A method for preparing low-allergenic peanut protein peptide

Abstract

The invention discloses a method for preparing low-allergenic peanut protein peptides, belonging to the technical field of food. The method comprises the following steps: irradiating a peanut protein solution with far-infrared rays, adding a composite microorganism into the peanut protein solution for fermentation, and obtaining low-allergenic peanut protein powder after centrifugation, solid-liquid separation, sterilization, and drying after the fermentation is completed. The invention destroys components such as DNA, RNA, and ribosomes in peanut protein cells through far-infrared irradiation technology, changes the high-level structure of peanut protein, causes the peanut protein to denature, and then cooperates with subsequent microbial fermentation steps to obtain highly active peanut protein peptides and reduce their allergenicity. Compared with existing methods such as enzymolysis, the technology has the advantages of simple process, obvious effect, safe operation, high efficiency and speed, green environmental protection, easy promotion, and the like.

Description

Technical Field

[0001] The invention relates to a method for preparing peanut protein peptides by taking peanut meal as raw material, in particular to a method capable of synchronously degrading aflatoxin in peanut meal and preparing low-allergenic peanut protein peptides, belonging to the field of food technology. Background Art

[0002] Compared with the widely used soybean protein, peanut protein has the advantage of containing fewer gastrointestinal bloating factors and anti-nutritional factors; compared with rapeseed and cottonseed proteins, it has the advantage of containing fewer toxic substances. However, peanut meal has a low utilization rate and is mainly used as feed. It is cheap and much lower than the price of soybean meal. The main factor limiting the price and wide application of peanut meal is that it is easily infected by aflatoxin and peanuts are rich in more than 30 kinds of proteins and are identified as one of the eight major allergic foods. Peanut allergy is an adverse immune response caused by the protein in peanuts, which can cause asthma, eczema, allergic rhinitis, gastrointestinal bleeding, diarrhea and other symptoms.

[0003] Aflatoxins are highly toxic and carcinogenic, seriously endangering the health of humans and animals. A dose of only 0.294 mg / kg can cause acute poisoning and death in sensitive animals. In addition, aflatoxin is stable in nature, and general processing methods cannot remove its toxicity. Over the years, scientists have been committed to using a variety of methods such as physical, chemical, and biological methods to remove aflatoxins. Traditional physical and chemical methods for removing aflatoxins include ozone, ammoniation, alkali, radiation, adsorption, and ultrafiltration-diafiltration. These methods all have some problems, such as causing nutritional loss in food and feed, affecting sensory quality, and some methods require expensive equipment. Secondly, the reaction mechanism of some methods is reversible, and the toxicity of aflatoxin will reappear. These have limited the application of traditional physical and chemical methods in actual production.

[0004] Peanut protein peptide is a kind of bioactive peptide, with a balanced and comprehensive amino acid composition, and high content of glutamic acid and aspartic acid. It has a good effect on promoting the development of human brain cells and increasing memory. At the same time, peanut protein peptide also has multiple physiological activities such as anti-oxidation, anti-fatigue, lowering blood pressure, and improving body immunity. In addition, peanut protein peptide products also retain the original unique flavor of peanuts, and have good water solubility and water retention. It has good application prospects in many fields such as medicine, health food, food additives, and condiments.

[0005] Traditional methods of reducing food allergenicity include heating, high pressure, microwave irradiation, fermentation, and enzymatic hydrolysis. The most commonly used method for producing low-allergenic foods is enzymatic hydrolysis, which acts on allergens mainly by breaking protein peptide bonds, hydrolyzing allergenic proteins into small peptide segments, reducing their molecular weight, or by changing the tertiary structure of allergen epitopes and removing some epitopes on the surface of allergen proteins, thereby achieving the purpose of reducing their allergenicity. At present, the process of enzymatic hydrolysis is mature and the cost is low, but some bitter peptides are easily produced during the hydrolysis process, which seriously affects the development and application of enzymatic hydrolysis in reducing the allergenicity of food proteins.

[0006] The research on biodegradation and removal of aflatoxins at home and abroad mainly focuses on the direct action of microbial strains on aflatoxins, and the use of enzymes produced by strains as preparations to act on aflatoxins. However, there are few studies on the degradation of aflatoxins in complex matrices such as peanut meal.

[0007] In summary, if a method can be developed to produce peanut protein peptides using peanut meal as raw material, while removing the allergenicity of peanut protein and degrading aflatoxin, the economic value of peanuts will be greatly improved, and it will have extremely broad application prospects. Summary of the invention

[0008] The purpose of the present invention is to provide a method for preparing low-allergenic peanut protein peptides. The prepared peanut protein peptides do not contain aflatoxin and have greatly reduced allergenicity. The method adopts a far-infrared irradiation technology coupled with a composite bacterial fermentation method to obtain the peanut protein peptides, which has excellent and stable effects, low industrial costs, and good application prospects.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for preparing a low-allergenic peanut protein peptide comprises the following steps:

[0011] 1) Mix 100-200 parts of peanut meal passed through a 40-60 mesh sieve, 200-300 parts of distilled water, and 5-10 parts of sodium chloride by weight, and stir them uniformly to obtain a peanut protein solution;

[0012] 2) Irradiate the peanut protein solution with far infrared rays for 20-40 minutes;

[0013] 3) Add the following microorganisms to the peanut protein solution: Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis, and ferment for 48-72 hours at 30-35°C, 150-200 r / min, and 1:(1-2) ventilation ratio, and obtain a fermentation liquid after the fermentation is completed;

[0014] 4) The fermentation liquid is centrifuged to separate the solid and liquid, and the obtained solid matter is sterilized and dried to obtain peanut protein peptide.

[0015] Furthermore, in step 2), the wavelength of the far infrared ray is 200-800 µm, the irradiation power is 345-355 W, and the temperature is 30-32 °C.

[0016] Furthermore, the amounts of the microorganisms added in step 3) are: based on the weight of the peanut protein solution, 80-100 CFU / g of Lactobacillus paracasei, 30-50 CFU / g of Bifidobacterium lactis, 50-80 CFU / g of Bacillus licheniformis, and 50-80 CFU / g of Bacillus subtilis.

[0017] Furthermore, the sterilization condition in step 4) is to place at 121° C. for 30-40 minutes.

[0018] The ventilation ratio described in the present invention refers to the ratio of the volume of air introduced into the fermentation tank per minute to the total volume of the fermentation liquid.

[0019] The Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis used in the technical solution of the present invention can be selected from products of any manufacturer on the market.

[0020] The method for preparing peanut protein peptide provided by the present invention has the following advantages:

[0021] 1) Far infrared irradiation technology changes the higher-level structure of peanut protein by destroying components such as DNA, RNA and ribosomes in peanut protein cells, causing the peanut protein to denature, and then cooperates with the subsequent microbial fermentation steps to obtain highly active peanut protein peptides and reduce their allergenicity. Compared with existing enzymatic hydrolysis methods, this technology has the advantages of simple process, obvious effect, safe operation, high efficiency and speed, green and environmental protection, and easy promotion;

[0022] 2) The Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis used in the fermentation process can produce aflatoxin degrading enzymes, hydrolytic proteases, etc. during the fermentation and growth process. The enzymes produced in the fermentation process of the composite bacteria are diverse and can decompose the denatured peanut protein after far-infrared irradiation into small molecular active peptides and amino acids, while degrading aflatoxin and reducing its allergenicity, eliminating the harm of aflatoxin to the utilization of peanut meal and reducing the limitation of allergenicity on the application of peanut protein active peptides. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0024] Example 1

[0025] A method for preparing a low-allergenic peanut protein peptide comprises the following steps:

[0026] 1) Mix 100 parts of peanut meal passed through a 40-60 mesh sieve, 200 parts of distilled water, and 5 parts of sodium chloride by weight, and stir them uniformly to obtain a peanut protein solution;

[0027] 2) Irradiate the peanut protein solution with far infrared light for 20 minutes, the wavelength of the far infrared light is 200-800µm, the power is 345-355W, and the temperature is 30-32℃;

[0028] 3) Add the following microorganisms to the peanut protein solution: Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis, and the added amounts are respectively: 80-100 CFU / g of Lactobacillus paracasei, 30-50 CFU / g of Bifidobacterium lactis, 50-80 CFU / g of Bacillus licheniformis and 50-80 CFU / g of Bacillus subtilis based on the weight of the peanut protein solution, and then ferment for 48 hours at 30-35° C., a rotation speed of 150 r / min and a ventilation ratio of 1:1, and obtain a fermentation liquid after the fermentation is completed;

[0029] 4) The fermentation liquid is centrifuged to separate the solid and the liquid, and the obtained solid matter is sterilized and dried to obtain peanut protein peptide A.

[0030] Circular dichroism detection showed that after far-infrared irradiation treatment, the proportion of α-helix and β-fold in peanut protein decreased significantly, from the original 23.1% and 26.7% to 12.6% and 15.9%, and the irregular curl increased significantly, from the original 37.6% to 46.5%. This shows that the allergenic proteins Arh1 and Arh3 in peanut protein have denatured, proving that far-infrared irradiation treatment can indeed reduce the allergenicity of peanut protein.

[0031] Example 2

[0032] A method for preparing a low-allergenic peanut protein peptide comprises the following steps:

[0033] 1) By weight, 150 parts of peanut meal passed through a 40-60 mesh sieve, 300 parts of distilled water, and 8 parts of sodium chloride are mixed and homogenized to obtain a peanut protein solution;

[0034] 2) Irradiate the peanut protein solution with far infrared light for 30 minutes, the wavelength of the far infrared light is 200-800µm, the power is 345-355W, and the temperature is 30-32℃;

[0035] 3) Add the following microorganisms to the peanut protein solution: Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis, and add 80-100 CFU / g of Lactobacillus paracasei, 30-50 CFU / g of Bifidobacterium lactis, 50-80 CFU / g of Bacillus licheniformis and 50-80 CFU / g of Bacillus subtilis, respectively, based on the weight of the peanut protein solution, and then ferment for 60 hours at 30-35° C., a rotation speed of 180 r / min and a ventilation ratio of 1:2, and obtain a fermentation liquid after the fermentation is completed;

[0036] 4) The fermentation liquid is centrifuged to separate the solid and the liquid, and the obtained solid matter is sterilized and dried to obtain peanut protein peptide B.

[0037] Circular dichroism detection showed that after far-infrared irradiation treatment, the proportion of α-helix and β-fold in peanut protein decreased significantly, from the original 23.1% and 26.7% to 12.1% and 15.3%, and the irregular curl increased significantly, from the original 37.6% to 47.2%. This shows that the allergenic proteins Arh1 and Arh3 in peanut protein have denatured, proving that far-infrared irradiation treatment can indeed reduce the allergenicity of peanut protein.

[0038] Example 3

[0039] A method for preparing a low-allergenic peanut protein peptide comprises the following steps:

[0040] 1) Mix 200 parts of peanut meal passed through a 40-60 mesh sieve, 300 parts of distilled water, and 10 parts of sodium chloride by weight, and stir them uniformly to obtain a peanut protein solution;

[0041] 2) Irradiate the peanut protein solution with far infrared light for 40 minutes, the wavelength of the far infrared light is 200-800µm, the power is 345-355W, and the temperature is 30-32℃;

[0042] 3) Add the following microorganisms to the peanut protein solution: Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis, and add 80-100 CFU / g of Lactobacillus paracasei, 30-50 CFU / g of Bifidobacterium lactis, 50-80 CFU / g of Bacillus licheniformis and 50-80 CFU / g of Bacillus subtilis, respectively, based on the weight of the peanut protein solution, and then ferment for 72 hours at 30-35° C., a rotation speed of 200 r / min and a ventilation ratio of 1:2, and obtain a fermentation liquid after the fermentation is completed;

[0043] 4) The fermentation liquid is centrifuged to separate the solid and the liquid, and the obtained solid matter is sterilized and dried to obtain peanut protein peptide C.

[0044] Circular dichroism detection showed that after far-infrared irradiation treatment, the proportion of α-helix and β-fold in peanut protein decreased significantly, from the original 23.1% and 26.7% to 11.8% and 15.1%, and the irregular curl increased significantly, from the original 37.6% to 46.6%. This shows that the allergenic proteins Arh1 and Arh3 in peanut protein have denatured, proving that far-infrared irradiation treatment can indeed reduce the allergenicity of peanut protein.

[0045] Using peanut protein powder purchased from Qingdao Changshou Food Co., Ltd. as product D, the raw material peanut meal, peanut protein peptides A, B and C obtained in Examples 1-3, and product D were tested as follows:

[0046] 1. Aflatoxin content test

[0047] After commissioning a third party to test, the aflatoxin content of each product is shown in Table 1.

[0048] Table 1 Aflatoxin content of each product

[0049]

[0050] Among them, ND means not detected.

[0051] As can be seen from Table 1, no aflatoxin was detected in the peanut protein peptides A, B and C prepared by the present invention, indicating that the method of the present invention can effectively degrade aflatoxin.

[0052] 2. Allergenicity Testing

[0053] Each product and the raw material peanut meal were subjected to ELISA, and the results are shown in Table 2.

[0054] Table 2 ELISA results of various products

[0055]

[0056] It can be seen from the data in Table 2 that the content of Arh1 and Arh3 that produce allergenicity in peanut protein is significantly reduced, indicating that the method of far-infrared irradiation and composite bacterial fermentation of the present invention can indeed significantly reduce the allergenicity of peanut protein.

[0057] 3. Molecular weight distribution test

[0058] The molecular weight distribution of peanut protein peptide product C was determined by size exclusion chromatography, and the results are shown in Table 3.

[0059] Table 3 Molecular weight distribution of peanut protein peptide C

[0060]

[0061] It can be seen from Table 3 that more than 96% of the peanut protein peptides obtained in Example 3 have a molecular weight of less than 2.0 kDa, and more than 92% have a molecular weight of less than 1.0 kDa. Therefore, the peanut protein peptides obtained by the method of the present invention are all oligopeptides.

[0062] 4. Functional properties of peanut protein peptide

[0063] Taking peanut protein peptide product C as an example and product D as a control, the functional properties of the peanut protein peptide prepared by the present invention were determined.

[0064] 4.1 Functional assay

[0065] 4.1.1 Solubility determination method

[0066] 1% (m / V) protein product was dissolved in deionized water, and the solution pH was adjusted to 7.0 with 1.0 mol / L NaOH or HCl. After stirring at room temperature for 1 hour, the solution was centrifuged at 3000 r / min for 20 minutes and the supernatant was collected. 20 mL of the supernatant was concentrated and the protein content was determined (protein conversion factor 6.25).

[0067] Protein solubility = protein content in supernatant / total protein content in raw material × 100%.

[0068] 4.1.2 Water absorption determination method

[0069] The method of Beuchat (1977) was used. 10 mL of 10% protein solution was transferred into a 15 mL centrifuge tube, vortexed for 2 min to ensure that all the protein was evenly dispersed, left to stand at room temperature for 30 min, and centrifuged at 3000 r / min for 20 min. The supernatant was discarded and weighed. The experiment was repeated 3 times.

[0070] Water absorption = (W2 - W1) / W0;

[0071] Where W0 - protein mass, g; W1 - centrifuge tube weight + protein mass, g; W2 - centrifuge tube weight + sediment mass, g.

[0072] 4.1.3 Oil absorption determination method

[0073] The method of Chakraborty (1986) was used. 1 g of protein (W0) was weighed into a 15 mL centrifuge tube, 10 mL (V1) of soybean oil was transferred into the tube, and the solution was mixed evenly using a vortex shaker. The solution was allowed to stand at room temperature for 30 min, and then centrifuged at 3000 r / min for 20 min. The upper layer was quickly and carefully transferred into a dry 10 mL measuring cylinder, and the volume (V2) was recorded.

[0074] Oil retention (mL / g) = (V1 - V2 ) / W0; the test was repeated 3 times.

[0075] 4.2 Results and Analysis

[0076] 4.2.1 Solubility

[0077] The solubility of product C reached 97%, while the solubility of product D was 57%.

[0078] 4.2.2 Water absorption

[0079] The water absorption of product C reaches 3.99 g / g, while the water absorption of product D is 2.85 g / g. The water absorption of the peanut protein peptide prepared by the method of the present invention is significantly better than that of commercially available products.

[0080] 4.2.3 Oil absorption

[0081] The oil absorption of product C reaches 4.48 mL / g, while the oil absorption of product D is 4.06 mL / g. The oil absorption of the peanut protein peptide prepared by the present invention is significantly improved compared with the commercially available products.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a low-allergenic peanut protein peptide, characterized in that: The steps include: 1) Mix 100-200 parts of peanut meal passed through a 40-60 mesh sieve, 200-300 parts of distilled water, and 5-10 parts of sodium chloride by weight, and stir them uniformly to obtain a peanut protein solution; 2) Irradiate the peanut protein solution with far infrared rays for 20-40 minutes; 3) Add the following microorganisms to the peanut protein solution: Lactobacillus paracasei, Bifidobacterium lactis, Bacillus licheniformis and Bacillus subtilis, and ferment for 48-72 hours at 30-35°C, 150-200 r / min, and 1:(1-2) ventilation ratio, and obtain a fermentation liquid after the fermentation is completed; 4) The fermentation liquid is centrifuged to separate the solid and liquid, and the obtained solid matter is sterilized and dried to obtain peanut protein peptide.

2. The method according to claim 1, characterized in that The far infrared ray in step 2) has a wavelength of 200-800 µm, an irradiation power of 345-355 W, and a temperature of 30-32 °C.

3. The method according to claim 1 or 2, characterized in that: The amounts of the microorganisms added in step 3) are: based on the weight of the peanut protein solution, 80-100 CFU / g of Lactobacillus paracasei, 30-50 CFU / g of Bifidobacterium lactis, 50-80 CFU / g of Bacillus licheniformis, and 50-80 CFU / g of Bacillus subtilis.

4. The method according to claim 1 or 2, characterized in that: The sterilization condition described in step 4) is to place at 121°C for 30-40 minutes.

5. The method according to claim 3, characterized in that: The sterilization condition described in step 4) is to place at 121°C for 30-40 minutes.

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