-20g when preparing plate or inclined plane, dissolving, adding sugar and water to 1000mL, and sterilizing at 121 deg.C for 20 min.
(II) method for detecting content of sensitizing substances
The detection of plant allergens before and after treatment adopts an indirect competitive enzyme-linked immunosorbent assay (ELISA).
The method comprises the following specific steps (taking soybean protein as an example):
(1) coating: diluting standard antigen to appropriate concentration, wherein the coating concentration of soybean total protein is 1 mug/mL, the coating concentration of beta-conglycinin is 0.5 mug/mL, the coating concentration of glycinin is 0.5 mug/mL, 100 muL is coated in a 96-hole enzyme label plate, and standing for 16h at 4 ℃;
(2) pre-reaction of competing antigen with antibody: the standard antigens (denatured and renatured extracted raw soybean meal protein, beta-conglycinin standard, glycinin standard) and denatured and renatured extracted sample protein were diluted with antibody diluent (containing 0.1% BSA), added to the reaction tube, and the corresponding diluted primary antibody was added in equal volume. The final dilution factor of the polyclonal antibody against the soybean total allergen is 1:50000, the final dilution factor of the polyclonal antibody against beta-conglycinin is 1:60000, the final dilution factor of the polyclonal antibody against glycinin is 1:800000, and the antigen-antibody mixture is mixed uniformly on a micro-oscillator and then placed at 4 ℃ for 16 h. Meanwhile, after the denatured and renaturated extraction reagent is properly diluted by the antibody diluent, a substituted sample is added into a reaction tube and mixed with the equal volume of the antibody to be used as a negative control, and after the denatured and renaturated extraction reagent is properly diluted by the antibody diluent, the substituted sample is used as a blank control;
(3) washing;
(4) and (3) sealing: adding blocking solution (phosphate buffer solution containing 1% BSA), sealing at 200 μ L/well in 37 deg.C incubator for 2 hr;
(5) washing;
(6) sample adding reaction: adding the antigen-antibody mixture, the negative control and the blank control which are subjected to the pre-reaction in the step (2) into an enzyme label plate, incubating for 2 hours at a constant temperature of 37 ℃ in a 100 mu L/hole manner;
(7) washing;
(8) adding an enzyme-labeled secondary antibody: diluting the secondary antibody by 5000 times, incubating the secondary antibody at 100 mu L/hole for 1h in a constant temperature box at 37 ℃;
(9) washing;
(10) color development: adding 100 μ L of TMB color developing solution into each well, and developing in a 37 deg.C incubator for 20min in the dark;
(11) termination of the assay: adding 50 mu L of 2mol/L sulfuric acid into each hole to terminate the reaction, and detecting OD in an enzyme-linked immunosorbent assay450A value;
(12) data processing: OD binding to antibody according to standard antigen450And (4) calculating the inhibition rate, and then taking the logarithm of the concentration of the standard antigen to draw a standard curve. And calculating the content of the soybean allergen in the soybean meal before and after fermentation according to a standard curve, and calculating the degradation rate of the soybean allergen.
The soybean allergen degradation rate (%) × (1-soybean allergen content in fermented soybean meal/soybean allergen content in raw soybean meal) × 100%.
(III) definition and determination method of enzyme activity
Protease: the protease has good hydrolysis effect on casein, whey protein, cereal protein, etc. The phosphotungstic acid and phosphomolybdic acid mixed reagent, namely a Folin-phenol reagent, is easy to be reduced by phenolic compounds under alkaline conditions to be in blue reaction. Since proteins contain amino acids having a phenol group (tyrosine, tryptophan, phenylalanine), the activity of proteases is indirectly measured by a chromogenic reaction in which a protease decomposes casein (substrate) to produce an amino acid containing a phenol group. The enzyme activity was defined as 1ml liquid protease, and 1min hydrolysis of casein at 37 ℃ (acidic pH 3.0, neutral pH 7.5, alkaline pH 10.5) yielded 1 μ g tyrosine as one enzyme activity unit. The activity units of the enzyme were calculated according to the following formula: activity of protease a × K × V/t × n U/g (ml), a: average OD values for parallel testing of samples; k: a light absorption constant; v: the total volume of the reaction reagents; t: carrying out enzymolysis reaction time; n: and (5) diluting the enzyme solution by total times.
And (3) cellulase: the cellulase hydrolyzes cellulose to generate reducing sugar such as cellobiose and glucose, can reduce nitro in 3, 5-dinitrosalicylic acid into orange amino compounds, and can express the activity of the enzyme by measuring the generation amount of the reduced products by a colorimetric method. The enzyme activity is defined as that 1mL of enzyme solution catalyzes the soluble starch to generate 1mg of reducing sugar under the conditions of 75 ℃ and pH 4.8 within 1 hour, and the enzyme amount is 1 enzyme activity unit.
Amylase: the amylase can catalyze and hydrolyze adjacent alpha-1, 4-glucosidic bonds of starch molecules to obtain decomposition products of glucose and maltose. The enzyme activity is defined as that 1mL of enzyme solution catalyzes the soluble starch to generate 1mg of glucose in 1 hour under the conditions of 70 ℃ and pH of 5.0, and the enzyme amount is 1 enzyme activity unit.
Example 1 degradation of allergens in plant tissue proteins by Bio-enzymatic Process
Enzymatic desensitization is the reduction of the effects of protein sensitization by altering the degree of enzymatic hydrolysis or protein structure of proteases. If the hydrolysis degree is proper, the epitope structure of the allergen can be changed, and the allergenicity is reduced; if the hydrolysis degree is not suitable, the internal allergen surface can be exposed, so that the allergenicity is improved; if extensively hydrolyzed, the protein may be hydrolyzed to small peptides or amino acids, reducing its allergenicity, but certain functional properties of the protein may be compromised. Enzymatic cross-linking can form covalent bonds among protein molecules or among internal polypeptide chains, lead to protein aggregation, and embed the antigen epitope exposed on the surface into the protein molecules, thereby eliminating the allergenicity of the protein molecules; however, the aggregation between protein molecules will form larger particles of protein, and it is also possible to increase the sensitization. Because the thixotropy of the soybean protein isolate is influenced and the swallowing difficulty of the soybean protein isolate is influenced after the soybean protein isolate is subjected to enzyme crosslinking, the method is not suitable for desensitization of the soybean protein isolate special for infant formula food.
When the enzyme is added, the degradation reaction conditions are as follows: the addition amount is 10-100U/g substrate, the reaction temperature is 25-37 ℃, and the reaction time is 24-60 h.
The selected proteins are respectively alkaline protease, acid protease, neutral protease, cellulase and amylase from microorganisms; animal derived trypsin. The complex enzyme is formed by compounding different enzymes in proportion. Preparing a complex enzyme: 0.3kg of acid protease, 0.1kg of alkaline protease, 0.1kg of neutral protease, 0.1kg of cellulase, 1.5kg of amylase and 0.5kg of trypsin.
When single protease is adopted to degrade the allergen in the plant tissue protein, the content of the allergen is degraded by 70-85%; when the compound enzyme is used for degrading the allergen in the plant tissue protein, the content of the allergen is degraded by 80-99%.
EXAMPLE 2 microbiological degradation of allergens in plant tissue proteins
The selected strains such as bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, aspergillus oryzae and the like are used for fermenting the soybean protein powder in a liquid state fermentation mode and fermenting the soybean protein in a solid state fermentation mode, and the degradation effect on the soybean allergen is obviously different.
The soybean protein is fermented by using bacillus subtilis, lactobacillus plantarum and saccharomyces cerevisiae, and the result shows that the content of Glym series allergen in the soybean protein is reduced by 75%. By fermenting the soybean protein with bacillus subtilis and aspergillus oryzae, the content of Glym series allergens in the soybean protein is reduced by 80 percent.
Through single-factor experiments and response surface design, the appropriate fermentation conditions for degrading the total soybean allergens by the lactobacillus plantarum liquid state fermentation soybean meal powder are determined as follows: the fermentation system is that 50mL fermentation liquor is placed in a 250mL triangular flask, the initial pH of a culture medium is 5.0, the concentration of soybean meal is 15%, and 1mL fermentation liquor with the concentration of 1 × 10 is inoculated8And sealing the bottle mouth of the lactobacillus plantarum suspension with gauze, fermenting for 60 hours in a shaking table at 37 ℃ at 100rpm, and measuring by indirect competitive ELISA to ensure that the degradation rate of the total soybean allergen can reach 97%. Meanwhile, the degradation rates of beta-conglycinin and glycinin are 99% and 92% respectively.
Through single factor experiments and response surface design, the proper fermentation conditions for Aspergillus oryzae solid state fermentation soybean meal to degrade the total soybean allergen are determined as follows: the fermentation system is that 20g of soybean total protein is put into a 250mL triangular flask, the ratio of material to water is 1:1.5, the fermentation temperature is 30 ℃, the initial pH of the culture medium is 6.0, and 1mL of the culture medium with the concentration of 2 multiplied by 10 is inoculated8And fermenting the Aspergillus oryzae spore suspension per mL for 120h, wherein the degradation rate of the soybean total allergen can reach 99% by indirect competitive ELISA determination. Meanwhile, the degradation rates of beta-conglycinin and glycinin are 99% and 99%, respectively.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for degrading allergen in plant tissue protein is characterized in that plant protein is used as a substrate, and microorganism and/or enzyme are added for degradation, the microorganism is bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, aspergillus oryzae or a mixture thereof, the enzyme is alkaline protease, neutral protease, acid protease, pepsin, chymotrypsin, amylase, cellulase, trypsin or a mixture thereof, and when the enzyme is added, the degradation reaction conditions are as follows: the addition amount is 10-100U/g substrate, the reaction temperature is 25-37 ℃, and the reaction time is 24-120 h; when the microorganism is added, the degradation reaction conditions are as follows: the addition amount is 20-200CFU/g substrate, the reaction temperature is 25-37 ℃, the rotation speed is 150-; when the enzyme and the microorganism are added simultaneously, the reaction conditions are as follows: adding microbial biomass 10-100CFU/g substrate, adding enzyme 10-50U/g substrate, reacting at 25-37 deg.C for 24-120 h.
2. The method of claim 1, wherein the vegetable protein includes but is not limited to soy tissue protein, peanut tissue protein, wheat tissue protein, pea tissue protein, or mixtures thereof.
3. The method of claim 1 or 2, wherein the vegetable protein includes but is not limited to including but not limited to β -conglycinin, soybean hydrophobin, soybean hull protein, soybean arrestin or mixtures thereof.
4. The method of claim 1, wherein the amount of protease added is 10-100U/g, the reaction conditions are 35-37 ℃, and the reaction time is 24-48 h.
5. The method according to claim 1, wherein when the complex enzyme is added, the adding amount is 10-100U/g substrate, the substrate comprises alkaline protease, neutral protease, acid protease, amylase and cellulase, and the mass ratio of the alkaline protease, the neutral protease, the acid protease, the amylase and the cellulase is 3: 1: 1: 10: 2, the reaction condition is 25-37 ℃, and the reaction time is 24-36 h.
6. The method of claim 1, wherein the mixed microorganism is added in an amount of 20-200CFU/g substrate, including bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, and aspergillus oryzae, wherein the ratio of the bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, and aspergillus oryzae is 3: 1: 2: 10, the reaction condition is 25-37 ℃, and the reaction time is 24-120 h.
7. The process of claim 1, wherein a single aspergillus oryzae is added in an amount of 40 to 300CFU/g substrate under reaction conditions of 25 to 37 ℃ for 24 to 120 hours.
8. The use of a method according to claim 1 for degrading allergens in plant tissue proteins in the food sector.
9. Use of a method according to claim 1 for degrading allergens in plant tissue proteins for the preparation of hypoallergenic plant protein meat.
10. The use as claimed in claim 9, wherein the vegetable protein meat ingredients include normal vegetable tissue protein and highly fibrillating protein.
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| CN113317388A (en) * | 2021-05-24 | 2021-08-31 | 好福(上海)食品科技有限公司 | Method for preparing hypoallergenic peanut protein powder by high-pressure steam and microbial fermentation |
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