阅读说明：本技术 一种低致敏性酪蛋白肽的生产方法及其应用 (Production method and application of low-sensitization casein peptide ) 是由 王中振 谢涛 谢骞 刘家生 于 2020-12-08 设计创作，主要内容包括：本发明提供了一种低致敏性酪蛋白肽的生产方法,包括以下步骤：(1)将酪蛋白加入纯水中,加热后保温搅拌,冷却至室温后转入超高压设备中,升压后保压,解压后得到酪蛋白溶液；(2)将瓜尔胶加入纯水中搅拌至混合均匀,加入甲基丙烯酸酐后用氢氧化钠溶液将体系的pH值调节至8-10,继续搅拌反应后加入复合蛋白酶,加热后保温搅拌得到复合蛋白酶溶液；(3)将酪蛋白溶液与复合蛋白酶溶液混合均匀后得到混合液,置于均质机中均质,取出后调节pH值至7,加热后酶解得到酶解液；(4)将酶解液加热灭酶,冷却至室温后离心得到上清液,将上清液冷冻干燥后得到低致敏性酪蛋白肽。本发明生产出的低致敏性酪蛋白肽的致敏性较低,苦味较小且稳定性较好。(The invention provides a production method of low-sensitization casein peptide, which comprises the following steps: (1) adding casein into pure water, heating, keeping the temperature, stirring, cooling to room temperature, transferring into an ultrahigh pressure device, pressurizing, maintaining the pressure, and decompressing to obtain a casein solution; (2) adding guar gum into pure water, stirring until the guar gum is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 8-10 by using a sodium hydroxide solution, continuously stirring for reaction, adding composite protease, heating, keeping the temperature, and stirring to obtain a composite protease solution; (3) uniformly mixing the casein solution and the compound protease solution to obtain a mixed solution, placing the mixed solution in a homogenizer for homogenization, taking out the mixed solution, adjusting the pH value to 7, heating and carrying out enzymolysis to obtain an enzymolysis solution; (4) heating the enzymolysis liquid to inactivate enzyme, cooling to room temperature, centrifuging to obtain supernatant, and freeze drying the supernatant to obtain the low-sensitization casein peptide. The low-sensitization casein peptide produced by the invention has low sensitization, small bitter taste and good stability.)

1. A production method of low-sensitization casein peptide is characterized in that: the method comprises the following steps:

(1) adding casein into pure water, heating to 75-90 ℃, keeping the temperature, stirring for 10-15 minutes, cooling to room temperature, transferring into an ultrahigh pressure device, increasing the pressure to 500MPa, maintaining the pressure for 10-15 minutes, and decompressing to obtain a casein solution;

(2) adding guar gum into pure water, stirring until the guar gum is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 8-10 by using a sodium hydroxide solution, continuously stirring for reaction for 4-5 hours, adding compound protease, heating to 45-55 ℃, preserving heat, and stirring for 0.5-1 hour to obtain a compound protease solution;

(3) uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) to obtain a mixed solution, placing the mixed solution in a homogenizer for homogenizing for 2 minutes, taking out the mixed solution, adjusting the pH value to 7, heating to 50-60 ℃, and carrying out enzymolysis for 2-3 hours to obtain an enzymolysis solution;

(4) heating the enzymolysis liquid obtained in the step (3) to 90-110 ℃, inactivating enzyme for 10-20 minutes, cooling to room temperature, centrifuging to obtain supernatant, and freeze-drying the supernatant to obtain the hypoallergenic casein peptide.

2. The method of claim 1, wherein the method comprises the steps of: in the step (1), the ratio of casein to pure water is (2-4) g:100 mL.

3. The method of claim 1, wherein the method comprises the steps of: in the step (1), the pressure increasing speed is 100 MPa/min, and the decompression time is 10 seconds.

4. The method of claim 1, wherein the method comprises the steps of: in the step (2), the proportion of the guar gum, the pure water, the methacrylic anhydride and the compound protease is 1g (150) -200 mL:4mL:50mg, and the compound protease consists of alkaline protease, trypsin and papain.

5. The method of claim 1, wherein the method comprises the steps of: in the step (2), the concentration of the sodium hydroxide solution is 1 mol/L.

6. The method of claim 1, wherein the method comprises the steps of: in the step (3), the mass ratio of the casein solution to the compound protease solution is (40-50): 1.

7. The method of claim 1, wherein the method comprises the steps of: in the step (3), the rotating speed of the homogenizer is 8000 rpm, and the pressure of the homogenizer is 60 MPa.

8. The method of claim 1, wherein the method comprises the steps of: in the step (4), the speed of centrifugation is 8000 rpm.

9. Hypoallergenic casein peptides obtainable by the production method according to any one of claims 1 to 8.

10. Use of the hypoallergenic casein peptide according to claim 9 for the preparation of a health food.

Technical Field

The invention relates to a production method and application of low-sensitization casein peptide.

Background

Casein is the protein with the highest content in milk, is mainly used as a food raw material or a microorganism culture medium, casein peptide (also called casein active peptide or casein active polypeptide) prepared by utilizing a protein enzymatic hydrolysis technology has various physiological effects of preventing mineral loss, preventing decayed teeth, resisting oxidation, preventing and treating osteoporosis and rickets, promoting animal in-vitro fertilization, regulating blood pressure, treating iron-deficiency anemia, magnesium-deficiency neuritis and the like, and particularly has the reputation of a mineral carrier due to the functional characteristics of promoting the high-efficient absorption of macroelements (Ca and Mg) and microelements (Fe, Zn, Cu, Cr, Ni, Co, Mn and Se), in particular, calcium ions are combined to form a soluble complex, on one hand, the calcium is effectively prevented from forming precipitates in the neutral or slightly alkaline environment of the small intestine, and on the other hand, the calcium can be absorbed by intestinal wall cells under the condition without VD. The existing casein peptide production method usually adopts protease to carry out enzymolysis on casein, has the problems of low enzymolysis degree and yield, and has high product sensitization, heavy bitter taste and poor stability, thereby limiting the application of the casein peptide in health-care food.

Chinese invention with application number CN01115249.4 discloses a process for producing casein active polypeptide by using crude cheese, which comprises pretreating crude cheese, adding alkaline solution into casein to convert and modify the casein into water-soluble casein, adding at least two proteolytic enzymes into the water-soluble casein to carry out enzymolysis respectively, and mixing the enzymolysis solutions to obtain casein active polypeptide. Although the invention improves the enzymolysis degree, the produced casein active polypeptide still has the problems of higher sensitization, heavier bitter taste and poor stability.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for producing low-sensitization casein peptide, wherein the produced low-sensitization casein peptide has low sensitization, small bitter taste and good stability.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a production method of low-sensitization casein peptide comprises the following steps:

(1) adding casein into pure water, heating to 75-90 ℃, keeping the temperature, stirring for 10-15 minutes, cooling to room temperature, transferring into an ultrahigh pressure device, increasing the pressure to 500MPa, maintaining the pressure for 10-15 minutes, and decompressing to obtain a casein solution;

(2) adding guar gum into pure water, stirring until the guar gum is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 8-10 by using a sodium hydroxide solution, continuously stirring for reaction for 4-5 hours, adding compound protease, heating to 45-55 ℃, preserving heat, and stirring for 0.5-1 hour to obtain a compound protease solution;

(3) uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) to obtain a mixed solution, placing the mixed solution in a homogenizer for homogenizing for 2 minutes, taking out the mixed solution, adjusting the pH value to 7, heating to 50-60 ℃, and carrying out enzymolysis for 2-3 hours to obtain an enzymolysis solution;

(4) heating the enzymolysis liquid obtained in the step (3) to 90-110 ℃, inactivating enzyme for 10-20 minutes, cooling to room temperature, centrifuging to obtain supernatant, and freeze-drying the supernatant to obtain the hypoallergenic casein peptide.

Further, in the step (1) of the present invention, the ratio of casein to pure water is (2-4) g:100 mL.

Further, in the step (1) of the present invention, the pressure increasing rate is 100 MPa/min, and the pressure decreasing time is 10 seconds.

Furthermore, in the step (2) of the invention, the proportion of the guar gum, the pure water, the methacrylic anhydride and the compound protease is 1g (150) -200 mL:4mL:50mg, and the compound protease consists of the alkaline protease, the trypsin and the papain.

Further, in the step (2) of the present invention, the concentration of the sodium hydroxide solution is 1 mol/L.

Further, in the step (3) of the invention, the mass ratio of the casein solution to the compound protease solution is (40-50): 1.

Further, in the step (3) of the present invention, the rotation speed of the homogenizer is 8000 rpm, and the pressure of the homogenizer is 60 MPa.

Further, in the step (4) of the present invention, the speed of centrifugation is 8000 rpm.

The invention also provides the low-sensitization casein peptide obtained by the production method and the application of the low-sensitization casein peptide in preparing health-care food.

Compared with the prior art, the invention has the following beneficial effects:

1) the protease used by the invention is a compound protease consisting of alkaline protease, trypsin and papain, the enzymolysis of the three proteases on different components in casein can be comprehensively exerted, guar gum and methacrylate react and then are mixed with the compound protease to prepare a compound protease solution, the reacted guar gum forms a coating on the compound protease, the stability of the compound protease is improved, the casein is subjected to moderate enzymolysis, most of allergens in the casein are eliminated, and the casein peptide which has low allergenicity and small bitter taste and can be applied to health-care food is obtained.

2) The casein is subjected to high-pressure treatment in the step (1), and the high-pressure treatment is favorable for accelerating the subsequent enzymolysis process and can improve the stability of the product.

3) The invention also carries out short-time homogenization treatment on the mixed solution consisting of the casein solution and the compound protease solution before the start of enzymolysis, and the homogenization treatment can further eliminate the allergen of the casein, reduce the allergenicity of the product and improve the oxidation resistance of the product.

Detailed Description

The present invention will be described in detail with reference to specific embodiments, and the exemplary embodiments and descriptions thereof herein are provided to explain the present invention but not to limit the present invention.

Example 1

A method for producing hypoallergenic casein peptides, comprising the steps of:

(1) adding casein into pure water, wherein the ratio of the casein to the pure water is 3g:100mL, heating to 80 ℃, keeping the temperature, stirring for 12 minutes, cooling to room temperature, transferring into an ultrahigh pressure device, increasing the pressure to 500MPa at the speed of 100 MPa/min, keeping the pressure for 12 minutes, and decompressing for 10 seconds to obtain a casein solution;

(2) adding guar gum into pure water, stirring until the mixture is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 9 by using a sodium hydroxide solution with the concentration of 1mol/L, continuously stirring for reacting for 4.5 hours, adding compound protease consisting of alkaline protease, trypsin and papain, heating to 50 ℃, keeping the temperature, and stirring for 0.8 hour to obtain a compound protease solution, wherein the proportion of the guar gum, the pure water, the methacrylic anhydride and the compound protease is 1g:180mL:4mL:50 mg;

(3) uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) in a mass ratio of 45:1 to obtain a mixed solution, placing the mixed solution in a homogenizer with the rotation speed of 8000 rpm and the pressure of 60MPa for homogenizing for 2 minutes, taking out, adjusting the pH value to 7, heating to 55 ℃, and carrying out enzymolysis for 2.5 hours to obtain an enzymolysis solution;

(4) and (4) heating the enzymolysis liquid obtained in the step (3) to 100 ℃, inactivating the enzyme for 15 minutes, cooling to room temperature, centrifuging at 8000 rpm to obtain supernatant, and freeze-drying the supernatant to obtain the hypoallergenic casein peptide.

Example 2

A method for producing hypoallergenic casein peptides, comprising the steps of:

(1) adding casein into pure water, wherein the ratio of the casein to the pure water is 2g:100mL, heating to 90 ℃, keeping the temperature, stirring for 11 minutes, cooling to room temperature, transferring into an ultrahigh pressure device, increasing the pressure to 500MPa at the speed of 100 MPa/min, keeping the pressure for 11 minutes, and decompressing for 10 seconds to obtain a casein solution;

(2) adding guar gum into pure water, stirring until the mixture is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 8.5 by using a sodium hydroxide solution with the concentration of 1mol/L, continuously stirring for reaction for 5 hours, adding compound protease consisting of alkaline protease, trypsin and papain, heating to 45 ℃, keeping the temperature, stirring for 1 hour to obtain a compound protease solution, wherein the proportion of the guar gum, the pure water, the methacrylic anhydride and the compound protease is 1g:150mL:4mL:50 mg;

(3) uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) in a mass ratio of 42:1 to obtain a mixed solution, placing the mixed solution in a homogenizer with the rotation speed of 8000 rpm and the pressure of 60MPa for homogenizing for 2 minutes, taking out, adjusting the pH value to 7, heating to 55 ℃, and carrying out enzymolysis for 2.5 hours to obtain an enzymolysis solution;

(4) heating the enzymolysis liquid obtained in the step (3) to 95 ℃, inactivating enzyme for 16 minutes, cooling to room temperature, centrifuging at 8000 rpm to obtain supernatant, and freeze-drying the supernatant to obtain the hypoallergenic casein peptide.

Example 3

A method for producing hypoallergenic casein peptides, comprising the steps of:

(1) adding casein into pure water, wherein the ratio of the casein to the pure water is 2.5g:100mL, heating to 85 ℃, keeping the temperature, stirring for 15 minutes, cooling to room temperature, transferring into an ultrahigh pressure device, increasing the pressure to 500MPa at the speed of 100 MPa/min, keeping the pressure for 15 minutes, and decompressing for 10 seconds to obtain a casein solution;

(2) adding guar gum into pure water, stirring until the mixture is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 8 by using a sodium hydroxide solution with the concentration of 1mol/L, continuously stirring for reacting for 4 hours, adding a compound protease consisting of alkaline protease, trypsin and papain, heating to 55 ℃, keeping the temperature, and stirring for 0.5 hour to obtain a compound protease solution, wherein the proportion of the guar gum, the pure water, the methacrylic anhydride and the compound protease is 1g:160mL:4mL:50 mg;

(3) uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) in a mass ratio of 40:1 to obtain a mixed solution, placing the mixed solution in a homogenizer with the rotation speed of 8000 rpm and the pressure of 60MPa for homogenizing for 2 minutes, taking out, adjusting the pH value to 7, heating to 60 ℃, and carrying out enzymolysis for 2 hours to obtain an enzymolysis solution;

(4) heating the enzymolysis liquid obtained in the step (3) to 90 ℃, inactivating enzyme for 20 minutes, cooling to room temperature, centrifuging at 8000 rpm to obtain supernatant, and freeze-drying the supernatant to obtain the hypoallergenic casein peptide.

Example 4

A method for producing hypoallergenic casein peptides, comprising the steps of:

(1) adding casein into pure water, wherein the ratio of the casein to the pure water is 4g:100mL, heating to 75 ℃, keeping the temperature and stirring for 10 minutes, cooling to room temperature, transferring into an ultrahigh pressure device, increasing the pressure to 500MPa at the speed of 100 MPa/min, keeping the pressure for 10 minutes, and decompressing for 10 seconds to obtain a casein solution;

(2) adding guar gum into pure water, stirring until the mixture is uniformly mixed, adding methacrylic anhydride, adjusting the pH value of a system to 10 by using a sodium hydroxide solution with the concentration of 1mol/L, continuously stirring for reacting for 4.5 hours, adding compound protease consisting of alkaline protease, trypsin and papain, heating to 50 ℃, keeping the temperature, and stirring for 0.7 hour to obtain a compound protease solution, wherein the proportion of the guar gum, the pure water, the methacrylic anhydride and the compound protease is 1g:200mL:4mL:50 mg;

(3) uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) in a mass ratio of 50:1 to obtain a mixed solution, placing the mixed solution in a homogenizer with the rotation speed of 8000 rpm and the pressure of 60MPa for homogenizing for 2 minutes, taking out, adjusting the pH value to 7, heating to 50 ℃, and carrying out enzymolysis for 3 hours to obtain an enzymolysis solution;

(4) heating the enzymolysis liquid obtained in the step (3) to 110 ℃, inactivating enzyme for 10 minutes, cooling to room temperature, centrifuging at 8000 rpm to obtain supernatant, and freeze-drying the supernatant to obtain the hypoallergenic casein peptide.

Reference example 1

Unlike example 1, step (1) is: adding casein into pure water, wherein the ratio of the casein to the pure water is 3g:100mL, heating to 80 ℃, keeping the temperature, stirring for 12 minutes, and cooling to room temperature to obtain a casein solution. I.e., lack of high pressure processing operations.

Reference example 2

Unlike example 1, step (3) is: uniformly mixing the casein solution obtained in the step (1) with composite protease (the using amount of the composite protease can be obtained by conversion according to the mass ratio in the embodiment 1) consisting of alkaline protease, trypsin and papain to obtain a mixed solution, placing the mixed solution in a homogenizer with the rotation speed of 8000 rpm and the pressure of 60MPa for homogenizing for 2 minutes, taking out the mixed solution, adjusting the pH value to 7, heating to 55 ℃, and carrying out enzymolysis for 2.5 hours to obtain an enzymolysis solution. I.e. step (2) is absent.

Reference example 3

Unlike example 1, step (3) is: uniformly mixing the casein solution obtained in the step (1) and the compound protease solution obtained in the step (2) in a mass ratio of 45:1 to obtain a mixed solution, adjusting the pH value of the mixed solution to 7, heating to 55 ℃, and carrying out enzymolysis for 2.5 hours to obtain an enzymolysis solution. I.e. lack of homogenization processing operations.

Comparative example

The comparative example is a specific embodiment of the chinese invention with application No. CN 01115249.4.

The first experimental example: sensitization test

The main allergen in casein is α s 1-casein, so the allergenicity can be judged by measuring α s 1-casein content in the product by enzyme linked immunosorbent assay, and then calculating α s 1-casein residue ratio, α s 1-casein residue ratio is α s 1-casein content in the product/α s 1-casein content in the original casein × 100%, the lower the α s 1-casein residue ratio indicates the lower the allergenicity of the product, the test results are shown in table 1:

TABLE 1

As can be seen from Table 1, the products obtained in examples 1 to 4 of the present invention all had significantly lower residual α s 1-casein ratios than the comparative examples, indicating that the products obtained in the present invention had low allergenicity, with the lowest allergenicity of example 1. The partial steps of reference examples 1 to 3 are different from those of example 1 in that the residual α s 1-casein ratio was increased in both reference examples 2 and 3, indicating that both step (2) and the homogenization procedure of the present invention can reduce the sensitization of the product.

Experiment example two: bitterness testing

The test method comprises the following steps: adding 0.1g casein peptide into 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100mL of distillation respectively, stirring and mixing uniformly to prepare standard solution, and defining the corresponding bitter value as 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 respectively. Preparing the product into a sample solution with the concentration of 0.01g/mL, selecting 20 students in food major to respectively compare the sample solution with the standard solution, grading the sample according to the bitterness value, and calculating the average bitterness value. Lower average bitterness values indicate less bitterness, and the results are shown in table 2:

	average bitterness
		Example 1	1.45
Example 2	1.7
		Example 3	1.65
Example 4	1.85
		Reference example 1	1.45
Reference example 2	3.1
		Reference example 3	1.5
Comparative example	5.8

TABLE 2

As can be seen from table 2, the average bitterness of the products obtained in examples 1 to 4 of the present invention was significantly lower than that of the comparative example, indicating that the products obtained in the present invention had less bitterness, with the bitterness of example 1 being minimal. The partial steps of reference examples 1 to 3 are different from those of example 1 in that the average bitterness of reference example 2 is significantly increased, indicating that step (2) of the present invention is effective in reducing the bitterness of the product.

Experiment example three: oxidation resistance test

The test method comprises the following steps: preparing a product into a sample solution with the concentration of 0.01g/mL, adding 500 mu L of the sample solution into 3.8mL of ABTS diluted working solution with the absorbance of 0.7 at 734nm, reacting for 8 minutes at room temperature, measuring the absorbance of the sample solution at 734nm, and calculating the ABTS free radical clearance rate, wherein the ABTS free radical clearance rate is (ABTS free radical clearance absorbance-sample solution absorbance)/(ABTS diluted working solution absorbance-distilled water absorbance). Higher ABTS radical scavenging indicates better oxidation resistance, and the test results are shown in table 3:

	ABTS radical scavenging ratio (%)
		Example 1	84.93
Example 2	84.62
		Example 3	84.76
Example 4	84.85
		Reference example 1	84.92
Reference example 2	84.93
		Reference example 3	80.54
Comparative example	78.89

TABLE 3

As can be seen from Table 3, the products obtained in examples 1-4 of the present invention all have significantly higher ABTS free radical scavenging rates than the comparative examples, indicating that the products obtained in the present invention have better oxidation resistance, with example 1 having the best oxidation resistance. The partial steps of reference examples 1-3 are different from example 1 in that the ABTS radical scavenging rate of reference example 3 is significantly decreased, indicating that the homogenization operation of the present invention is effective in improving the oxidation resistance of the product.

Experimental example four: stability test

The test method comprises the following steps: respectively weighing 1g of sample, filling the sample into a transparent plastic packaging bag, measuring ABTS free radical clearance rate according to the method of the third experimental example after standing for 30 days at normal temperature, and then calculating the ABTS free radical clearance rate retention rate, wherein the ABTS free radical clearance rate retention rate is ABTS free radical clearance rate/original ABTS free radical clearance rate multiplied by 100 percent after standing for 30 days. Higher ABTS radical scavenging retention indicates better stability, and the test results are shown in table 4:

TABLE 4

As can be seen from Table 4, the ABTS free radical scavenging rate retention rates of the products obtained in examples 1-4 of the present invention are significantly higher than those of the comparative examples, which indicates that the products obtained in the present invention have better stability, wherein the product obtained in example 1 has the best stability. The reference examples 1-3 are different from example 1 in part of the steps, wherein the ABTS free radical clearance retention rate of reference example 1 is obviously reduced, which shows that the high-pressure operation of the invention can effectively improve the stability of the product.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.