Method for removing antigen protein from soybean meal

Abstract

The invention discloses a method for removing antigen protein from soybean meal, which comprises the following steps: putting a soybean meal raw material into a digester, and carrying out two-stage temperature-controlled cooking: in an initial heating stage, raising the temperature in the digester from normal temperature to 80 DEG C at a heating rate of 5-10 DEG C / min for about 10-15 minutes; and a high-temperature cooking stage: continuously raising the temperature to 110-121 DEG C, and keeping the temperature for 20-30 minutes. In the high-temperature cooking process, the temperature fluctuation range should be controlled within + / -1 DEG C, and the pressure in the cooking pot is maintained at 0.1-0.15 MPa. According to the method for removing the antigen protein from the soybean meal provided by the invention, the structure of the antigen protein is denatured and inactivated by controlling conditions such as specific temperature, time, pressure and the like, so that the antigenicity of the antigen protein is reduced, and subsequent production or application requirements are met.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen protein removal, and particularly to a method for removing antigen proteins from soybean meal. Background Art

[0002] Soybeans contain 36%-56% protein and are one of the richest sources of plant protein. Due to its high protein content and reasonable amino acid structure, soybean meal has become the most widely used plant protein feed resource in feed industrial production. However, soybean meal contains antigen proteins that may cause adverse reactions in animals (especially young animals), such as intestinal damage and diarrhea. After antigen proteins enter the animal intestine, they will trigger an immune response, resulting in damage to the intestinal mucosal barrier, shortening of villi, hyperplasia of crypts, and a decrease in digestive enzyme activity, ultimately leading to diarrhea. Among them, glycinin and β-conglycinin will reduce the expression of divalent metal ion transporter (DMT1) and small peptide transporter (PEPT1) in intestinal epithelial cells, and at the same time enhance the activity of Na+-K+-ATPase, resulting in blocked absorption of minerals and small peptides, and further causing symptoms such as malabsorption and diarrhea.

[0003] Among the plant protein antinutritional factors in soybean meal, it mainly includes lectin, trypsin inhibitor, soybean antigen protein, etc. Among them, lectin and trypsin inhibitor are sensitive to heat and can be eliminated by high-temperature heating during the feed production process. However, soybean antigen protein is a type of antinutritional factor with strong thermal stability and is difficult to degrade or inactivate at high temperatures. Moreover, during the high-temperature removal process, the temperature needs to be well controlled to avoid protein carbonization or Maillard reaction caused by too high temperature, which not only destroys the amino acid structure but may also generate harmful substances such as acrylamide. At the same time, it is also necessary to avoid too low temperature, which cannot effectively remove antigen proteins. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for removing antigen proteins from soybean meal. By controlling specific conditions such as temperature, time, and pressure, the structure of antigen proteins is denatured and inactivated, thereby reducing their antigenicity to meet the subsequent production or application requirements.

[0005] The technical solution adopted by the method for removing antigen proteins from soybean meal disclosed by the present invention is as follows:

[0006] A method for removing antigen proteins from soybean meal, putting the soybean meal raw material into a cooking pot and performing two-stage temperature-controlled cooking:

[0007] Initial heating stage: Raise the temperature in the cooking pot from room temperature to 80°C at a heating rate of 5-10°C / min, and the heating time is about 10-15 minutes;

[0008] High-temperature cooking stage: Continue to heat up to 110 - 121 °C and maintain at this temperature for 20 - 30 minutes. During the high-temperature cooking process, the temperature fluctuation range should be controlled within ±1 °C, and the pressure inside the cooking pot is maintained at 0.1 - 0.15 MPa.

[0009] As a preferred solution, the cooking pot is internally heated by introducing high-temperature steam, and a stirring device is provided inside the cooking pot to stir the soybean meal raw material during the heating process.

[0010] As a preferred solution, the cooking pot is correspondingly provided with an exhaust valve, which is controlled by the coordinated adjustment of the steam intake and the exhaust valve to ensure that the pressure is stable within the set range, and the pressure fluctuation shall not exceed ±0.02 MPa.

[0011] As a preferred solution, before cooking, it is necessary to measure the initial moisture content of the raw material. According to the initial moisture content of the raw material, adjust the moisture of the raw material, adjust the added water, and control the final cooking moisture at 30 - 45%.

[0012] As a preferred solution, the spray humidification method is adopted, and filtered pure water is used to spray evenly on the surface of the soybean meal raw material in a stirring state until the required moisture content is reached.

[0013] As a preferred solution, the raw material after cooking should be sampled and tested to check whether the antigen protein removal rate reaches the expected standard.

[0014] As a preferred solution, after the cooking is completed, the soybean meal raw material treated by high temperature and high pressure can also be subjected to enzymatic hydrolysis treatment.

[0015] As a preferred solution, the enzymatic hydrolysis treatment includes:

[0016] Pretreatment before enzymatic hydrolysis: The soybean meal raw material treated by high temperature and high pressure needs to be cooled to 50 - 60 °C, and the soybean meal raw material is crushed and sieved through a 60-mesh sieve;

[0017] Selection and preparation of enzymes: Alkaline protease and papain are used;

[0018] Preparation of each enzyme solution: Weigh the enzyme powder according to the enzyme addition amount of 2 - 4% w / w, dissolve it with warm water at 40 °C, and prepare a 5 - 10% enzyme solution;

[0019] Setting of enzymatic hydrolysis reaction conditions:

[0020] Mass ratio of soybean meal to water: 1:3 - 1:5;

[0021] pH adjustment: Use 1 mol / L NaOH or HCl to adjust to the optimal pH range of the enzyme;

[0022] Temperature control: Control the temperature in a water bath or jacketed enzymolysis tank to maintain at 50 - 60 °C;

[0023] Stirring speed: Mechanical stirring at 30 - 60 rpm to ensure the material is uniform and no foam is generated:

[0024] Enzyme addition method:

[0025] Step - by - step addition: First add alkaline protease and hydrolyze for 2 hours, then supplement papain and continue to hydrolyze for 2 hours;

[0026] One - time addition: Mix the enzymes, add the mixture of alkaline protease:papain = 2:1 to the reaction; The total reaction time is 4 - 6 hours, and samples are taken every 30 minutes to detect the degree of hydrolysis (DH) or antigen residue;

[0027] Inactivation of enzyme and termination of reaction: Raise the temperature to 85 °C and maintain for 10 - 15 minutes to inactivate the protease. After enzyme inactivation, quickly cool to below 40 °C to avoid residual enzyme activity or Maillard reaction;

[0028] Post - treatment of enzymolysis liquid: Filter, collect the supernatant, and the solid residue can be returned for secondary enzymolysis or used as a fermentation substrate.

[0029] The beneficial effect of the method for removing antigenic protein from soybean meal disclosed in the present invention is as follows: Put the soybean meal raw material into a steaming pot, perform high - temperature and high - pressure treatment through the steaming pot, and during the heating process, adopt the method of two - stage temperature - controlled steaming. First, uniformly heat the soybean meal raw material to preliminarily soften the tissue of the soybean meal raw material, preparing for subsequent high - temperature treatment. Then raise the temperature while maintaining the pressure state in the steaming pot, so that the structure of the antigenic protein in the soybean meal raw material denatures and inactivates under high - temperature and high - pressure conditions, thereby reducing its antigenicity to meet the requirements of subsequent production or application. Specific embodiments

[0030] The following further elaborates and explains the present invention with specific embodiments:

[0031] A method for removing antigenic protein from soybean meal, put the soybean meal raw material into a steaming pot and perform two - stage temperature - controlled steaming:

[0032] Initial heating stage: Raise the temperature in the steaming pot from room temperature to 80 °C at a heating rate of 5 - 10 °C / min, and the heating time is about 10 - 15 minutes. The main purpose of this stage is to uniformly heat the raw material, preliminarily soften the raw material tissue, and prepare for subsequent high - temperature treatment;

[0033] High-temperature cooking stage: Continue to raise the temperature to 110 - 121 °C and maintain it at this temperature for 20 - 30 minutes. During the high-temperature cooking process, the temperature fluctuation range should be controlled within ±1 °C, and the pressure in the cooking pot should be maintained at 0.1 - 0.15 MPa. This temperature range can effectively destroy the higher-order structure of antigen proteins and make them denatured and inactivated.

[0034] The cooking pot is internally heated by introducing high-temperature steam, and a stirring device is provided inside the cooking pot to stir the soybean meal raw materials during the heating process, enabling them to be fully heated. Moreover, heating the soybean meal raw materials by means of high-temperature steam can maintain a high pressure inside the cooking pot while heating and can also supplement water vapor.

[0035] The cooking pot is correspondingly equipped with an exhaust valve, which is controlled by coordinating the regulation of the steam intake and the exhaust valve to ensure that the pressure is stably within the set range, and the pressure fluctuation shall not exceed ±0.02 MPa. Ensure that the pressure is stably within the set range to prevent equipment damage or safety accidents caused by excessive pressure, and at the same time avoid the influence of too low pressure on the cooking effect.

[0036] In the above solution, before cooking, a professional moisture analyzer (such as an infrared moisture analyzer or an oven method moisture analyzer) is required to measure the initial moisture content of the soybean meal raw materials, and the measurement result should be accurate to ±0.01%. According to the initial moisture content of the soybean meal raw materials and the cooking process requirements, the moisture of the soybean meal raw materials is adjusted. The initial moisture of the soybean meal should generally be 10% - 13%, and an appropriate amount of water needs to be calculated and added in a certain proportion for adjustment to control the final cooking moisture at 30 - 45%.

[0037] If the moisture content of the soybean meal raw materials is lower than the lower limit of the process requirements, the spray humidification method can be adopted. Use filtered pure water and spray it evenly on the surface of the soybean meal raw materials in the form of a spray, spraying while stirring until the required moisture content is reached.

[0038] If the moisture content is too low, it may cause the soybean meal raw materials to be over-dried during the cooking process, resulting in uneven heat transfer during the cooking process, affecting the removal effect of antigen proteins, and affecting their quality and subsequent processing performance. If the moisture content is too high, it may pose higher requirements for the subsequent drying process and increase the processing cost of the product.

[0039] The raw materials after cooking should be sampled and tested to check whether the antigen protein removal rate reaches the expected standard. The raw materials after cooking should be sampled and tested to check whether the antigen protein removal rate reaches the expected standard, and the removal rate should not be less than 90%. If the test result is qualified, the raw materials can be processed or packaged subsequently; if it is unqualified, the reasons need to be analyzed, and the cooking process parameters should be adjusted and then cooked again.

[0040] Among them, the enzyme-linked immunosorbent assay (ELISA) method was used to detect the content of antigen proteins in the raw materials before and after cooking. This method has the advantages of high sensitivity, strong specificity, good repeatability, etc., and can accurately measure the change in the content of antigen proteins in the raw materials.

[0041] Preparation of test samples: 0.3 grams of raw material samples before and after cooking were respectively taken and added to an appropriate amount of extraction buffer, and antigen protein extraction was carried out under specific temperature and time conditions. After the extract was pretreated by centrifugation, filtration, etc., it was used for subsequent ELISA detection.

[0042] ELISA detection steps: According to the requirements of the kit instructions, operations such as sample addition, incubation, washing, color development, and termination of the reaction were carried out in sequence. Finally, the absorbance value was measured at 450 nm using an enzyme-labeled instrument, and the content of antigen proteins in the sample was calculated according to the standard curve.

[0043] Example

[0044] The pretreated raw materials were loaded into an 8m 3 cooking pot at a rate of 2240 kg per batch (the raw materials were added to cooking pots of different volumes according to the same proportion), the lid of the cooking pot was closed, and it was ensured that the lid was tightly sealed for subsequent high-temperature and high-pressure cooking.

[0045] Based on the initial content of antigen proteins in the raw materials before cooking, after cooking treatment, the removal rate of antigen proteins should reach more than 90%, and the content of antigen proteins in the treated raw materials should not be higher than 10 mg / g.

[0046] Each batch of cooked raw materials needs to be sampled and tested. When sampling, a multi-point sampling method should be adopted, and the sampling quantity should not be less than 500 g. Three parallel samples should be set up for testing.

[0047] The results are as follows:

[0048]

[0049] Thus, it can be seen that after cooking, the glycinin and β-conglycinin in the soybean meal raw materials are significantly reduced.

[0050] Different treatment methods result in incomplete removal of antigen proteins

[0051] The pretreated soybean meal raw materials were loaded into an 8m 3 cooking pot at a rate of 2240 kg per batch (the raw materials were added to cooking pots of different volumes according to the same proportion), and the lid of the cooking pot was closed according to the proportion and it was ensured that the lid was tightly sealed.

[0052] In the method of spray humidification, filtered pure water is used and sprayed in a uniform spray form while stirring on the surface of the soybean meal raw material until the moisture content reaches 10% or 20%.

[0053] Heat up to 110 ± 1 °C and maintain at this temperature for 20 minutes. During the high-temperature cooking process, the temperature fluctuation range should be controlled within ±1 °C, and the pressure in the cooking pot is maintained at 0.1 MPa.

[0054] Based on the initial content of antigenic proteins in the raw material before cooking, after cooking treatment, the removal rate of antigenic proteins should reach over 90%, and the content of antigenic proteins in the treated raw material should not be higher than 10 mg / g.

[0055] The raw material after each batch of cooking treatment needs to be sampled and tested. When sampling, the method of multi-point sampling should be adopted, and the sampling quantity should not be less than 500 g. When conducting the test, three parallel samples should be set up for testing.

[0056]

[0057] It can be seen from the above treatment methods that after treatment with existing conventional means, the soybean globulin and β-conglycinin cannot be effectively removed.

[0058] After adopting the technical solution of this embodiment, after cooking is completed, the soybean meal raw material after high-temperature and high-pressure treatment can also be subjected to enzymatic hydrolysis treatment.

[0059] The enzymatic hydrolysis treatment includes:

[0060] Pretreatment before enzymatic hydrolysis: The soybean meal raw material after high-temperature and high-pressure treatment needs to be cooled to 50 - 60 °C to avoid destroying the enzyme activity at high temperature. The soybean meal raw material is crushed and sieved through a 60-mesh sieve to increase the surface area to improve the enzymatic hydrolysis efficiency;

[0061] Selection and preparation of enzymes: Alkaline protease and papain are used. Alkaline protease is used to target and cut the hydrophilic region of antigenic proteins, while papain destroys the disulfide bonds to completely degrade the antigenic epitopes;

[0062] Preparation of each enzyme solution: Weigh the enzyme powder according to the enzyme addition amount of 2 - 4% w / w, dissolve it with warm water at 40 °C, and prepare a 5 - 10% enzyme solution;

[0063] Setting of enzymatic hydrolysis reaction conditions:

[0064] Mass ratio of soybean meal to water: 1:3 - 1:5;

[0065] pH adjustment: Use 1 mol / L NaOH or HCl to adjust to the optimal pH range of the enzyme;

[0066] Temperature control: Control the temperature in a water bath or jacketed enzymatic hydrolysis tank to maintain at 50 - 60°C;

[0067] Stirring speed: Mechanical stirring at 30 - 60 rpm to ensure the material is uniform and no foam is generated:

[0068] Enzyme addition method:

[0069] Stepwise addition: First add alkaline protease and hydrolyze for 2 hours, then supplement papain and continue to hydrolyze for 2 hours;

[0070] One-time addition: Mix the enzymes and add them to the reaction at a ratio of alkaline protease:papain = 2:1; The total reaction time is 4 - 6 hours, and samples are taken every 30 minutes to detect the degree of hydrolysis (DH) or the residual amount of antigen;

[0071] Inactivation of enzyme and termination of reaction: Raise the temperature to 85°C and maintain for 10 - 15 minutes to inactivate the protease. After enzyme inactivation, quickly cool to below 40°C to avoid residual enzyme activity or Maillard reaction;

[0072] Post-treatment of enzymatic hydrolysate: Filter, collect the supernatant, and the solid residue can be returned for secondary enzymatic hydrolysis or used as a fermentation substrate.

[0073] The enzymatic hydrolysis process needs to achieve efficient removal of antigenic proteins through precise enzyme species selection, segmented hydrolysis strategy, and dynamic parameter regulation, further removing antigenic proteins in soybean meal raw materials and improving the removal rate of antigenic proteins.

[0074] The present invention provides a method for removing antigenic proteins from soybean meal. The soybean meal raw material is put into a cooking pot and subjected to high-temperature and high-pressure treatment through the cooking pot. During the heating process, a two-stage temperature-controlled cooking method is adopted. First, the soybean meal raw material is evenly heated to preliminarily soften the tissue of the soybean meal raw material in preparation for subsequent high-temperature treatment. Then the temperature is raised while maintaining the pressure state in the cooking pot, so that the structure of the antigenic proteins in the soybean meal raw material undergoes denaturation and inactivation under high-temperature and high-pressure conditions, thereby reducing its antigenicity to meet the requirements of subsequent production or application.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for removing antigenic proteins from soybean meal, characterized in that: Put the soybean meal raw material into the cooking pot and perform two-stage temperature-controlled cooking: Initial heating stage: the temperature in the cooking pot is raised from room temperature to 80°C at a heating rate of 5-10°C / min, and the heating time is about 10-15 minutes; High temperature cooking stage: continue to heat up to 110-121°C and maintain at this temperature for 20-30 minutes. During the high temperature cooking process, the temperature fluctuation range should be controlled within ±1°C, and the pressure in the cooking pot is maintained at 0.1-0.15MPa.

2. The method for removing antigenic protein from soybean meal according to claim 1, characterized in that: The cooking pot is heated internally by introducing high-temperature steam, and a stirring device is arranged inside the cooking pot to stir the soybean meal raw material during the heating process.

3. The method for removing antigenic protein from soybean meal according to claim 2, characterized in that: The cooking pot is provided with an exhaust valve, which is controlled by controlling the steam intake and the coordinated adjustment of the exhaust valve to ensure that the pressure is stable within the set range and the pressure fluctuation does not exceed ±0.02MPa.

4. The method for removing antigenic protein from soybean meal according to claim 2, characterized in that: Before cooking, the initial moisture content of the raw materials needs to be measured, and the moisture content of the raw materials is adjusted according to the initial moisture content of the raw materials, and the added water is adjusted to control the moisture content of the final cooking to 30-45%.

5. The method for removing antigenic protein from soybean meal according to claim 4, characterized in that: The spray humidification method uses filtered pure water in a uniform spray form, which is sprayed on the surface of the soybean meal raw material while stirring until the required moisture content is reached.

6. The method for removing antigenic protein from soybean meal according to claim 1, characterized in that: The raw materials after cooking should be sampled and tested to check whether the antigen protein removal rate meets the expected standards.

7. The method for removing antigenic protein from soybean meal according to claim 1, characterized in that: After the cooking is completed, the soybean meal raw material treated with high temperature and high pressure can also be subjected to enzymatic hydrolysis.

8. The method for removing antigenic protein from soybean meal according to claim 7, characterized in that: Enzymatic treatment includes: Pretreatment before enzymatic hydrolysis: The soybean meal raw material after high temperature and high pressure treatment needs to be cooled to 50-60℃, and the soybean meal raw material is crushed and passed through a 60-mesh sieve; Selection and preparation of enzymes: alkaline protease and papain are used; Preparation of each enzyme solution: weigh enzyme powder according to the enzyme addition amount of 2-4% w / w, dissolve it in 40℃ warm water, and prepare 5-10% enzyme solution; Enzymatic reaction conditions: Mass ratio of soybean meal to water: 1:3-1:5; pH adjustment: Use 1 mol / L NaOH or HCl to adjust to the optimal pH range for the enzyme; Temperature control: water bath or sandwich enzymatic hydrolysis tank temperature control, maintain 50-60℃; Stirring speed: Mechanical stirring 30-60rpm to ensure that the material is uniform and does not produce foam: Enzyme addition method: Add in steps: first add alkaline protease to hydrolyze for 2 hours, then add papain to continue hydrolyzing for 2 hours; One-time addition: Mix enzymes and add alkaline protease to papain in a ratio of 2:1 to the reaction; total reaction time is 4-6 hours, and samples are taken every 30 minutes to test the degree of hydrolysis (DH) or antigen residue; Inactivate the enzyme and terminate the reaction: heat to 85°C and maintain for 10-15 minutes to inactivate the protease. After inactivating the enzyme, quickly cool to below 40°C to avoid residual enzyme activity or Maillard reaction; Post-treatment of the enzymatic hydrolysate: filtration is performed, and the supernatant is collected. The solid residue can be returned to the secondary enzymatic hydrolysis or used as a fermentation substrate.

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