A method for preparing a hypoallergenic milk protein base

Abstract

The application discloses a preparation method of a low-sensitization milk protein base, and relates to the technical field of milk product processing. The preparation method of the milk protein base comprises the following steps: (1) preheating fresh milk, adding 0.1-0.4% of protease, 0.02-0.05% of vitamin B1 and 0.02-0.08% of catechin by weight of the fresh milk, performing constant-temperature oscillation enzymolysis for 60-80 min, and then performing enzyme inactivation treatment to obtain a first treatment liquid; (2) preheating the first treatment liquid, adding 2-8% of aspergillus oryzae extract by weight of the first treatment liquid, uniformly mixing and stirring, performing constant-temperature water bath for 4-7 h, and then performing enzyme inactivation treatment to obtain a second treatment liquid; and (3) performing ultrafiltration on the second treatment liquid, collecting a filtrate, and concentrating and drying the filtrate to obtain the low-sensitization milk protein base. The application can improve the enzymolysis efficiency, effectively removes the sensitization protein in the milk by using a small amount of enzyme, and has a remarkable de-bittering effect, and is suitable for promotion.

Description

Technical Field

[0001] This invention relates to the field of dairy product processing technology, and in particular to a method for preparing a low-allergenic milk protein base. Background Technology

[0002] Cow's milk is a primary source of nutrition for infants and young children, but it is listed by the Food and Agriculture Organization of the United Nations as one of the eight major food allergens. The incidence of cow's milk protein allergy in infants and young children is approximately 2%-5%. Allergic reactions to cow's milk can cause vomiting, abdominal pain, diarrhea, hives, and even anaphylactic shock. The main allergenic proteins in cow's milk are β-lactoglobulin, α-lactalbumin, and casein. To reduce the risk of cow's milk allergy in infants and young children, desensitization treatment of cow's milk is necessary.

[0003] In existing technologies, desensitization methods for cow's milk mainly include heating, high pressure, glycosylation, and enzymatic hydrolysis. Enzymatic hydrolysis, using proteases, achieves the best results without affecting the nutritional value of the milk. However, the hydrophobic small peptides produced by enzymatic hydrolysis have varying degrees of bitterness, affecting the taste, and the cost is relatively high. Patent application number 201510131763.4 discloses a low-allergenic milk protein powder and its preparation method. This method uses a complex protease (flavor protease, alkaline protease, and neutral protease) combined with ultrasound to desensitize cow's milk. However, the enzymatic hydrolysis efficiency is low, the amount of enzyme used is large, and the cost is high, making it unsuitable for desensitizing large quantities of cow's milk. Therefore, how to debitter the enzymatic hydrolysis products and how to improve enzymatic hydrolysis efficiency and reduce enzyme usage are urgent problems that need to be solved. Summary of the Invention

[0004] To address the above shortcomings, this invention provides a method for preparing a low-allergenic milk protein base, which can improve enzymatic hydrolysis efficiency, effectively remove allergenic proteins in cow's milk with a small amount of enzyme, and has a significant debittering effect, making it suitable for widespread application. The specific technical solution is as follows:

[0005] A method for preparing a low-allergenic milk protein base includes the following steps:

[0006] (1) After preheating the fresh milk, add 0.1-0.4% of the weight of the fresh milk of protease, 0.02-0.05% of vitamin B1 and 0.02-0.08% of catechin, and hydrolyze at a constant temperature for 60-80 minutes. Then, perform enzyme inactivation treatment to obtain the first treatment solution.

[0007] (2) After preheating the first treatment solution, add 2-8% of Aspergillus oryzae extract by weight of the first treatment solution and mix and stir evenly. Then, perform enzyme inactivation treatment to obtain the second treatment solution.

[0008] (3) The second treatment liquid is subjected to ultrafiltration, the filtrate is collected, concentrated and dried to obtain the low-allergenic milk protein base.

[0009] Furthermore, the preheating temperature is 40-50°C.

[0010] Furthermore, the protease is one of an alkaline protease, a neutral protease, or a trypsin.

[0011] Furthermore, the enzyme inactivation treatment involves heating to 80-90°C, holding for 5-10 minutes, and then naturally cooling to room temperature.

[0012] Furthermore, the preparation method of the Aspergillus oryzae extract is as follows:

[0013] S1. Prepare the culture medium: 20-40 g / L corn flour, 8-15 g / L beef extract, 5-15 g / L apple peel, 0.01-0.05 g / L potassium dihydrogen phosphate and 0.05-0.15 g / L calcium chloride. Adjust the pH to 7-8 and sterilize at 121℃ for 20-30 min.

[0014] S2. Inoculate the above culture medium with 5-10% of the weight of Aspergillus oryzae, and culture in a shaker at 30-45℃ for 40-60h. Centrifuge, filter, and take the permeate.

[0015] S3. Take half of the permeate and add 100-200 times the weight of physiological saline to the permeate and stir well. Incubate in a water bath at 30-35℃ for 20-30 minutes, centrifuge, take the supernatant, concentrate and dry to obtain the first extract.

[0016] S4. After filtering the other half of the permeate, add 1-2 times the volume of anhydrous ethanol to the filtrate, let stand until a precipitate forms, take the precipitate and dry it to obtain the second extract.

[0017] S5. Mix the first extract and the second extract to obtain the Aspergillus oryzae extract.

[0018] Furthermore, the apple peel is dried and pulverized into powder before use.

[0019] Furthermore, the ultrafiltration is performed using a ceramic membrane with a pore size of 0.015-0.02 μm.

[0020] This invention provides the use of the low-allergenic milk protein base material prepared by the above method in the preparation of low-allergenic foods.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention improves enzymatic hydrolysis efficiency, enabling the effective removal of allergenic proteins in milk with a high removal rate using only a small amount of enzyme. Vitamin B1 activates proteases, increasing their activity and accelerating the hydrolysis process. Catechins, acting as protease activity protectants, bind to proteases to prevent oxidation and subsequent activity reduction during hydrolysis, thus improving protease activity stability and slowing down enzyme inactivation. Therefore, vitamin B1 enhances enzyme activity, while catechins stabilize it; their combined use effectively improves the efficiency of enzymatic hydrolysis of allergenic proteins, resulting in a higher removal rate and more significant effects.

[0023] 2. The present invention exhibits significant debittering effects. The Aspergillus oryzae extract prepared by the method of the present invention contains flavor protease and pectin. The flavor protease can further degrade bitter substances into flavor amino acids, removing bitterness while enhancing milk flavor. Pectin can polymerize some bitter substances, achieving the purpose of removing bitterness. Therefore, the Aspergillus oryzae extract can effectively remove bitterness and increase flavor compounds. Detailed Implementation

[0024] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0025] The fresh milk used in the embodiments and comparative examples of this invention was obtained from buffalo milk from Xijiang Farm in Guangxi; the protease was purchased from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.; and Aspergillus oryzae was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0026] Example 1

[0027] A method for preparing a low-allergenic milk protein base includes the following steps:

[0028] (1) After preheating fresh milk to 40°C, add 0.1% alkaline protease, 0.02% vitamin B1 and 0.02% catechin by weight of milk, and hydrolyze at a constant temperature for 60 minutes. Then, perform enzyme inactivation treatment, that is, heat to 80°C, keep for 5 minutes, and cool naturally to room temperature to obtain the first treatment solution.

[0029] (2) After preheating the first treatment solution to 40°C, add 2% of the weight of Aspergillus oryzae extract of the first treatment solution and mix and stir evenly. Then, perform enzyme inactivation treatment by heating to 80°C, holding for 5 minutes, and then naturally cooling to room temperature to obtain the second treatment solution.

[0030] The preparation method of Aspergillus oryzae extract is as follows:

[0031] S1. Preparation of culture medium: 20 g / L corn flour, 8 g / L beef extract, 5 g / L apple peel, 0.01 g / L potassium dihydrogen phosphate and 0.05 g / L calcium chloride, adjust pH to 7, and sterilize at 121℃ for 20 min; the apple peel should be dried and pulverized into powder before use.

[0032] S2. Inoculate the above culture medium with 5% of the culture medium weight of Aspergillus oryzae, culture in a shaker at 30°C for 40 h, centrifuge, filter, and take the permeate;

[0033] S3. Take half of the permeate and add 100 times the weight of physiological saline to the permeate and stir well. Incubate in a water bath at 30°C for 20 minutes, centrifuge, take the supernatant, concentrate and dry to obtain the first extract.

[0034] S4. After filtering the other half of the permeate, add 1 volume of anhydrous ethanol to the filtrate, let stand until a precipitate forms, take the precipitate and dry it to obtain the second extract.

[0035] S5. Mix the first extract and the second extract to obtain the Aspergillus oryzae extract.

[0036] (3) The second treatment liquid is ultrafiltered using a ceramic membrane with a pore size of 0.015 μm, the filtrate is collected, concentrated and dried to obtain low-allergenic milk protein base.

[0037] Example 2

[0038] A method for preparing a low-allergenic milk protein base includes the following steps:

[0039] (1) After preheating fresh milk to 50°C, add 0.4% of neutral protease, 0.05% of vitamin B1 and 0.08% of catechin by weight of milk, and hydrolyze at a constant temperature for 80 minutes. Then, perform enzyme inactivation treatment, that is, heat to 90°C, maintain for 10 minutes, and cool naturally to room temperature to obtain the first treatment solution.

[0040] (2) After preheating the first treatment solution to 50°C, add 8% of the weight of Aspergillus oryzae extract of the first treatment solution and mix and stir evenly. Then, perform enzyme inactivation treatment by heating to 90°C, holding for 10 minutes, and then naturally cooling to room temperature to obtain the second treatment solution.

[0041] The preparation method of Aspergillus oryzae extract is as follows:

[0042] S1. Preparation of culture medium: 40 g / L corn flour, 15 g / L beef extract, 15 g / L apple peel, 0.05 g / L potassium dihydrogen phosphate and 0.15 g / L calcium chloride, adjust pH to 8, and sterilize at 121℃ for 30 min; the apple peel should be dried and pulverized into powder before use.

[0043] S2. Inoculate the above culture medium with 10% of the culture medium weight of Aspergillus oryzae, culture in a shaker at 45°C for 60 h, centrifuge, filter, and take the permeate;

[0044] S3. Take half of the permeate and add 200 times the weight of physiological saline to the permeate and stir well. Incubate in a water bath at 35°C for 30 minutes, centrifuge, take the supernatant, concentrate and dry to obtain the first extract.

[0045] S4. After filtering the other half of the permeate, add 2 times the volume of anhydrous ethanol to the filtrate, let stand until a precipitate forms, take the precipitate and dry it to obtain the second extract.

[0046] S5. Mix the first extract and the second extract to obtain the Aspergillus oryzae extract.

[0047] (3) The second treatment liquid is ultrafiltered using a ceramic membrane with a pore size of 0.02 μm, the filtrate is collected, concentrated and dried to obtain low-allergenic milk protein base.

[0048] Example 3

[0049] A method for preparing a low-allergenic milk protein base includes the following steps:

[0050] (1) After preheating fresh milk to 45°C, add 0.2% trypsin, 0.03% vitamin B1 and 0.05% catechin by weight of milk, and hydrolyze at a constant temperature for 70 minutes. Then, perform enzyme inactivation treatment, that is, heat to 85°C, maintain for 8 minutes, and cool naturally to room temperature to obtain the first treatment solution.

[0051] (2) After preheating the first treatment solution to 45°C, add 6% of the weight of Aspergillus oryzae extract of the first treatment solution and mix and stir evenly. Then, perform enzyme inactivation treatment by heating to 85°C, holding for 8 minutes, and then naturally cooling to room temperature to obtain the second treatment solution.

[0052] The preparation method of Aspergillus oryzae extract is as follows:

[0053] S1. Preparation of culture medium: 30 g / L corn flour, 12 g / L beef extract, 10 g / L apple peel, 0.02 g / L potassium dihydrogen phosphate and 0.09 g / L calcium chloride, adjust pH to 7.2, and sterilize at 121℃ for 25 min; the apple peel should be dried and pulverized into powder before use.

[0054] S2. Inoculate the above culture medium with 5% of the culture medium weight of Aspergillus oryzae, culture in a shaker at 40°C for 50 h, centrifuge, filter, and take the permeate;

[0055] S3. Take half of the permeate and add 150 times the weight of physiological saline to the permeate and stir well. Incubate in a water bath at 32°C for 25 minutes, centrifuge, take the supernatant, concentrate and dry to obtain the first extract.

[0056] S4. After filtering the other half of the permeate, add 1.5 times the volume of anhydrous ethanol to the filtrate, let it stand until a precipitate forms, take the precipitate and dry it to obtain the second extract.

[0057] S5. Mix the first extract and the second extract to obtain the Aspergillus oryzae extract.

[0058] (3) The second treatment liquid is ultrafiltered using a ceramic membrane with a pore size of 0.018 μm, the filtrate is collected, concentrated and dried to obtain low-allergenic milk protein base.

[0059] I. Comparative Examples 1 to 4 were set up to demonstrate the desensitization effect of the method of the present invention.

[0060] Comparative Example 1: Same as Example 1, but without the addition of vitamin B1 and catechins in step (1).

[0061] Comparative Example 2: Same as Example 1, but without the addition of vitamin B1 in step (1).

[0062] Comparative Example 3: Same as Example 1, but without the addition of catechins in step (1).

[0063] Comparative Example 4: The method disclosed in patent application number 201810252433.4 was used for treatment, namely, desensitization by a combination of ultrasound, protease and glycosylation.

[0064] The antigenicity of the protein bases prepared in Examples 1 to 3 and Comparative Examples 1 to 4 was determined, and the results are shown in Table 1. The antigenicity was determined by diluting the bases.

[0065] The antigenicity assay was performed using an indirect competitive ELISA method: (1) Antigen coating: α-lactoglobulin, β-lactoglobulin and casein were diluted with coating solution and added to the wells of the ELISA plate. The plate was incubated overnight at 4°C. (2) Antigen and antibody premixing: The diluted test sample and an equal volume of diluted antiserum were added to the reaction tube. At the same time, a reaction tube containing only antiserum without sample was used as a non-competitive tube. The plate was incubated overnight at 4°C. (3) Washing: The plate was brought to room temperature the next day. The liquid in the wells was discarded. Each well was washed 4 times with PBST for 3 minutes each time and then patted dry on absorbent paper. (4) Blocking: Gelatin was added to each well. The plate was incubated at 37°C for 1 hour. The plate was brought to room temperature. The blocking solution was discarded. The plate was washed 4 times with PBST for 3 minutes each time and then patted dry on absorbent paper. (5) Antigen-antibody reaction: The antigen-antibody premix was added to the ELISA plate. The plate was placed in a humidified chamber and incubated at 37°C for 1 hour. After the reaction, the liquid in the wells was discarded. The plate was washed 4 times with PBST for 3 minutes each time and then patted dry on absorbent paper. (6) Add enzyme-labeled anti-antibody: Add diluted HRP-goat anti-rabbit IgG to each well, place in a humidified chamber, and incubate at 37°C for 1 h. After the reaction, discard the liquid in the well, wash 4 times with PBST for 3 min each time, and pat dry on absorbent paper. (7) Color development: Add HRP substrate solution to each well, place in a humidified chamber, and incubate at 37°C for 20 min. A yellow color will appear. (8) Stop the reaction: Add sulfuric acid to each well to stop the reaction. (9) Colorimetric analysis: Measure the absorbance of the liquid in each well at 490 nm using an ELISA reader. (10) Calculation of antigen reduction rate: Antigen reduction rate (%) = (A 样品 -A 原乳 / A 无竞争 -A 原乳 )×100.

[0066] Table 1. Antigenicity of each group of protein-based materials

[0067] Implementation α-lactoglobulin antigen reduction rate (%) β-lactoglobulin antigen reduction rate (%) Casein antigen reduction rate (%) Example 1 92 91 94 Example 2 98 94 99 Example 3 95 92 97 Comparative Example 1 70 70 75 Comparative Example 2 88 82 89 Comparative Example 3 85 80 82 Comparative Example 4 79 74 80

[0068] Example 2 showed the best clearance effect on sensitizing proteins such as α-lactoglobulin, β-lactoglobulin, and casein. Data from Examples 1 and 4 (Comparative Example 4) show that the method of this invention is superior to existing technologies; that is, the combined enzymatic hydrolysis effect of protease + vitamin B1 + catechin is better than the combination of ultrasound + protease + glycosylation. Data from Examples 1 to 3 (Comparative Examples 1 to 3) show that vitamin B1 and catechin in this invention have a synergistic effect, improving the efficiency of enzymatic hydrolysis of sensitizing proteins, resulting in a higher clearance rate and more significant effect.

[0069] II. Comparative Examples 5 and 6 are set up to demonstrate the debittering effect of the method of the present invention.

[0070] Comparative Example 5: Same as Example 1, but without step S3 in the preparation method of Aspergillus oryzae extract, i.e., the first extract is not prepared.

[0071] Comparative Example 6: Same as Example 1, but without step S4 in the preparation method of Aspergillus oryzae extract, i.e., no second extract is prepared.

[0072] Comparative Example 7: Same as Example 1, but without Aspergillus oryzae extract.

[0073] The bitterness of the protein base materials prepared in Examples 1 to 3 and Comparative Examples 5 to 7 was determined, and the results are shown in Table 2. The method for determining the bitterness is as follows: the bitterness of each group of protein base materials was quantitatively scored. Twenty people were selected to score the diluted protein base materials of each group at the same time, and the average score was calculated. The average score is the bitterness value. The scoring rules are: no bitterness (0-0.9 points), slightly bitter (1-2.9 points), quite bitter (3-3.9 points), and bitter (4-5 points).

[0074] Table 2. Bitterness level of each group of protein-based materials

[0075] Group Bitterness value Description of bitterness Example 1 0.80 No bitter taste Example 2 0.71 No bitter taste Example 3 0.77 No bitter taste Comparative Example 5 3.10 rather bitter Comparative Example 6 2.41 Slightly bitter Comparative Example 7 4.88 bitter

[0076] Table 2 shows that the Aspergillus oryzae extract prepared by the present invention can further degrade or polymerize bitter substances to achieve the purpose of removing bitterness. The resulting protein base has a low bitterness value, which is acceptable to the general public.

[0077] III. Calculation using enzyme dosage.

[0078] Patent application number 201810252433.4 discloses a method of desensitization using a combination of ultrasound, protease, and glycosylation, with 150,000-250,000 U of alkaline protease added per kilogram of the mixture. The enzyme dosage used in this invention is 0.1-0.4% of the weight of fresh milk, i.e., 0.01-0.04 kg of enzyme (i.e., 10,000-40,000 mg) per kg of milk. Assuming 1 mg of enzyme is equivalent to 0.4 U, the enzyme dosage used in this application is 4,000-16,000 U per kilogram of milk, which is less than the enzyme dosage used in the prior art.

[0079] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preparing a low-allergenic milk protein base, characterized in that, Includes the following steps: (1) After preheating the fresh milk, add 0.1-0.4% of the weight of the fresh milk of protease, 0.02-0.05% of vitamin B1 and 0.02-0.08% of catechin, and hydrolyze at a constant temperature for 60-80 minutes. Then, perform enzyme inactivation treatment to obtain the first treatment solution. (2) After preheating the first treatment solution, add 2-8% of Aspergillus oryzae extract by weight of the first treatment solution and mix and stir evenly. Then, perform enzyme inactivation treatment to obtain the second treatment solution. The preparation method of the Aspergillus oryzae extract is as follows: S1. Prepare the culture medium: 20-40 g / L corn flour, 8-15 g / L beef extract, 5-15 g / L apple peel, 0.01-0.05 g / L potassium dihydrogen phosphate and 0.05-0.15 g / L calcium chloride. Adjust the pH to 7-8 and sterilize at 121℃ for 20-30 min. S2. Inoculate the culture medium with 5-10% of the weight of Aspergillus oryzae, and culture in a shaker at 30-45℃ for 40-60 hours. Centrifuge, filter, and collect the permeate. S3. Take half of the permeate and add 100-200 times the weight of physiological saline to the permeate and stir well. Incubate in a water bath at 30-35℃ for 20-30 minutes, centrifuge, take the supernatant, concentrate and dry to obtain the first extract. S4. After filtering the other half of the permeate, add 1-2 times the volume of anhydrous ethanol to the filtrate, let stand until a precipitate forms, take the precipitate and dry it to obtain the second extract. S5. Mix the first extract and the second extract to obtain the Aspergillus oryzae extract; (3) The second treatment liquid is subjected to ultrafiltration, the filtrate is collected, concentrated and dried to obtain the low-allergenic milk protein base.

2. The method for preparing a low-allergenic milk protein base according to claim 1, characterized in that, The preheating temperature is 40-50℃.

3. The method for preparing a low-allergenic milk protein base according to claim 1, characterized in that, The protease is one of alkaline protease, neutral protease, or trypsin.

4. The method for preparing a low-allergenic milk protein base according to claim 1, characterized in that, The enzyme inactivation treatment involves heating to 80-90℃, holding for 5-10 minutes, and then naturally cooling to room temperature.

5. The method for preparing a low-allergenic milk protein base according to claim 1, characterized in that, The apple peel is dried and pulverized into powder before use.

6. The method for preparing a low-allergenic milk protein base according to claim 1, characterized in that, The ultrafiltration is performed using a ceramic membrane with a pore size of 0.015-0.02 μm.

7. The application of the low-allergenic milk protein base material as described in claim 1 in the preparation of low-allergenic foods.