Whey protein composition, its preparation method and application

Abstract

The present invention relates to the field of protein processing technology and discloses a whey protein composition, its preparation method, and applications. The whey protein composition of the present invention comprises whey protein and β-casein, with the mass ratio of β-casein to whey protein being 4.5:95.5 or greater. The whey protein composition provided by the present invention is primarily composed of a novel whey protein component that has slower digestion properties than other commercially available whey proteins. The whey protein composition is a whey protein concentrate that is rich in β-casein and can be further micronized and complexed with natural polysaccharides, which can slow digestion, promote muscle synthesis, and provide sufficient nutrition.

Description

[Technical field]

[0001] This application claims priority from a Chinese patent application bearing application number 202011068591.8 and entitled "Whey protein composition, its preparation method and application" filed with the China Patent Office on September 29, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of protein processing technology, specifically to a whey protein composition, its preparation method and application. [Background technology]

[0003] Whey protein, known as the king of proteins, is a protein extracted from milk. It is highly nutritious, easily digested and absorbed, and contains a variety of active ingredients, and is recognized as one of the high-quality protein supplements for the human body.

[0004] Whey protein is the most nutritious of all kinds of proteins. Whey protein belongs to high-quality complete proteins and is also an animal protein. It contains eight kinds of amino acids required by the human body, and the composition ratio is compatible, close to the ratio required by the human body, and is an essential essence for human growth, development, anti-aging and other vital activities. At the same time, whey protein is easy to digest and absorb, and many experimental studies have proven that ingesting whey protein concentrate can promote humoral immunity and cellular immunity, stimulate the human immune system, and prevent the occurrence of chemically induced cancer. In addition, whey protein has a low content of fat and lactose, but contains β-lactoglobulin, α-lactalbumin, immunoglobulin, and many other active ingredients. Due to these active ingredients, whey protein can have many health functions beneficial to the human body, so it is considered to be one of the high-quality protein sources required by the human body.

[0005] From a nutritional point of view, foods derived from animal protein contain substances harmful to the human body, such as excess saturated fat and cholesterol, and excessive intake may increase body fat and cholesterol, leading to the development of cardiovascular disease. These problems can be avoided by taking protein powder to supplement protein. In addition, taking protein powder is convenient, has a high absorption rate, and can reduce the burden on the stomach, so taking protein powder is the best choice for protein supplementation. And whey protein is the most important choice for protein powder.

[0006] Whey protein is generally considered to be able to prevent muscle fatigue and increase muscle efficiently because it is fast in digestion and absorption rate after oral intake and contains a lot of branched chain amino acids such as valine, leucine, isoleucine, etc. However, if this release characteristic can be delayed, it can further promote active synthesis of muscle, and therefore it can be widely applied to many fields such as prevention of muscle atrophy in the elderly, muscle repair after exercise, slimming needs, and growth and development of children.

[0007] Slow-release whey currently relies on the development of "micellar whey" and an undisclosed polymerization process. WheyXR (WXR), a commercially available ingredient from Glanbia, is said to be a polymerized whey with slow-release properties. It is believed that the large whey aggregates formed may restrict pepsin access to the proteins, slowing digestion in the stomach. Whey proteins tend to pass through the stomach and into the small intestine rapidly, so more technological means are needed to slow digestion. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in view of the above circumstances, and aims to provide a whey protein composition having slower digestive properties when passing through the gastrointestinal tract. Another object of the present invention is to provide a whey protein composition capable of slowly releasing leucine. Another object of the present invention is to provide a whey protein composition which can significantly improve viscosity in the stomach and delay the time it takes to enter the intestinal tract. Another object of the present invention is to provide a whey protein composition capable of reducing the digestion rate of β-lactoglobulin. Another object of the present invention is to provide the application of said whey protein composition in the preparation of dietary supplements, muscle synthesis promoters and dairy products, and the preparation method thereof. [Means for solving the problem]

[0009] In order to achieve the above object, the present invention provides the following technical aspects. In one aspect, there is provided a whey protein composition comprising whey protein and β-casein, wherein the mass ratio of β-casein to whey protein is 4.5:95.5 or more. In a specific embodiment of the present invention, the whey protein and β-casein are mainly derived from cow's milk, but those selected from raw milk of other origins can also be used. The whey protein composition according to the present invention may be a mixture that meets a standard prepared by directly processing raw milk, or a mixture in which β-casein and whey protein are blended in a predetermined ratio and additives are added.

[0010] Preferably, the mass ratio of β-casein to whey protein is (4.5:95.5) to (50:50). More preferably, the mass ratio of β-casein to whey protein is (9:91) to (20:80). More preferably, the mass ratio of β-casein to whey protein is (9:90) to (17:83). In a specific embodiment of the present invention, the mass ratio of β-casein to whey protein may be selected from 4.6:95.4, 5:95, 9.4:90.6, 10:90, 15:85, 16.6:83.4, and 17:83.

[0011] Other than the direct use of β-casein and whey protein in the formulation, in the present invention, β-casein is dissolved from casein micelles by a specific process (including keeping at low temperature for a certain period of time) and then incorporated into whey, and its unique molecular chaperone effect is utilized to form a specific bond with α-lactalbumin and β-lactoglobulin in the whey protein, thereby protecting the whey protein from rapid digestion.Compared with commercial products such as standard whey protein concentrate, the composition of the present invention has the property of slowing down the release rate of leucine, whether it is prepared by processing raw milk or is formulated in a proportion and supplemented with additives.

[0012] At the same time, analysis of the residual amount of β-lactoglobulin in compositions containing different ratios of β-casein and whey protein showed that compositions with a ratio of 4:96 or higher showed a slower digestion rate of β-lactoglobulin than standard whey protein concentrate.

[0013] In the present invention, it has been found that the degree of combination of β-casein and whey protein can be further enhanced by the micronization process on the whey protein, thereby further achieving the effect of slowly releasing leucine, and that the viscosity of gastric juice during digestion in an in vitro digestion model can be increased, which can prevent the release of protein into the intestine and delay the digestion process. Compared with the micronization process at pH 6.5, the micronization process at pH 7.5 has a more significant effect in these respects, and the composition of the present invention can achieve the above effects by micronization whether it is prepared by processing raw milk or by adding additives at a certain ratio. In addition, the micronization process can improve other physical properties of the mixture, including particle size distribution, solubility, stability, viscosity, etc. In the present invention, it has been surprisingly found that the micronization of standard whey concentrated protein alone without adding β-casein may accelerate the digestion rate of whey protein, which indicates that the addition of β-casein can reduce such a phenomenon of too fast digestion rate and further delay the digestion process. According to the above experimental conclusions, the whey protein composition according to the present invention is preferably a micronized whey protein composition.

[0014] Preferably, the atomization is performed by high-temperature, high-speed shearing at a pH value of 6.5 to 9.0. More preferably, the atomization is performed at a high temperature of 80 to 100°C, a high shear rate of 6000 to 20000 rpm, and a processing time of 3 to 10 minutes, and even more preferably at a high temperature of 91 to 99°C, a high shear rate of 8000 to 15000 rpm, and a processing time of 4 to 8 minutes. Among these, the parameters of the granulation process are preferably pH 7.5, temperature 95°C, time 5 minutes, and shear rate 9600 rpm. After the completion of the atomization, the mixture is rapidly cooled to 15°C.

[0015] In a further optimization aspect, the present invention further adds polysaccharides that have more binding sites due to the natural viscosity advantage and the presence of polyhydroxyl groups, which further binds to whey proteins rich in β-casein, and can enhance the viscosity characteristics of the mixture or delay the release rate of leucine. Alginate and κ-carrageenan have higher viscosities in gastric juice in an in vitro digestion model than other measured polysaccharides. Among them, κ-carrageenan is 30 to 50 times higher than other polysaccharides. In addition, the effect of adding polysaccharides under micronization at pH 7.5 is better than the effect of adding polysaccharides under micronization at pH 6.5.

[0016] The analytical experiment on leucine release showed that potassium alginate could reduce the release rate of leucine from micronized β-casein-rich whey protein during in vitro digestion, and the addition of κ-carrageenan could not significantly reduce the release rate of leucine from micronized β-casein-rich whey protein at the intestinal stage, but since κ-carrageenan could lead to a significant increase in the viscosity of gastric juice, it is expected that the rate at which whey protein enters the intestinal tract would be slowed down after supplementation of κ-carrageenan, which could help delay the release of free leucine into the small intestine in vivo.

[0017] In the digestion experiment, compared with the standard whey concentrated protein, commercial products and their polysaccharide-added control products, the hydrolysis degree of the whey protein composition of the present invention, which is micronized and added with the same polysaccharides, is lower than that of each control product, which indicates that the whey protein composition of the present invention has a better sustained-release effect; similarly, the effect of the experimental group at pH 7.5 micronization is better than that of the experimental group at pH 6.5 micronization.

[0018] Preferably, the polysaccharides described in the present invention are one or more selected from carrageenan, alginate, chitosan, carboxymethylcellulose, and tragacanth gum, and the amount of polysaccharides added is 0.1-0.3%. According to the above experimental results, in a specific embodiment, κ-carrageenan and / or potassium alginate can be added to achieve excellent effects in different digestive tracts.

[0019] The present invention also provides a method for preparing a whey protein composition by processing raw milk, comprising the steps of: treating skimmed raw milk at a low temperature of 0 to 10°C (allowing it to stand for 10 to 120 hours, preferably 10 to 24 hours); and then filtering the resulting permeate through a membrane of 20 to 200 nm or more, and concentrating the permeate by ultrafiltration to remove lactose, thereby obtaining a whey protein composition containing whey protein and β-casein.

[0020] β-casein is released from casein micelles at low temperatures and concentrated in the supernatant of skim milk. The reason is that as the temperature decreases, the strength of the hydrophobic effect decreases, and therefore β-casein is free to dissolve from the micelles into the supernatant. β-casein can form its own micelles, but the diameter of the micelles does not exceed 20-30 nm. Therefore, a ceramic or polymeric membrane with a diameter of 20-200 nm (preferably 50 nm) is sufficient to block casein micelles, while allowing whey proteins and β-casein to pass through and enter the permeate. The ratio of β-casein to whey proteins contained in the supernatant protein remaining after concentrating the permeate by ultrafiltration (10 KDa) to remove lactose is (4.5:95.5) to (20:80).

[0021] As a result of SDS-PAGE gel electrophoresis, the ratio of β-casein to whey protein obtained by treating milk at 10°C, 4°C and 2°C was estimated using a chemiluminescence imager to be 4.6:95.4 at 10°C, 9.4:90.6 at 4°C, and 16.6:83.4 at 2°C. Therefore, according to the preparation method of the present invention, whey protein containing 5 to 20% β-casein can be easily obtained. In order to realize a whey protein composition with a higher ratio of β-casein, it can be carried out by blending.

[0022] According to the above explanation of the beneficial effects of the atomization process, the preparation method according to the present invention can further increase the operation process of atomization, which is high-temperature, high-speed shearing at a pH value of 6.5 to 9.0, followed by rapid cooling to prevent protein denaturation at high temperature for a long time. Preferably, the high temperature is 80 to 100°C, the high-speed shearing speed is 6000 to 20000 rpm, and the treatment time is 3 to 10 min. More preferably, the high temperature is 91 to 99°C, the high-speed shearing speed is 8000 to 15000 rpm, and the treatment time is 4 to 8 min. Among them, the parameters of the atomization process are preferably pH 7.5, temperature 95°C, time 5 min, and shear rate 9600 rpm. After the completion of atomization, the mixture is rapidly cooled to 15°C.

[0023] Preferably, in the micronization process, a concentrated solution is obtained by a concentration process (protein concentration is 4% or more), which is then subjected to a micronization process and further concentrated to a total solid content of 20% or more, and if necessary, spray-dried to form a powder, or not sprayed into a dry powder, or made into a composition in other forms.

[0024] In a specific embodiment of the present invention, the micronization process involves concentrating the ultrafiltered filtrate at 55°C until a protein concentration of 5%-10% is reached. The concentrate is adjusted to pH 7.5 by adding HCl or NaOH, and treated at 95°C for 5 min, while simultaneously high-speed shearing at 9600 rpm. After this process, the micronized protein system is rapidly cooled to 15°C, and then further concentrated until a total solid content of 20-25% is reached, and finally spray-dried (maintaining an inlet temperature of 200°C and an outlet temperature of 90-100°C).

[0025] After the microparticulation, one or more polysaccharides selected from carrageenan, alginate, chitosan, carboxymethylcellulose, and tragacanth gum may be added and mixed in an amount of 0.1 to 0.3%.

[0026] According to the above preparation method, the present invention also provides a whey protein composition prepared by said preparation method.Because the whey protein composition provided in the present invention has multiple technical effects of slowing digestion, and has a better effect of slowing digestion than the same kind of products on the market, the present invention further provides the use of the whey protein composition in the preparation of whey protein products having slow-digesting and / or slow-release amino acids, where these products can be dietary supplements, muscle synthesis promoters, or dairy products.

[0027] Therefore, the present invention also provides the use of the whey protein composition in the preparation of a dietary supplement, a muscle synthesis promoter or a dairy product, wherein the whey protein composition may be a mixture prepared by processing raw milk (e.g., the above-mentioned preparation process) that meets the standards, a mixture of β-casein and whey protein in a predetermined ratio with the addition of additives, or a mixture obtained by further micronization and / or the addition of polysaccharides.

[0028] According to the above-mentioned use, the present invention provides a nutritional supplement, a muscle synthesis promoter or a dairy product. The names of these products are specifically determined according to the application field, but they all have a common feature of containing the whey protein composition described in the present invention, and may contain food additives and / or nutrients that can be added to food according to the requirements of the product as other ingredients. Nutrients include, but are not limited to, vitamins and minerals.

[0029] In a specific embodiment of the present invention, the present invention provides a nutritional supplement in the form of a nutritional bar, comprising the whey protein composition of the present invention, further comprising glucose syrup, glycerin, maltodextrin, coconut oil, and lecithin.

[0030] The present invention also provides a whey protein composition, specifically in powder form, comprising the whey protein composition of the present invention, and further comprising lactose, fructose, glucose, cream, vitamins, minerals, and lecithin, where the vitamins include, but are not limited to, vitamins A, C, D, and E. The minerals include, but are not limited to, calcium salts, iron salts, zinc salts, and magnesium salts, such as phosphates or sulfates thereof.

[0031] In the digestion experiment of a specific formulated product, the present invention has the result that different food formulas have a strong effect on the release rate of amino acids. The powdered formula has a slower digestion rate of leucine than the solid bar formula. In the same food formula, the product added with the whey protein composition of the present invention has a leucine release effect close to that of the commercially available control product.

[0032] From the above technical aspects, it can be seen that the whey protein composition provided by the present invention is mainly composed of novel whey protein components and has slower digestion characteristics than other whey proteins on the market. In addition, the whey protein composition of the present invention is a whey protein concentrate that is rich in β-casein and can be further micronized and complexed with natural polysaccharides, which can delay digestion, promote muscle synthesis, and provide sufficient nutrition. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows an exemplary flow chart for the preparation of a whey protein composition according to the present invention. [Diagram 2] FIG. 2 shows the protein electrophoretic patterns of a whey protein composition according to the present invention obtained under different temperature processing conditions. [Diagram 3] FIG. 3 is a graph showing leucine release from a β-casein-rich whey protein composition and a control group in an artificially simulated digestive fluid. [Figure 4] FIG. 4 is a graph showing the amount of leucine released by the whey protein composition according to the present invention and the control group in response to an artificial simulated digestive fluid. [Diagram 5] FIG. 5 shows a viscosity curve when the whey protein composition according to the present invention is prepared into a solution with a protein concentration of 5% and then subjected to a microparticulation treatment. [Figure 6] FIG. 6 shows the viscosity curves of a whey protein composition without micronization treatment and a control product in an artificial simulated digestive fluid. [Figure 7] FIG. 7 shows the viscosity curves of the whey protein composition according to the present invention and a control product in an artificial simulated digestive fluid. [Figure 8] FIG. 8 shows the viscosity curves of whey protein compositions according to the present invention complexed with alginate and carrageenan in an artificial simulated digestive fluid. [Figure 9] FIG. 9 is a graph showing the amount of leucine released from the whey protein composition of the present invention complexed with potassium alginate in an artificial simulated digestive fluid. [Figure 10]FIG. 10 is a graph showing the amount of leucine released from the whey protein composition of the present invention complexed with carrageenan in an artificial simulated digestive fluid. [Figure 11] FIG. 11 is a graph showing the amount of whey protein remaining in an artificially simulated digestive fluid of a whey protein composition according to the present invention obtained under different temperature treatment conditions. [Figure 12] FIG. 12 is a bar graph showing the relative extent of protein hydrolysis of whey protein compositions according to the present invention digested by simulated gastric and intestinal fluids, respectively. [Figure 13] FIG. 13 is a bar graph showing the degree of protein hydrolysis before and after whey protein compositions according to the present invention were digested by artificial digestive fluid. [Figure 14] FIG. 14 is a graph showing leucine release in artificial simulated digestive fluids after application of a whey protein composition according to the present invention to a formulation. [Figure 15] FIG. 15 shows non-reducing (NR) and reducing (R) gel electrophoresis diagrams of a whey protein composition according to the present invention. [Figure 16] FIG. 16 shows curves illustrating the effect of complexation with different polysaccharides on the viscosity of whey protein in artificial simulated digestive fluid. [Figure 17] FIG. 17 shows curves illustrating the effect of whey protein when complexed with different concentrations of carrageenan on the viscosity in artificial simulated digestive fluid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention provides a whey protein composition, its preparation method and application, which can be realized by those skilled in the art by referring to the present specification and appropriately modifying the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The whey protein composition, its preparation method and application of the present invention have been described in the preferred embodiments, but it is obvious to those skilled in the art that the whey protein composition, its preparation method and application described herein can be improved, modified and combined to realize and apply the technology of the present invention without departing from the content, spirit and scope of the present invention.

[0035] The important point about the whey protein composition of the present invention is that, compared to a conventional whey protein concentrate and a commercial product as a reference, the whey protein composition of the present invention has three improvements or any combination thereof: 1) concentration of β-casein by a low-temperature (0-10°C) membrane filtration process, 2) improvement of properties such as particle size distribution, solubility, stability, viscosity, leucine release, etc. by a micronization process, and 3) further increase in viscosity and delay of leucine release by the use of specific polysaccharides (κ-carrageenan and / or potassium alginate) without causing gelation phenomenon.

[0036] In the comparative experiments according to the present invention, the experimental conditions were the same except for the differences between the groups, unless otherwise specified. The whey protein composition provided by the present invention, its preparation method and applications are further described below. EXAMPLES

[0037] Example 1: Preparation of the whey protein composition with skimmed raw milk 1. Production of whey protein raw material rich in β-casein For raw milk, after degreasing and pasteurization, for example, it was left standing at a low temperature of 4°C for 24 h, and β-casein was released from casein micelles and concentrated in the supernatant of skim milk. While blocking casein micelles using a 50-nm ceramic membrane or polymer membrane, whey protein and β-casein were passed through into the permeate. Then, the permeate was concentrated by ultrafiltration with a 10 KDa cut-off to remove lactose, and the supernatant protein was left as a raw material for whey protein rich in β-casein. An exemplary flowchart is shown in Figure 1.

[0038] 2. Micronization The retentate in Step 1 was concentrated at 55°C until the protein concentration reached 5%. The concentrate, adjusted to pH 7.5 by adding 1M HCl or 40% NaOH, was subjected to high-speed shearing at 9600 rpm while being treated at 95°C for 5 min. After that process, the micronized protein system was rapidly cooled to 15°C. Then, it was further concentrated so that the total solid content became 20 - 25%, and finally spray drying (maintaining the inlet temperature at 200°C and the outlet temperature at 90 - 100°C) was performed.

[0039] 3. Addition of Polysaccharides The optimal polysaccharide and addition ratio for improving the viscosity of the above raw material during gastric digestion were determined by experiments. The viscosity in the stomach was highest with 0.3% κ-carrageenan, but the viscosity was also enhanced with 0.3% alginate and maintained during total digestion in the intestinal tract. Finally, it was discovered that whey protein complexed with polysaccharides micronized under pH 7.5 exhibited an excellent sustained-release effect different from whey protein concentrate.

[0040] Example 2: Raw materials of whey protein rich in β-casein obtained at different temperatures According to Step 1 of Example 1, milk was processed by adjusting the temperature to 10°C, 4°C, and 2°C to obtain raw materials of whey protein rich in β-casein. The results of estimating the ratio of β-casein to whey protein using a chemiluminescent imager are shown in Figure 2.

[0041] 2, it was found that the ratio was 4.6:95.4 at 10° C., 9.4:91.6 at 4° C., and 16.6:83.4 at 2° C. Therefore, in the present invention, a whey protein composition falling within the range of 4.5:94.5 to 17:83 could be easily obtained at a low temperature of 2 to 10° C., and a whey protein composition falling within the range of 4.5:94.5 to 20:80 could be expected to be obtained at 0 to 10° C.

[0042] Example 3: In vitro digestion of whey proteins rich in β-casein The purpose of the experiment: to study the changes in beta-casein protein and whey protein before and after digestion, and to select a protein source with a low leucine release rate.

[0043] Experimental Method: The target protein was enzymatically hydrolyzed according to standard static in vitro digestion method using relevant digestive enzymes, and the leucine content in the digestive fluid after digestion was detected using a Shimadzu LCMS 2010 EV system.

[0044] Experimental Grouping: Control group: WPC-Whey Protein Concentrate (WPC392, Fonterra), a commercially available product, WXR-WheyXR Experimental Group 1: (BCNWP 6.5) Whey sample containing β-casein and adjusted to pH 6.5 produced by membrane filtration (prepared according to the method of step 1 of Example 1) Experimental Group 2: (BCNWP 7.5) Whey sample produced by membrane filtration containing β-casein and adjusted to pH 7.5 (prepared according to the method of step 1 of Example 1) Experimental group 3: (ComBCNWP7.5) Whey sample mixed with β-casein and whey protein WPC392 (10:90) and adjusted to pH 7.5 The results shown in Figure 3 indicate that in the two control samples, leucine was 1.4-1.6 mM after digestion by intestinal fluid for 120-240 min, while in the three experimental groups, the leucine released after enzymatic hydrolysis in the digestion step by intestinal fluid was only 0.2-0.4 mM, and the digestion rate in the three experimental groups was much slower.

[0045] Example 4: In vitro digestion of whey proteins rich in β-casein Aim of the experiment: To compare the effect of β-casein protein on the digestion rate of micronized and non-micronized whey protein.

[0046] Experimental Method: The target protein was enzymatically hydrolyzed according to standard static in vitro digestion method using relevant digestive enzymes, and the leucine content in the digestive fluid after digestion was detected using a Shimadzu LCMS 2010 EV system.

[0047] Experimental Grouping: Control group: WPC: whey protein concentrate (WPC392, Fonterra) Experimental Group 1: (MWP6.5) Whey protein concentrate micronized at pH 6.5 Experimental Group 2: (MWP7.5) Whey protein concentrate micronized at pH 7.5 Experimental Group 3: (MBCNWP6.5) β-casein-rich whey raw material micronized at pH 6.5 (prepared according to the method of steps 1-2 of Example 1) Experimental Group 4: (MBCNWP7.5) β-casein-rich whey raw material micronized at pH 7.5 (prepared according to the method of steps 1-2 of Example 1) Experimental Group 5: (ComBCNWP7.5) A whey sample rich in β-casein mixed with β-casein and whey protein WPC392 (10:90) and then micronized at pH 7.5

[0048] The results shown in FIG. 4 show that experimental groups 1 and 2 had a faster leucine release rate than the control group, indicating that micronization accelerated the digestion of accelerated whey protein; experimental groups 3 and 4 had a slower digestion rate than experimental groups 1 and 2, indicating that β-casein was able to reduce the digestion rate of micronized whey protein; and experimental group 5 had an amino acid release rate close to that of experimental groups 3 and 4, indicating that mixing β-casein with whey protein in the ratio of experimental groups 3 and 4 also had the effect of slowly releasing leucine.

[0049] Example 5: Viscosity study of whey protein samples with different casein contents Experimental Purpose: Foods that "thicken" with gastric juices tend to slow stomach emptying and can significantly slow digestion. To identify which types of samples slow digestion, we compared the viscosity of samples and controls.

[0050] Experimental method: A solution with a total protein content of 5% was prepared, and the viscosity was detected within 180 seconds under the same conditions when the total protein concentration was 5% using MCR301 rheometer and cone plate, and then subjected to a digestion experiment with simulated artificial gastric fluid for 2 hours.

[0051] Experimental Grouping: Control: (WPI 6.5 / 7.5) (Whey protein isolate, Fonterra WPI 895, the same WPI was used in the experiment.) Whey protein isolate micronized at pH 6.5 or 7.5. Experimental group 1: (15 / 6.5 and 15 / 7.5) Whey protein samples prepared by keeping skim milk at 15℃ for 24h and micronizing the sample solution at pH 6.5 and 7.5, respectively. The ratio of β-casein to whey protein was approximately 4:96. Experimental group 2: (10 / 6.5 and 10 / 7.5) Whey protein samples prepared by keeping skim milk at 10℃ for 24h and micronizing the sample solution at pH 6.5 and 7.5, respectively. The ratio of β-casein to whey protein was approximately 5:95. Experimental group 3: (4 / 6.5 and 4 / 7.5) Whey protein samples prepared by keeping skim milk at 4℃ for 24h and micronizing the sample solution at pH 6.5 and 7.5, respectively. The ratio of β-casein to whey protein was approximately 10:90.

[0052] The experimental results shown in Figure 5 show that compared to the control sample WPI, all the experimental samples showed higher viscosity. The lower the storage temperature of skim milk, the higher the viscosity of the prepared 5% protein solution. Among the two adjusted pH levels, the protein solution at pH 7.5 was found to have a higher viscosity.

[0053] Example 6: Viscosity studies of whey protein samples enriched with β-casein Objective of the experiment: To determine the effect of atomization on viscosity. Experimental Method: MCR301 rheometer and cone plate were used to detect the viscosity in artificial simulated gastric and intestinal fluids.

[0054] Experimental Grouping: Control group 1: WPC (whey protein concentrate, Fonterra WPC392), competitive products WXR and WheyRX Control group 2: Micellar whey (MW) Control group 3: (MWP6.5, MWP7.5) Whey protein WPC392 micronized at pH 6.5 and 7.5, respectively Experimental Group 1: (BCNWP6.5 and BCNWP7.5) Whey samples containing β-casein and adjusted to pH 6.5 and pH 7.5, respectively, produced by membrane filtration (prepared according to the method in step 1 of Example 1). Experimental Group 2: (MBCNWP6.5, MBCNWP7.5) β-casein-rich whey protein micronized at pH 6.5 and 7.5, respectively (prepared according to the method of steps 1-2 of Example 1)

[0055] The experimental results are shown in Figures 6 and 7, and show that the viscosity of the atomized experimental group 2 in gastric juice was significantly higher than that of the non-atomized experimental group 1. In experimental group 2, it was found that the viscosity of the atomized raw material at pH 7.5 in gastric juice was significantly higher than that of the atomized raw material at pH 6.5.

[0056] Example 7: In vitro digestion experiment of raw materials with added polysaccharides --viscosity test Aim of the experiment: To determine the effect of polysaccharides on improving viscosity. Experimental method: After adding polysaccharides at a ratio of 0.3% w / w and mixing uniformly, the artificial simulated gastric and intestinal fluid viscosities were detected using an MCR301 rheometer and a cone plate.

[0057] Experimental Grouping: Control group 1: (WXR + ALG) Commercially available WXR + potassium alginate Control group 2: (WXR + KCG) Commercially available WXR + κ-carrageenan Experimental Group 1: (MBCNWP6.5+ALG) β-casein-rich whey micronized at pH 6.5 + potassium alginate (prepared according to steps 1-3 of Example 1) Experimental Group 2: (MBCNWP7.5+ALG) β-casein-rich whey micronized at pH 7.5 + potassium alginate (prepared according to steps 1-3 of Example 1) Experimental Group 3: (MBCNWP6.5+KCG) β-casein-rich whey micronized at pH 6.5 + κ-carrageenan (prepared according to steps 1-3 of Example 1) Experimental Group 4: (MBCNWP7.5+KCG) β-casein-rich whey micronized at pH 7.5 + κ-carrageenan (prepared according to steps 1-3 of Example 1)

[0058] The experimental results shown in FIG. 8 show that experimental groups 2 and 4 formed complexes with polysaccharides at pH 7.5 and had higher viscosities than the control group complexed with polysaccharides at pH 6.5.

[0059] Example 8: In vitro digestion experiment of raw materials with added polysaccharides--release of amino acids Aim of the experiment: To determine the effect of the two selected ingredients on the leucine release rate. Experimental method: After polysaccharides were added at a rate of 0.3% w / w and mixed uniformly, the target proteins were enzymatically hydrolyzed according to standard static in vitro digestion method using relevant digestive enzymes, and the content of leucine in the digestive juice after digestion was detected using a Shimadzu LCMS 2010 EV system.

[0060] Experimental Grouping: Control group 1: WPC (whey protein concentrate, Fonterra WPC392) Experimental Group 1: (MBCNWP6.5) β-casein-rich whey micronized at pH 6.5 (prepared according to steps 1-2 of Example 1) Experimental Group 2: (MBCNWP7.5) β-casein-rich whey micronized at pH 7.5 (prepared according to steps 1-2 of Example 1) Experimental Group 3: (MBCNWP6.5+ALG) β-casein-rich whey micronized at pH 6.5 + potassium alginate (prepared according to steps 1-3 of Example 1) Experimental Group 4: (MBCNWP7.5+ALG) β-casein-rich whey micronized at pH 7.5 + potassium alginate (prepared according to steps 1-3 of Example 1) Experimental Group 5: (MBCNWP6.5+KCG) β-casein-rich whey micronized at pH 6.5 + κ-carrageenan (prepared according to steps 1-3 of Example 1) Experimental Group 6: (MBCNWP7.5+KCG) β-casein-rich whey micronized at pH 7.5 + κ-carrageenan (prepared according to steps 1-3 of Example 1)

[0061] The experimental results showed that potassium alginate was able to reduce the release rate of amino acids during the in vitro digestion process of micronized β-casein-rich whey protein (Figure 9). Although the addition of κ-carrageenan failed to significantly reduce the release rate of leucine in the intestinal tract from fine whey proteins rich in β-casein (Figure 10), because κ-carrageenan can strongly increase the viscosity of gastric juice, supplementation with κ-carrageenan is expected to further slow down the rate at which whey proteins enter the intestinal tract and thus help delay the release of free leucine into the small intestine in vivo.

[0062] Example 9: Digestion experiment--Analysis of remaining amount of β-lactoglobulin in whey protein Purpose of the experiment: To compare the degree of digestion and determine the rate of slow digestion. Experimental method: A solution with a total protein content of 10% was prepared and then subjected to in vitro digestion. Size exclusion (SCE-HPLC) and reversed-phase high performance liquid chromatography (RP-HPLC) were used to measure the amount of residual β-lactoglobulin before digestion, after digestion with gastric juice, and after digestion with intestinal juice.

[0063] Experimental Grouping: Control: (WPI 6.5 / 7.5) (Whey protein isolate, Fonterra WPI 895, the same WPI was used in this study) Whey protein isolate micronized at pH 6.5 or 7.5 Experimental group 1: (15 / 6.5 and 15 / 7.5) Whey protein samples prepared by keeping skim milk at 15°C for at least 10h and micronizing the sample solution at pH 6.5 or 7.5, respectively (however, the ratio of β-casein to whey protein was about 4:96, and the sample was prepared according to the method of steps 1-2 of Example 1, and the temperature, low-temperature treatment time and pH parameters were changed accordingly). Experimental Group 2: (10 / 6.5 and 10 / 7.5) Skim milk was kept at 10°C for at least 10h to prepare a sample solution, which was then micronized at pH 6.5 or 7.5, respectively. (However, the ratio of β-casein to whey protein was approximately 5:95, and the sample was prepared according to the method of steps 1-2 of Example 1. The temperature, low-temperature treatment time and pH parameters were changed accordingly.) Experimental group 3: (4 / 6.5 and 4 / 7.5) Skim milk was kept at 4°C for at least 10 hours to prepare a sample solution, which was then micronized at pH 6.5 or 7.5. (However, the ratio of β-casein to whey protein was approximately 10:90, and the sample was prepared according to the method of steps 1-2 of Example 1. The temperature, low-temperature treatment time, and pH parameters were changed accordingly.)

[0064] The experimental results shown in Figure 11 showed that as the temperature decreased, the proportion of β-casein and whey protein increased, and the proportion of undigested β-lactoglobulin in whey protein gradually increased after in vitro digestion, which explained why the experimental group with a higher proportion of β-casein showed a slower digestion rate than the standard whey protein control group.

[0065] Compared to the amount left in the standard whey protein controls (WPI / 6.5 and WPI / 7.5), the whey protein compositions with a β-casein to whey protein ratio of about 5:95 (precisely 4.6:95.4) or compositions with a higher β-casein ratio left more β-lactoglobulin at the end of digestion than the controls. Thus, whey protein compositions according to the present invention can show a slower digestion rate when the β-casein to whey protein mass ratio is 4.6:95.4 or more.

[0066] Example 10: Digestion Experiments--Degree of Hydrolysis Analysis 1 Aim of the experiment: To compare the degree of hydrolysis and determine the rate of slow digestion. Experimental method: The degree of hydrolysis of proteins digested with gastric juice and intestinal juice was measured according to the method of J. Adler-Nissen (1979).

[0067] Experimental Grouping: Control group 1: (WXR+ALG-G / I) Commercially available WXR+0.3% potassium alginate was digested in artificially simulated gastric fluid / intestinal fluid, respectively. Control group 2: (WXR+KCG-G / I) Commercially available WXR+0.3% κ-carrageenan was digested in artificially simulated gastric fluid / intestinal fluid, respectively. Experimental Group 1: (MBCNWP6.5+ALG-G / I) β-casein-rich whey protein micronized at pH 6.5 with the addition of 0.3% potassium alginate (prepared according to steps 1-3 of Example 1) was digested in artificially simulated gastric and intestinal fluids. Experimental Group 2: MBCNWP7.5+ALG-G / I: β-casein-rich whey protein micronized at pH 7.5 with the addition of 0.3% potassium alginate (prepared according to steps 1-3 of Example 1) was digested in artificially simulated gastric and intestinal fluids. Experimental Group 3: (MBCNWP6.5+KCG-G / I) β-casein-rich whey protein micronized at pH 6.5 with the addition of 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1) was digested in artificially simulated gastric and intestinal fluids. Experimental Group 4: (MBCNWP7.5+KCG-G / I) β-casein-rich whey protein micronized at pH 7.5 with the addition of 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1) was digested in artificially simulated gastric and intestinal fluids.

[0068] The experimental results shown in FIG. 12 show that the hydrolysis degree in gastric juice was about 30% in experimental groups 1 and 2, the hydrolysis degree in gastric juice was over 35% in the ALG-added control group, the hydrolysis degree in intestinal juice was about 40% in experimental groups 1 and 2, the hydrolysis degree in intestinal juice was close to 55% in the ALG-added control group, the hydrolysis degree in gastric juice was also about 30% in experimental groups 3 and 4, the hydrolysis degree in gastric juice was over 35% in the KCG-added control group, the hydrolysis degree in intestinal juice was about 40% in experimental groups 3 and 4, and the hydrolysis degree in intestinal juice was close to 50% in the KCG-added control group. This indicates that the sustained release effect of experimental groups 1, 2, 3, and 4 was significant. In addition, the hydrolysis degree of the samples micronized at pH 7.5 was lower than that at pH 6.5 in experimental groups 1, 2, 3, and 4, which indicates that the sustained release effect of the samples micronized at pH 7.5 is even better.

[0069] Example 11: Digestion Experiment--Degree of Hydrolysis Analysis 2 Aim of the experiment: To compare the degree of hydrolysis and determine the rate of slow digestion. Experimental method: The degree of hydrolysis of proteins digested in gastric juice and intestinal juice was measured according to the trinitrobenzenesulfonic acid (TNBS) method described by J. Adler-Nissen (1979).

[0070] Experimental Grouping: Control group 1: WPC (whey protein concentrate, Fonterra WPC392) Control group 2: WXR (WheyRX, a competing product) Experimental Group 1: (MBCNWP6.5+ALG) β-casein-rich whey protein micronized at pH 6.5 with the addition of 0.3% potassium alginate (prepared according to steps 1-3 of Example 1) was digested in an artificially simulated digestive fluid. Experimental Group 2: (MBCNWP7.5+ALG) β-casein-rich whey protein micronized at pH 7.5 with the addition of 0.3% potassium alginate (prepared according to steps 1-3 of Example 1) was digested in an artificially simulated digestive fluid. Experimental Group 3: (MBCNWP6.5+KCG) β-casein-rich whey protein micronized at pH 6.5 with the addition of 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1) was digested in an artificially simulated digestive fluid. Experimental Group 4: (MBCNWP7.5+KCG) β-casein-rich whey protein micronized at pH 7.5 with the addition of 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1) was digested in an artificially simulated digestive fluid.

[0071] The experimental results shown in Figure 13 show that the hydrolysis degree of the samples in experimental groups 1, 2, 3 and 4 did not exceed 41%, the hydrolysis degree of control group 1 exceeded 44%, and the hydrolysis degree of control group 2 exceeded 42%, indicating that the sustained release effect of experimental groups 1, 2, 3 and 4 was significant; and in experimental groups 1, 2, 3 and 4, the hydrolysis degree of the samples micronized at pH 7.5 was lower than that at pH 6.5, indicating that the sustained release effect of the samples micronized at pH 7.5 was even better.

[0072] Example 12: Digestion experiment of samples added to the formulation--release of amino acids Experimental objective: To determine the effect of different formulation aspects on digestion rate. Experimental Method: The final product was prepared according to the formulation listed in Table 1 below, and the relevant digestive enzymes were used to enzymatically hydrolyze the target protein according to the standard static in vitro digestion method, and the content of leucine in the digestive fluid after digestion was detected using a Shimadzu LCMS 2010 EV system.

[0073] [Table 1]

[0074] Experimental Grouping: Control group 1: (P1 + WXR) Powdered formulation P1 + commercial WXR sustained-release whey ingredient Control group 2: (P2 + WXR) Solid bar formulation P2 + commercial WXR sustained-release whey ingredient Experimental Group 1: (P1 + MBCNWP7.5) Powdered formulation P1 + β-casein-rich whey raw material micronized at pH 7.5 with the addition of 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1) Group 2: (P2+MBCNWP7.5) solid bar formulation + pH 7.5 micronized β-casein rich whey ingredient supplemented with 0.3% κ-carrageenan (prepared according to methods steps 1-3 of Example 1).

[0075] The experimental results shown in FIG. 14 show that different food formulations have a strong effect on the release rate of amino acids, the digestion rate of the powder formulation is slower than that of the solid bar formulation, and in the case of the same food formulation, the leucine release effect of the whey protein composition of the present invention added is close to that of the WXR control sample.

[0076] Example 13: Non-reducing (NR) and reducing (R) SDS-PAGE gel electrophoresis experiments of different protein samples

[0077] Experimental Grouping: 1. Milk 2. Whey protein isolated from milk of group 1 3. β-casein-rich whey protein prepared by whey protein of group 2 (UF / DAF filter solution had a total solids concentration of 0.2%) 4. Whey protein micronized at pH 6.5 (similar to whey protein in Group 2) 5. Whey protein micronized at pH 7.5 (similar to whey protein in Group 2) 6. β-casein-rich whey protein micronized at pH 6.5 (similar to whey protein in group 3, total solids concentration was 10%) 7. β-casein-rich whey protein micronized at pH 7.5 (similar to whey protein in group 3, total solids concentration was 10%) 8. Whey protein rich in β-casein micronized at pH 6.5 (same as group 6, powder) 9. Whey protein rich in β-casein micronized at pH 7.5 (same as group 7, powder)

[0078] The results shown in Figure 15 show that non-reducing (NR) gel electrophoresis shows only proteins that are not aggregated with other proteins, whereas reducing (R) gel electrophoresis shows all proteins by disrupting the polymerization reaction between proteins. Lanes 4, 6, and 8 show the samples micronized at pH 6.5, and the bottom band (representing α-lactalbumin) in the non-reducing gel was almost the same as that of the non-micronized control sample, lane 3. The second-lowest band (representing β-lactoglobulin) almost disappeared, indicating that β-lactoglobulin was almost completely aggregated after the micronization treatment.

[0079] Lanes 5, 7 and 9 show micronization at pH 7.5, where the bottom band was less clear and the second band was slightly stronger than in lanes 4, 6 and 8, indicating that more α-lactalbumin and less β-lactoglobulin were aggregated. Thus, the figure allows one to observe the different mechanisms of whey protein micronization using the two pHs.

[0080] Example 14: Viscosity test of whey protein with different polysaccharides in artificial simulated digestive fluid The viscosity of WPC392 (WP) protein solutions was determined in artificial gastric fluid (without enzymes) with 8% protein, 0.2% or 0.3% by weight of selected polysaccharides. The viscosity results in artificial gastric fluid (GJ) at constant temperature (Temp) with potassium alginate (ALG), carboxymethylcellulose (CMC), tragacanth gum (TGH) or xanthan gum (XTH) are shown in Figure 16.

[0081] At the same time, the viscosity of WPC392 protein solutions containing 0.1 to 0.3% carrageenan by weight, with a protein concentration of 8%, was measured in artificial gastric fluid (without enzymes). Here, "Pure" refers to 0.1% carrageenan by weight. The results are shown in Figure 17.

[0082] 16 and 17, it was found that the viscosity of κ-carrageenan in the in vitro digestion model gastric juice was significantly higher than that of the other measured polysaccharides, and was about 30 to 50 times that of the other polysaccharides. In addition, the viscosity of potassium alginate in the in vitro digestion model gastric juice was significantly higher than that of the other measured polysaccharides.

[0083] It should be noted that the above description is only a preferred embodiment of the present invention, and those skilled in the art may make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications are also considered to be within the protection scope of the present invention.

Claims

1. A whey protein composition for reducing the digestion rate of whey protein and the release rate of leucine, comprising β-casein and whey protein, the mass ratio of β-casein being 4.5% or more relative to the total mass of β-casein and whey protein, the whey protein composition is a micronized whey protein composition, and the micronization is performed by high speed shear at 6000-20000 rpm under high temperature of 80-100° C. and pH value of 6.5-9.0; The whey protein composition further comprises a polysaccharide, wherein the polysaccharide is one or more selected from the group consisting of carrageenan, alginate, chitosan, carboxymethylcellulose, tragacanth gum, and mixtures thereof.

2. 2. The whey protein composition according to claim 1, wherein the mass ratio of β-casein to the total mass of β-casein and whey protein is 4.5% to 50%.

3. A method for preparing a whey protein composition, comprising the steps of subjecting skim milk to low-temperature treatment at 0-10°C for 10-24 hours, filtering the skim milk through a membrane of 20 nm or more, and concentrating the permeate by ultrafiltration to remove lactose, thereby obtaining a whey protein composition containing whey protein and β-casein, The method further comprises the step of atomizing the whey protein composition, the atomization being by high speed shear at 6000-20000 rpm under elevated temperature of 80-100°C at a pH value of 6.5-9.0; The method further comprises the step of adding a polysaccharide, wherein the polysaccharide is one or more selected from carrageenan, alginate, chitosan, carboxymethylcellulose, tragacanth gum, and mixtures thereof.

4. 3. Use of the whey protein composition according to claim 1 or 2 in the preparation of a dietary supplement, a muscle synthesis promoter or a dairy product.

5. 3. Use of a whey protein composition according to claim 1 or 2 in the preparation of a whey protein product having slowly digested and / or slowly released amino acids.

6. 3. A nutritional supplement, muscle synthesis promoter or dairy product comprising the whey protein composition according to claim 1 or 2 and a food additive and / or nutrient that can be added to food.

7. 7. The nutritional supplement, muscle synthesis promoter or dairy product of claim 6, which is a nutritional bar comprising the whey protein composition of claim 1 or 2, glucose syrup, glycerin, maltodextrin, coconut oil and lecithin.

8. 7. The nutritional supplement, muscle synthesis promoter or dairy product according to claim 6, which is a powdered dairy product comprising the whey protein composition according to claim 1 or 2, lactose, fructose, glucose, cream, vitamins, minerals and lecithin.

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