A hydrolyzed whey protein modified complex, its preparation method and application

Abstract

The present application relates to a kind of hydrolyzed whey protein modified complex and its preparation method and application, belong to the field of special medical food research, the preparation method includes: whey protein is dissolved in water to obtain whey protein solution;2'-fucosyl lactose, maltose and fructose are added to the obtained whey protein solution, mixed uniformly, to obtain mixed solution, the protease includes alkaline protease and papain;The pH of mixed solution is adjusted, first in 45~55 ℃ water bath condition is reacted 2~3h, then in 60~65 ℃ water bath condition is reacted 3~4h, freeze-drying is obtained freeze-dried powder;The obtained freeze-dried powder is reacted under the condition that 55~65 ℃, relative humidity is 79-82%, to obtain hydrolyzed whey protein modified complex.The preparation process of the present application is simple and efficient, and the obtained hydrolyzed whey protein modified complex can effectively alleviate the IgE increase caused by allergy.

Description

Technical Field

[0001] This invention belongs to the field of special medical food research technology, and relates to a hydrolyzed whey protein modified complex, its preparation method and application. Background Technology

[0002] Whey protein is a protein extracted from milk. It is highly nutritious, easily digestible and absorbed, and contains various bioactive components, making it a recognized source of high-quality protein for the human body. Whey protein mainly includes α-lactalbumin, β-lactoglobulin, serum albumin, and immunoglobulins. Hydrolyzed whey protein is a hydrolysis product of whey protein. Studies have shown that specific proteases can hydrolyze whey protein, releasing bioactive peptides distributed in its primary structure, thus enabling it to exert specific functional effects, such as preventing allergies, improving digestibility and absorption, and antibacterial properties. Compared to regular whey protein, hydrolyzed whey protein not only has better solubility, higher digestibility and absorption, and is more acid- and heat-resistant, but also possesses various physiological regulatory functions, making it suitable for the development of functional products and specialized medical products.

[0003] Glycosylation is a Maillard reaction that covalently links carbohydrates to the α- and ε-phase atoms of a protein molecule to form glycosylated proteins. Glycosylation can improve some functional properties of whey proteins, such as solubility, emulsifying properties, and thermal stability. However, the properties of glycosylated products vary depending on the type of carbohydrate, and the changes in the properties of whey protein glycosylated products from different types of sugars have not been systematically studied.

[0004] With the continuous development of society and the industrial application of whey protein, higher requirements are being placed on whey protein, and strengthening and improving the functional properties of whey protein is conducive to expanding its application scope. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a modified hydrolyzed whey protein complex. Its preparation involves innovative improvements to existing modification methods, resulting in a simple and efficient preparation process. The modified hydrolyzed whey protein complex can effectively alleviate IgE elevation caused by allergies and improve allergy symptoms induced by whey protein.

[0006] Another object of the present invention is to provide the application of the above-mentioned hydrolyzed whey protein modified complex in the preparation of special medical foods with protein formulations.

[0007] Another object of the present invention is to provide a protein component formulation food for special medical purposes.

[0008] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention claims protection for a hydrolyzed whey protein modified complex, prepared by a method comprising the following steps:

[0010] Step 1: Dissolve whey protein in water to obtain a whey protein solution; wherein the whey protein solution contains 6-9% whey protein by mass.

[0011] Step 2: Add protease and carbohydrates to the whey protein solution obtained in Step 1, mix thoroughly to obtain a mixture; the mass ratio of protease, carbohydrates, and whey protein is 4-7:10-15:25-30.

[0012] Step 3: Adjust the pH of the mixture obtained in Step 2 to 9-11, react it in a water bath at 45-55℃ for 2-3 hours, then react it in a water bath at 60-65℃ for 3-4 hours, and freeze dry to obtain freeze-dried powder.

[0013] Step 4: React the lyophilized powder obtained in Step 3 at 55-65℃ and 79-82% relative humidity for 8-10 hours to obtain the hydrolyzed whey protein modified complex.

[0014] In step one above, the whey protein solution has a mass percentage of 8%.

[0015] In step two above, the mass ratio of the protease, carbohydrate compound, and whey protein is 5:12:28.

[0016] In step two above, the protease includes alkaline protease and papain, and the mass ratio of alkaline protease to papain is 1-3:6-7, preferably 2:7.

[0017] In step two above, the carbohydrate compounds are 2'-fucosylated lactose, maltose, and fructose, and the mass ratio of the three is 1-2:3-5:2-3, preferably 1:3:2.

[0018] In step three above, the pH of the mixture is adjusted to 9, and the mixture is reacted for 2 hours in a 50°C water bath, then reacted for 3 hours in a 65°C water bath, and then freeze-dried to obtain freeze-dried powder.

[0019] In step four above, the freeze-dried powder is reacted at 60°C and 79% relative humidity for 10 hours.

[0020] Secondly, the present invention seeks protection for the use of the above-mentioned hydrolyzed whey protein modified complex in the preparation of special medical foods with protein formulations.

[0021] Thirdly, this invention claims protection for a protein component formulation food for special medical purposes, comprising the above-mentioned hydrolyzed whey protein modified complex. Further, it also includes soy peptide powder, edible flavoring, steviol glycosides, and sucralose; wherein the amount of the hydrolyzed whey protein modified complex added is 590 g / kg, and the amount of the soy peptide powder added is 405 g / kg.

[0022] Beneficial Effects: This invention combines enzymatic hydrolysis and glycosylation to modify whey protein in a one-step process. The resulting hydrolyzed whey protein-modified complex effectively alleviates IgE elevation caused by allergies and improves whey protein-induced allergy symptoms. In this invention, protease and carbohydrate compounds are added together during the modification process, and a two-stage water bath treatment is performed at different temperatures. During this process, the protease can stably exert its enzymatic hydrolytic effect. The better alleviating effect of this invention compared to other modification methods may be due to the simultaneous presence of carbohydrate compounds, which can, to a certain extent, prevent changes in the spatial structure of enzyme molecules, thus ensuring enzyme activity stability. Furthermore, compared to conventional dry-heat glycosylation, this invention requires no pretreatment, has a shorter reaction time, and a more controllable reaction process, greatly improving modification efficiency.

[0023] Enzymatic hydrolysis with proteases can alleviate allergic reactions induced by whey protein. Studies have also shown that 2'-fucosylated lactose can improve allergic reactions. This application utilizes 2'-fucosylated lactose in the glycosylation modification process, further enhancing its improving effect. The hydrolyzed whey protein modified complex prepared using the method of this invention can be used to prepare special medical foods with protein formulations. These can serve as a high-quality protein source. Furthermore, the modified whey protein exhibits enhanced and optimized protein function, expanding its application areas. This has significant practical implications for the research and development of related special medical purpose foods, especially low-allergenic infant formula. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0025] Raw materials: alkaline protease (enzyme activity 1.15 million U / g), papain (enzyme activity 11,000 U / g).

[0026] In the following embodiments, unless otherwise specified, the raw materials or reagents used are all commercially available products, and the technical means used are all conventional operating methods.

[0027] Example 1

[0028] A modified hydrolyzed whey protein complex is prepared as follows:

[0029] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 8%;

[0030] Step 2: Add protease and carbohydrates to the whey protein solution obtained in Step 1, mix well to obtain a mixture; the mass ratio of protease, carbohydrates and whey protein is 5:12:28.

[0031] The protease includes alkaline protease and papain, with a mass ratio of 2:7; the carbohydrate compounds are 2'-fucosylated lactose, maltose, and fructose, with a mass ratio of 1:3:2.

[0032] Step 3: Adjust the pH of the mixture to 9, react it in a 50°C water bath for 2 hours, then react it in a 65°C water bath for 3 hours, and freeze-dry it to obtain freeze-dried powder.

[0033] Step 4: The lyophilized powder obtained in Step 3 is reacted at 60°C and maintained at a relative humidity of 79% with saturated KBr for 10 hours to obtain the hydrolyzed whey protein modified complex.

[0034] Example 2

[0035] A modified hydrolyzed whey protein complex is prepared as follows:

[0036] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 6%;

[0037] Step 2: Add protease and carbohydrates to the whey protein solution obtained in Step 1, mix well to obtain a mixture; the mass ratio of protease, carbohydrates and whey protein is 4:15:25.

[0038] The protease includes alkaline protease and papain, with a mass ratio of 2:7; the carbohydrate compounds are 2'-fucosylated lactose, maltose, and fructose, with a mass ratio of 1:3:2.

[0039] Step 3: Adjust the pH of the mixture obtained in Step 2 to 11, react it in a 45°C water bath for 2 hours, then react it in a 60°C water bath for 3 hours, and freeze-dry it to obtain freeze-dried powder.

[0040] Step 4: The lyophilized powder obtained in Step 3 is reacted at 65°C and maintained at a relative humidity of 79% with saturated KBr for 10 hours to obtain the hydrolyzed whey protein modified complex.

[0041] Example 3

[0042] A modified hydrolyzed whey protein complex is prepared as follows:

[0043] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 9%;

[0044] Step 2: Add protease and carbohydrates to the whey protein solution obtained in Step 1, mix well to obtain a mixture; the mass ratio of protease, carbohydrates and whey protein is 7:10:30.

[0045] The protease includes alkaline protease and papain, with a mass ratio of 2:7; the carbohydrate compounds are 2'-fucosylated lactose, maltose, and fructose, with a mass ratio of 1:3:2.

[0046] Step 3: Adjust the pH of the mixture obtained in Step 2 to 9, react it in a 45°C water bath for 2 hours, then react it in a 60°C water bath for 3 hours, and freeze-dry it to obtain freeze-dried powder.

[0047] Step 4: The lyophilized powder obtained in Step 3 is reacted at 55°C and maintained at a relative humidity of 79% with saturated KBr for 10 hours to obtain the hydrolyzed whey protein modified complex.

[0048] Comparative Example 1: enzymatic hydrolysis followed by glycosylation.

[0049] A modified hydrolyzed whey protein complex is prepared as follows:

[0050] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 8%;

[0051] Step 2: Add protease to the whey protein solution obtained in Step 1, adjust the pH of the solution to 9, react in a 50°C water bath for 3 hours, inactivate the enzyme with boiling water, and then cool to room temperature to obtain the enzymatic hydrolysate.

[0052] Step 3: Add carbohydrate compounds to the enzymatic hydrolysate, mix well, let stand for 3 hours, freeze dry to obtain lyophilized powder, and react at 60°C and 79% relative humidity with saturated KBr for 25 hours to obtain hydrolyzed whey protein modified complex.

[0053] In the above steps, the mass ratio of the protease, carbohydrates, and whey protein is 5:12:28. The protease includes alkaline protease and papain, with a mass ratio of 2:7; the carbohydrates are 2'-fucosylated lactose, maltose, and fructose, with a mass ratio of 1:3:2.

[0054] Comparative Example 2: Glycosylation followed by enzymatic hydrolysis

[0055] A modified hydrolyzed whey protein complex is prepared as follows:

[0056] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 8%;

[0057] Step 2: Add carbohydrate compounds to the whey protein solution obtained in Step 1, mix well, let stand for 3 hours, freeze dry to obtain lyophilized powder, and react the lyophilized powder at 60°C and 79% relative humidity with saturated KBr for 25 hours to obtain modified whey protein.

[0058] Step 3: Dissolve the modified whey protein in deionized water, then add protease, mix well, adjust the pH of the solution to 9, react in a 50°C water bath for 3 hours, inactivate the enzyme with boiling water, and cool to room temperature to obtain the enzymatic hydrolysate; freeze-dry to obtain the hydrolyzed whey protein modified complex.

[0059] In the above steps, the mass ratio of the protease, carbohydrates, and whey protein is 5:12:28. The protease includes alkaline protease and papain, with a mass ratio of 2:7; the carbohydrates are 2'-fucosylated lactose, maltose, and fructose, with a mass ratio of 1:3:2.

[0060] Comparative Example 3 (Glycosylation Only)

[0061] A modified hydrolyzed whey protein complex is prepared as follows:

[0062] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 8%;

[0063] Step 2: Add a carbohydrate compound to the whey protein solution obtained in Step 1, mix well, let stand for 3 hours, freeze dry to obtain a lyophilized powder, and react the lyophilized powder at 60°C and 79% relative humidity with saturated KBr for 25 hours to obtain modified whey protein.

[0064] Comparative Example 4 (enzymatic hydrolysis, mixed with sugars, without glycosylation)

[0065] A modified hydrolyzed whey protein complex is prepared as follows:

[0066] Step 1: Dissolve whey protein in water to prepare a whey protein solution with a mass concentration of 8%;

[0067] Step 2: Add protease to the whey protein solution obtained in Step 1, adjust the pH of the solution to 9, react in a 50°C water bath for 3 hours, inactivate the enzyme with boiling water, and then cool to room temperature to obtain the enzymatic hydrolysate.

[0068] Step 3: Add carbohydrate compounds to the enzymatic hydrolysate, mix well, let stand for 3 hours, and freeze dry to obtain the hydrolyzed whey protein modified complex.

[0069] In the above steps, the mass ratio of the protease, carbohydrates, and whey protein is 5:12:28. The protease includes alkaline protease and papain, with a mass ratio of 2:7; the carbohydrates are 2'-fucosylated lactose, maltose, and fructose, with a mass ratio of 1:3:2.

[0070] Comparative Example 5

[0071] It is basically the same as Example 1, except that the carbohydrate compound does not contain 2'-fucosylated lactose.

[0072] Comparative Example 6

[0073] It is basically the same as Example 1, except that the mass ratio of 2'-fucosylated lactose, maltose and fructose in the carbohydrate compound is 5:3:2.

[0074] Comparative Example 7

[0075] The process is basically the same as in Example 1, except that the mass ratio of 2'-fucosylated lactose, maltose and fructose in the carbohydrate compound is 4:3:2.

[0076] Comparative Example 8

[0077] The method is basically the same as in Example 1, except that the mass ratio of 2'-fucosylated lactose, maltose and fructose in the carbohydrate compound is 3:3:2.

[0078] The modified proteins prepared in Examples 1-3 and Comparative Examples 1-8 were subjected to performance tests.

[0079] I. Allergy Efficacy Test

[0080] Sixty healthy 1-week-old BALB / c mice were randomly divided into 13 groups of 5 mice each. Each group received the modified protein prepared according to Examples 1-3 and Comparative Examples 1-8, administered once daily at a dose of 0.5 g / kg dissolved in water via gavage. A blank control group and a control group were also included; the blank control group received an equal volume of water via gavage, while the control group received whey protein via gavage. After one week of continuous gavage, the mice were sacrificed, and serum was collected to detect the IgE content. The results are shown in Table 1.

[0081] Table 1. IgE content in the serum of mice in each group.

[0082] Group IgE content (ng / mL) Blank group 65.23±1.81 control group 106.24±2.35 Example 1 71.68±4.26* Example 2 77.65±1.89* Example 3 75.98±2.63* Comparative Example 1 82.64±1.74 Comparative Example 2 79.67±3.10 Comparative Example 3 96.23±1.63 Comparative Example 4 84.26±1.98 Comparative Example 5 90.17±2.41 Comparative Example 6 85.23±2.56 Comparative Example 7 83.45±1.32 Comparative Example 8 80.23±2.66

[0083] As shown in Table 1 above, the control group administered whey protein by gavage had the highest serum IgE content, while the mice administered the hydrolyzed whey protein modified complex prepared in Examples 1-3 by gavage showed a significant decrease in serum IgE content, indicating that the method of this invention can alleviate the problem of elevated IgE levels caused by allergies after modifying whey protein. Although Comparative Examples 1 and 2 also involved hydrolysis and glycosylation of whey protein, the modification methods differed from those of this invention, and the results showed a slight increase in serum IgE content compared to Examples 1-3. Comparative Example 3 only underwent glycosylation treatment, and the IgE content was lower than that of mice administered whey protein by gavage, indicating that glycosylation has a certain preventive effect against allergies. Comparative Example 4 involved mixing whey protein after enzymatic hydrolysis with carbohydrate compounds, resulting in a lower IgE content compared to mice administered whey protein by gavage, but an increased IgE content compared to mice administered the hydrolyzed whey protein modified complex by gavage. Compared to Example 1, Comparative Example 5, which removed 2'-fucosylation lactose from the carbohydrate compound, showed a significantly higher IgE content, indicating that 2'-fucosylation lactose plays an important role in alleviating allergies. Comparative Examples 5-7, compared to Example 1, all increased the content of 2'-fucosylation lactose. The results showed that the IgE content gradually increased after the 2'-fucosylation lactose content increased, indicating that the anti-allergic effect of 2'-fucosylation lactose in the complex is only effective within a certain range.

[0084] Example 4: Protein Component Formula Food for Special Medical Purposes (Dongzeding)

[0085] The protein (amino acid) component has the following ingredients: hydrolyzed whey protein powder (hydrolyzed whey protein modified complex prepared in Example 1), soybean peptide powder, edible flavor, steviol glycosides and sucralose.

[0086] This product uses hydrolyzed whey protein powder (added at 590g / kg) and soybean peptide powder (405g / kg) as protein sources. Each 100g of this product can provide 330kcal of energy and has a protein content of approximately 80%.

[0087] The nutritional information for this product is shown in Table 2.

[0088] Table 2 Nutritional Information

[0089] Nutritional components per 100g per 100kJ Energy (kJ) 1380 100 Protein (g) 80 5.8 Fat (g) 0 0 Carbohydrates (g) 1.2 0.1 Sodium (mg) 1110 80.4

[0090] Process flow: Packaging material preparation → Packaging material feeding → Raw and auxiliary materials → Raw and auxiliary material preparation → Raw and auxiliary material feeding → Ingredient premixing → Material receiving and feeding → Mixing → Inner packaging → Outer packaging → Boxing → Warehousing.

[0091] The resulting product is in powder form and is suitable for people aged 10 and above who need to supplement protein due to specific diseases or medical conditions.

[0092] For oral administration. The method of consumption and dosage should be determined under the guidance of a doctor or clinical nutritionist, based on the age, weight, and medical condition of the target population. Dissolve each sachet (15g) in 100mL of warm water, stirring to obtain the standard reconstituted solution.

[0093] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A method for preparing a hydrolyzed whey protein modified complex, characterized by: The enzymolysis and glycosylation are combined, the whey protein is modified by a one-step method, the protease and the saccharide compound are added in the modification process, the stability of the enzyme activity is ensured by the saccharide compound, and the modification process comprises the following steps: In step one, the whey protein is dissolved in water to obtain a whey protein solution; wherein the mass percentage of the whey protein in the whey protein solution is 6-9%. In step two, the protease and the saccharide compound are added into the whey protein solution obtained in step one, and are uniformly mixed to obtain a mixed solution; the mass ratio of the protease, the saccharide compound and the whey protein is 4-7:10-15:25-30. The protease comprises alkaline protease and papain, and the mass ratio of the alkaline protease to the papain is 1-3:6-7; the saccharide compound is 2'-fucosyllactose, maltose and fructose, and the mass ratio of the three is 1-2:3-5:2-3. In step three, the pH of the mixed solution obtained in step two is adjusted to 9-11, the mixed solution is first reacted under the condition of a water bath at 45-55 DEG C for 2-3 h, and then is reacted under the condition of a water bath at 60-65 DEG C for 3-4 h, and is freeze-dried to obtain a freeze-dried powder. In step four, the freeze-dried powder obtained in step three is reacted under the condition of 55-65 DEG C and a relative humidity of 79-82% for 8-10 h to obtain a hydrolyzed whey protein modified compound.

2. The method for preparing a hydrolyzed whey protein modified complex according to claim 1, characterized in that: In step one, the mass percentage of the whey protein solution is 8%.

3. The method of claim 1, wherein the hydrolyzed whey protein modified complex is prepared by: In step two, the mass ratio of the protease, the saccharide compound and the whey protein is 5:12:

28.

4. The method of claim 1, wherein the hydrolyzed whey protein modified complex is prepared by: In the protease, the mass ratio of the alkaline protease to the papain is 2:

7.

5. The method of claim 1, wherein the hydrolyzed whey protein modified complex is prepared by: In the saccharide compound, the mass ratio of 2'-fucosyllactose, maltose and fructose is 1:3:

2.

6. The method of claim 1, wherein the hydrolyzed whey protein modified complex is prepared by: In step three, the pH of the mixed solution is adjusted to 9, the mixed solution is first reacted under the condition of a water bath at 50 DEG C for 2 h, and then is reacted under the condition of a water bath at 65 DEG C for 3 h, and is freeze-dried to obtain a freeze-dried powder.

7. The method of claim 1, wherein the hydrolyzed whey protein modified complex is prepared by: In step four, the freeze-dried powder is reacted under the condition of 60 DEG C and a relative humidity of 79% for 10 h.