Whey protein composition, method of preparation thereof, and use thereof

Abstract

The present invention relates to the field of protein processing technology and discloses a whey protein composition, a method for manufacturing the same, and uses thereof. The whey protein composition of the present invention comprises whey protein and β-casein, wherein the mass ratio of β-casein to whey protein is 4.5:95.5 or higher. The whey protein composition provided by the present invention is mainly composed of novel whey protein components that have characteristics of slower digestion than other whey proteins currently on the market. The whey protein composition is a β-casein-rich whey protein concentrate to which micronized and complex natural polysaccharides may be added, and can acquire the function of delaying digestion to promote muscle synthesis and provide sufficient nutrition.

Description

Technology Field Cross-reference regarding related applications

[0001] The present application claims priority to Chinese patent application No. 202011068591.8, titled “Whey Protein Composition, and Preparation Method Therefor and Use Therefor,” filed with the Chinese Intellectual Property Office on September 29, 2020, the entire contents of which are incorporated herein by reference. Technology field

[0002] The present disclosure relates to the field of protein processing technology, and in particular to whey protein compositions, methods for preparing the same, and uses thereof. Background Technology

[0003] Whey protein, known as the "king of proteins," is a protein extracted from milk. It is characterized by its high nutritional value, easy digestion and absorption, and the presence of various active ingredients. It is recognized as one of the high-quality protein supplements for the human body.

[0004] Among various proteins, whey protein possesses the highest nutritional value. It is a high-quality complete protein and is also an animal protein. It contains the eight types of amino acids required by the human body in reasonable proportions close to the ratios needed by the body. It is an indispensable substance for human life activities such as growth, development, and anti-aging. Furthermore, whey protein is easily digested and absorbed, and numerous experimental studies have proven that consuming whey protein concentrate promotes humoral and cellular immunity, stimulates the human immune system, and can prevent the development of chemically induced cancer. Additionally, while whey protein is low in fat and lactose, it contains β-lactoglobulin, α-lactalbumin, immunoglobulins, and various other active ingredients. It is these active ingredients that enable whey protein to possess many health-beneficial functions for the human body. Therefore, it is considered one of the essential sources of high-quality protein for the human body.

[0005] From a nutritional perspective, foods derived from animal protein contain components harmful to the human body, such as excessive saturated fatty acids and cholesterol. Excessive consumption can easily lead to an increase in body fat and cholesterol, potentially causing cardiovascular disease. These issues can be avoided by consuming protein powder to supplement protein. Furthermore, protein powder is easy to consume, has a high absorption rate, and reduces the burden on the stomach. Therefore, consuming protein powder is the best choice for protein supplementation. Whey protein is the primary factor people consider when selecting protein powder.

[0006] Whey protein is generally considered to be highly effective for preventing muscle fatigue and strengthening muscles because it has a short time from oral intake to digestion and absorption and contains large amounts of branched-chain amino acids such as valine, leucine, and isoleucine. However, if these release characteristics can be slowed down, positive muscle synthesis can be promoted more effectively, so it is widely used in many fields such as preventing muscle atrophy in the elderly, muscle recovery and weight loss after exercise in humans, and growth and development in children.

[0007] The current status of sustained-release whey depends on the development of "micelle whey" and undisclosed polymerization processes. WheyXR (WXR), an ingredient sold by Glanbia, is claimed to be polymerized whey with sustained-release properties. The large whey aggregates formed are believed to limit pepsin's ability to reach proteins and slow down digestion in the stomach. Since whey proteins tend to move rapidly through the stomach to the small intestine, more technical means are required for slow digestion. The problem to be solved

[0008] Accordingly, the objective of the present invention is to provide a whey protein composition having slower digestion characteristics when passing through the gastrointestinal tract.

[0009] Another objective of the present invention is to provide a whey protein composition capable of slowly releasing leucine.

[0010] Another objective of the present invention is to provide a whey protein composition that can significantly increase viscosity in the stomach and delay the time it takes to enter the intestinal tract.

[0011] Another objective of the present invention is to provide a whey protein composition capable of reducing the digestibility of β-lactoglobulin.

[0012] Another objective of the present invention is to provide the use of the whey protein composition in the manufacture of nutritional supplements, muscle synthesis promoters, and dairy products, and a method for manufacturing the whey protein composition. means of solving the problem

[0013] To achieve the above objective, the present invention provides the following technical solution.

[0014] The whey protein composition comprises whey protein and β-casein, with a mass ratio of β-casein to whey protein of 4.5:95.5 or higher. The whey protein and β-casein used in specific embodiments of the present invention are primarily derived from milk, but raw milk extracted from other sources is not excluded. The whey protein composition of the present invention may be a qualified mixture prepared by directly processing raw milk, or it may be a mixture in which β-casein and whey protein are blended and added in the appropriate ratio.

[0015] Preferably, the mass ratio of β-casein to whey protein is (4.5:95.5)-(50:50); additionally preferably, the mass ratio of β-casein to whey protein is (9:91)-(20:80), and more preferably, the mass ratio of β-casein to whey protein is (9:90)-(17:83). In a specific embodiment of the present invention, the mass ratio of β-casein to whey protein may be 4.6:95.4, 5:95, 9.4:90.6, 10:90, 15:85, 16.6:83.4, or 17:83.

[0016] In addition to directly mixing β-casein and whey protein, the present invention may further utilize a specific treatment (including maintaining a low temperature for a certain period) to elute β-casein from casein micelles and introduce it into the whey. Due to the unique molecular chaperone effect of β-casein, it forms a unique combination with α-lactalbumin and β-lactoglobulin within the whey protein, thereby protecting the whey protein from rapid digestion. The composition of the present invention is characterized by a reduced leucine release rate compared to commercially available products, whether prepared by processing raw milk or by blending and adding raw materials in proportion.

[0017] In addition, when analyzing the β-lactoglobulin residues in compositions with different ratios of β-casein and whey protein, it was found that only compositions with a ratio of 4:96 or higher had a slower β-lactoglobulin digestion rate compared to standard whey protein concentrate.

[0018] Based on the whey protein described above, the present invention achieves the effect that the micronization process further strengthens the sufficient binding of β-casein and whey protein, thereby additionally slowly releasing leucine, and in vitro ( in vitroIt was discovered that increasing the viscosity of gastric juice in the digestion of a digestion model can prevent proteins from being released into the intestines and delay the digestion process. Compared to the micronization process at pH 6.5, the micronization process at pH 7.5 has a more pronounced effect in this regard. Furthermore, the composition of the present invention can achieve the above effects through micronization, whether it is prepared by processing raw milk or by adding raw materials in the appropriate proportions. Additionally, the micronization process can improve other physical properties of the mixture, including particle size distribution, solubility, stability, and viscosity. Surprisingly, the present invention discovered that when a standard whey protein concentrate undergoes the micronization process without being combined with β-casein, the digestion rate of the whey protein increases; this demonstrates that the addition of β-casein can alter this excessively rapid digestion rate phenomenon and further slow down the digestion process. Based on the above experimental results, the whey protein composition of the present invention is preferably a micronized whey protein composition.

[0019] Preferably, pulverization is performed under high temperature and high-speed shearing at a pH value of 6.5-9.0; wherein the high temperature is 80-100°C, the high-speed shearing is performed at 6000-20000 rpm, and the pulverization time is 3-10 minutes. More preferably, the high temperature is 91-99°C, the high-speed shearing is performed at 8000-15000 rpm, and the pulverization time is 4-8 minutes. Among these, preferred parameters for the pulverization process are pH 7.5, temperature 95°C, duration 5 minutes, and shearing rate 9600 rpm. After pulverization is completed, the materials are rapidly cooled to 15°C.

[0020] As an additional solution optimization, the present invention adds polysaccharides based on the above. Polysaccharides possess unique viscosity advantages and have more binding sites due to the presence of polyhydroxyl groups, thereby providing conditions for additional binding with β-casein-rich whey protein, which can further improve the viscosity of the mixture or delay the release rate of leucine. Alginate and κ-carrageenan in the gastric juice of an in vitro digestion model have higher viscosity than other polysaccharides tested, where the viscosity of κ-carrageenan is 30 to 50 times that of other polysaccharides. Furthermore, adding polysaccharides to micronization at pH 7.5 has a better effect than adding polysaccharides to micronization at pH 6.5.

[0021] According to leucine release analysis, potassium alginate can reduce the rate of leucine release from β-casein-rich undifferentiated whey protein during in vitro digestion. Although the addition of κ-carrageenan did not significantly reduce the rate of leucine release from β-casein-rich undifferentiated whey protein in the intestinal tract, considering that κ-carrageenan can significantly increase the viscosity of gastric juice, it is expected that whey protein may enter the intestinal tract more slowly after supplementation with κ-carrageenan, which will help delay the release of free leucine in the body's small intestine.

[0022] In digestion experiments, compared to standard whey protein concentrates, commercially available products, and their polysaccharide-added controls, the micronized whey protein composition of the present invention with the same polysaccharide added has a lower degree of hydrolysis than each control, which indicates that the whey protein composition of the present invention has a superior sustained-release effect. Similarly, the micronized experimental group at pH 7.5 always has a better effect than the micronized experimental group at pH 6.5.

[0023] Preferably, the polysaccharide of the present invention is selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, tragacanth gum, and mixtures thereof, and the amount of said polysaccharide added is 0.1 to 0.3%. According to the experimental results described above, in certain embodiments, κ-carrageenan and / or potassium alginate may be added to achieve excellent effects in different digestive tracts.

[0024] In addition, the present invention further provides a method for producing a whey protein composition by processing raw milk, comprising processing skimmed raw milk at a low temperature of 0 to 10°C (10 to 120 hours, preferably 10 to 24 hours), then performing filtration using a membrane of 20 to 200 nm or larger, and concentrating the permeate by ultrafiltration to remove lactose to obtain a whey protein composition containing whey protein and β-casein.

[0025] β-casein is released from casein micelles and concentrated in the supernatant of skim milk at low temperatures, because as the temperature decreases, the strength of hydrophobic interactions decreases, allowing β-casein to freely elute from the micelles and enter the supernatant. β-casein can form its own micelles with a diameter of 20-30 nm or less. Therefore, a ceramic or polymer membrane of 20–200 nm (preferably 50 nm) is sufficient to retain casein micelles while allowing whey protein and β-casein to pass through and enter the permeate. The permeate is then concentrated via ultrafiltration (10 kDa) to remove lactose, and the remaining supernatant protein contains β-casein and whey protein in a ratio of (4.5:95.5)–(20:80).

[0026] SDS-PAGE gel electrophoresis results, evaluated using a chemiluminescence imaging device, show that the β-casein and whey protein obtained after treating milk at 10°C, 4°C, and 2°C have the following ratios: 4.6:95.4 at 10°C; 9.4:90.6 at 4°C and 16.6:83.4 at 2°C. Thus, the manufacturing method of the present invention can easily obtain whey protein containing β-casein in an amount of 5 to 20%. If a whey protein composition containing a higher proportion of β-casein is to be achieved, it can be obtained by a compounding method.

[0027] According to the description of the beneficial effects obtainable by the above-mentioned pulverization process, the manufacturing method of the present invention may further include a pulverization process, wherein pulverization is performed under high temperature and high-speed shearing at a pH value of 6.5 to 9.0, and then rapidly cooled to prevent long-term denaturation of the protein at the high temperature. Preferably, the high temperature is 80 to 100°C, the high-speed shearing is performed at 6,000 to 20,000 rpm, and the pulverization time is 3 to 10 minutes. More preferably, the high temperature is 91 to 99°C, the high-speed shearing is performed at 8,000 to 15,000 rpm, and the pulverization time is 4 to 8 minutes. Among these, preferred parameters for the pulverization process are pH 7.5, temperature 95°C, duration 5 minutes, and shearing speed 9,600 rpm. After pulverization is completed, the materials are rapidly cooled to 15°C.

[0028] Preferably, the pulverization process is carried out by performing concentration (protein concentration of 4% or more) to obtain a concentrate, then performing a pulverization treatment, then further concentrating the resulting mixture so that the total solid content reaches 20% or more, and then spray-drying the obtained material into a powder, or making the obtained material into a powder without spray-drying, or, if necessary, preparing the obtained material into a different form of composition.

[0029] In a specific embodiment of the present invention, the micronization process is carried out by concentrating the retained material after ultrafiltration at 55°C until the protein concentration reaches 5%-10%, adding HCl or NaOH to adjust the pH of the concentrate to 7.5, processing the concentrate at 95°C for 5 minutes while performing high-speed shearing at 9600 rpm, then rapidly cooling the micronized protein system to 15°C, then further concentrating the obtained mixture until the total solid content reaches 20-25%, and finally spray-drying the mixture at an inlet temperature of 200°C and an outlet temperature maintained between 90-100°C.

[0030] After micronization, additional polysaccharides may be added and mixed, said polysaccharides are selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, tragacanth gum, and mixtures thereof, and are added in an amount of 0.1-0.3%.

[0031] According to the manufacturing method provided above, the present invention further provides a whey protein composition prepared by said method. Since the whey protein composition provided by the present invention has various slow digestion technical effects and has a better slow digestion effect than similar products on the market, the present invention further provides a use of the whey protein composition in the manufacture of a whey protein product having slow digestion and / or slow release of amino acids. The product may be a nutritional supplement, a muscle synthesis promoter, or a dairy product.

[0032] Accordingly, the present invention further provides a use of a whey protein composition in the manufacture of nutritional supplements, muscle synthesis promoters, or dairy products. The whey protein composition may be a qualified mixture prepared by processing raw milk as in the manufacturing process mentioned above; or a mixture in which β-casein and whey protein are mixed in proportion and added; or a mixture in which micronized and / or polysaccharides are additionally added.

[0033] According to the above application, the present invention provides a nutritional supplement, a muscle synthesis promoter, or a dairy product. The name of the product is specifically determined according to the field of application, but as a common feature, all contain the whey protein composition described in the present invention, and other ingredients may be added as needed, along with excipients and / or nutrients that can be added to food. Nutrients include, but are not limited to, vitamins, minerals, etc.

[0034] In a specific embodiment of the present invention, the present invention provides a nutritional supplement, which is provided in the form of a nutritional bar, and further comprises glucose syrup, glycerin, maltodextrin, coconut oil, and lecithin in addition to the whey protein composition of the present invention.

[0035] Furthermore, the present invention more specifically provides a dairy product provided in the form of a powder and comprises the whey protein composition of the present invention and lactose, fructose, glucose, cream, vitamins, minerals, and lecithin. The vitamins include but are not limited to vitamins A, C, D, and E, and the minerals include but are not limited to calcium salts, iron salts, zinc salts, and magnesium salts, for example, their phosphates or sulfates.

[0036] In digestion experiments of specific formula products, the present invention obtained results showing that different formulas strongly affect the release rate of amino acids, wherein the digestion rate of leucine in powder formulations is slower than that of solid bar formulations. Under the same food formulation, the whey protein composition of the present invention has a leucine release effect close to the leucine release effect of commercially available control samples.

[0037] From the above technical solution, it can be seen that the whey protein composition provided by the present invention is mainly composed of a novel milk protein component that has a slower digestion rate than other whey proteins on the market. A whey protein concentrate rich in β-casein, which can be undifferentiated and mixed with natural polysaccharides, can delay digestion, promote muscle synthesis, and provide adequate nutrition. Brief explanation of the drawing

[0038] FIG. 1 shows an exemplary manufacturing flowchart of a whey protein composition of the present invention;

[0039] FIG. 2 shows a protein electrophoresis graph of the whey protein composition of the present invention obtained by processing at different temperatures;

[0040] Figure 3 shows the leucine release curves of a β-casein-rich whey protein composition and a control group after being digested with an artificially simulated digestive fluid;

[0041] FIG. 4 shows the leucine release curves of the whey protein composition of the present invention and the control group after being artificially digested with a simulated digestive fluid;

[0042] FIG. 5 shows the viscosity curve when the whey protein composition of the present invention is formulated into a 5% protein concentrate solution and a micronization process is performed;

[0043] Figure 6 shows the viscosity curves of an undifferentiated whey protein composition and a control sample after being digested by an artificially simulated digestive fluid;

[0044] FIG. 7 shows the viscosity curves of the whey protein composition of the present invention and a control sample after being digested by an artificially simulated digestive fluid;

[0045] FIG. 8 shows the viscosity curves of the whey protein composition of the present invention after being combined with alginate and carrageenan, respectively, and then digested with an artificially simulated digestive fluid;

[0046] FIG. 9 shows the leucine release curve after combining the whey protein composition of the present invention with potassium alginate and digesting it with an artificially simulated digestive fluid;

[0047] FIG. 10 shows the leucine release curve after mixing the whey protein composition of the present invention with carrageenan and digesting it with an artificially simulated digestive fluid;

[0048] FIG. 11 shows the residual amount curve of whey protein in the whey protein composition of the present invention obtained by processing at different temperatures and digesting with an artificially simulated digestive fluid;

[0049] FIG. 12 is a histogram showing the relative degree of protein degradation of the whey protein composition of the present invention after digestion by artificial gastric juice and intestinal juice, respectively.

[0050] FIG. 13 is a histogram showing the degree of protein degradation of the whey protein composition of the present invention before and after digestion with an artificial digestive liquid.

[0051] Figure 14 shows the leucine release curve after applying the whey protein composition of the present invention to a formulation and digesting it with an artificially simulated digestive fluid.

[0052] Figure 15 shows non-reduced (NR) and reduced (R) gel electrophoresis images of the whey protein composition of the present invention.

[0053] Figure 16 shows the viscosity curve of whey protein affected by different complex polysaccharides in an artificially simulated digestive fluid.

[0054] Figure 17 shows the viscosity curve of whey protein affected by mixing different concentrations of carrageenan in an artificially simulated digestive fluid. Specific details for implementing the invention

[0055] The present invention discloses a whey protein composition, a method for preparing the same, and uses. Those skilled in the art can learn from the contents of this application and can appropriately improve process parameters to realize the present invention. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The whey protein composition, the method for preparing the same, and uses of the present invention have been described through preferred embodiments, and those skilled in the art will clearly be able to make modifications or appropriate changes and combinations to the whey protein composition, the method for preparing the same, and uses described herein without departing from the content, spirit, and scope of the present invention in order to implement and apply the technology of the present invention.

[0056] Compared to conventional whey protein concentrates and commercially available products, the main contents of the whey protein composition of the present invention comprise three improvements or any combination thereof, namely: 1) concentration of β-casein by a membrane filtration process at low temperatures (0-10°C); 2) improvement of properties such as particle size distribution, solubility, stability, viscosity, and leucine release by micronization treatment; and 3) specific polysaccharides (κ-carrageenan and / or potassium alginate) that do not cause gelation but further increase viscosity and delay the release of leucine.

[0057] In the comparative experiments performed in the present invention, unless otherwise noted, additional experimental conditions are maintained identically, except for significant differences between each group.

[0058] The whey protein composition provided by the present invention, the method of preparing the same, and the use thereof are described in detail below. Example 1: Preparation of a whey protein composition using skimmed milk

[0059] 1. Preparation of β-casein-rich whey protein raw materials

[0060] Raw milk was skimmed and pasteurized, then left at a low temperature, such as 4°C, for 24 hours to allow β-casein to be released from casein micelles and concentrated in the supernatant of the skimmed milk. A 50 nm ceramic membrane or polymer membrane was used to maintain the casein micelles, allowing whey protein and β-casein to pass through and enter the permeate. The permeate was then concentrated by ultrafiltration at 10 kDa to remove lactose. The remaining supernatant protein was a whey protein substance rich in β-casein. Refer to Figure 1 for an exemplary flowchart.

[0061] 2. Differentiation

[0062] The residue obtained in Step 1 was concentrated at 55°C to achieve a protein concentration of 5%. The concentrate was adjusted to pH 7.5 using 1M HCl or 40% NaOH and processed at 95°C for 5 minutes under high-speed shearing at 9600 rpm. After this step, the micronized protein system was rapidly cooled to 15°C. The mixture was then further concentrated to reach a total solid content of 20-25% and finally spray-dried at an inlet temperature of 200°C and an outlet temperature maintained between 90-100°C.

[0063] 3. Addition of polysaccharides

[0064] The optimal type of polysaccharide and addition ratio to increase the viscosity of the above raw material during the gastric digestion stage were determined through experiments. 0.3% κ-carrageenan showed maximum viscosity in the stomach, and 0.3% alginate also increased viscosity and maintained viscosity throughout the digestion process in the digestive tract. Finally, it was confirmed that whey protein finely divided at pH 7.5 and combined with polysaccharides exhibited an excellent sustained-release effect distinct from whey protein concentrate. Example 2: β-casein-rich whey protein raw materials obtained at different temperatures

[0065] According to the method of Step 1 of Example 1, milk was processed at temperatures of 10°C, 4°C, and 2°C to obtain a β-casein-rich whey protein raw material. The ratio of β-casein to whey protein was estimated using a chemiluminescence imaging device. The results are shown in Figure 2.

[0066] It can be clearly seen in FIG. 2 that the ratio is 4.6:95.4 at 10℃; 9.4:91.6 at 4℃; and 16.6:83.4 at 2℃. Therefore, the present invention can easily obtain a whey protein composition having a ratio in the range of 4.5:94.5 to 17:83 at low temperatures of 2-10℃, and can expect to obtain a whey protein composition having a ratio in the range of 4.5:94.5 to 20:80 at 0-10℃. Example 3: In vitro digestion of β-casein-rich whey protein

[0067] Experimental Objective: To study the changes in β-casein protein and whey protein before and after digestion, and to select protein sources with a low leucine release rate.

[0068] Experimental method: The target protein was enzymatically digested using the relevant digestive enzyme according to a standardized static in vitro digestion method, and the leucine content in the digested solution was detected using a Shimadzu LCMS 2010 EV system.

[0069] Experimental Grouping:

[0070] Control group: WPC represents whey protein concentrate (WPC392, Fonterra); WXR represents commercially available WheyXR.

[0071] Experimental group 1: BCNWP6.5 represents a whey sample produced by membrane filtration containing β-casein when the pH was adjusted to 6.5 (prepared according to Step 1 of Example 1);

[0072] Experimental group 2: BCNWP7.5 represents a whey sample produced by membrane filtration containing β-casein when the pH was adjusted to 7.5 (prepared according to Step 1 of Example 1);

[0073] Experimental Group 3: ComBCNWP7.5 represents a whey sample produced by mixing β-casein and whey protein WPC392 (10:90) when the pH was adjusted to 7.5;

[0074] The results are shown in Figure 3. Leucine in the two control samples reached 1.4-1.6 mM after 120-240 minutes of digestion by intestinal fluid, whereas leucine released in the three experimental groups was only 0.2-0.4 mM after enzymatic hydrolysis during the intestinal digestion stage, indicating that the digestion rate in the three experimental groups was lower. Example 4: In vitro digestion of β-casein-rich whey protein

[0075] Experimental Objective: To compare the effect of β-casein protein on the digestibility of undifferentiated whey protein and undifferentiated whey protein.

[0076] Experimental method: According to a standardized static in vitro digestion method, the target protein was enzymatically digested using the relevant digestive enzyme, and the leucine content in the digested solution was detected using a Shimadzu LCMS 2010 EV system.

[0077] Experimental Grouping:

[0078] Control group: WPC: Whey protein concentrate (WPC392, Fonterra);

[0079] Experimental Group 1: MWP6.5 represents micronized whey protein concentrate at pH 6.5;

[0080] Experimental Group 2: MWP7.5 represents micronized whey protein concentrate at pH 7.5;

[0081] Experimental group 3: MBCNWP6.5 represents a β-casein-rich whey raw material finely differentiated at pH 6.5 (prepared according to the method of Step 1-2 of Example 1);

[0082] Experimental group 4: MBCNWP7.5 represents a β-casein-rich whey raw material finely differentiated at pH 7.5 (prepared according to the method of step 1-2 of Example 1);

[0083] Experimental Group 5: ComBCNWP7.5 represents a β-casein-rich whey sample produced by mixing β-casein and whey protein WPC392 (10:90) and micronized at pH 7.5;

[0084] The results are shown in Figure 4. In experimental groups 1 and 2, the leucine release rate was faster than in the control group, indicating that micronization can promote the digestion of whey protein; in experimental groups 3 and 4, the digestibility was slower than in experimental groups 1 and 2, indicating that β-casein can reduce the digestibility of micronized whey protein; the amino acid release rate in experimental group 5 was similar to that of experimental groups 3 and 4, suggesting that the effect of slowing leucine release can also be obtained by mixing β-casein and whey protein in the ratios of experimental groups 3 and 4. Example 5: Viscosity test of whey protein samples with different casein contents

[0085] Experimental Objective: Foods "concentrated" in gastric juice tend to delay gastric emptying and, possibly, significantly delay digestion. The purpose of this experiment is to determine the types of samples that delay digestion by comparing the viscosity of the samples with a control group.

[0086] Experimental method: A solution with a total protein content of 5% was prepared. Using an MCR301 rheometer and a cone plate, viscosity at a total protein concentration of 5% was measured under the same conditions within 180 seconds, and then a 2-hour digestion experiment was performed on simulated artificial gastric juice.

[0087] Experimental Grouping:

[0088] Control group: WPI6.5 / 7.5 (whey protein isolated, Fonterra WPI895, the same WPI used in this experiment) represents undifferentiated whey protein isolated at pH 6.5 / 7.5.

[0089] Experimental group 1: 15 / 6.5 and 15 / 7.5 represent whey protein samples prepared by micronizing sample solutions at pH 6.5 / 7.5, respectively, which were prepared by keeping skim milk at 15°C for 24 hours, where the ratio of β-casein to whey protein was approximately 4:96.

[0090] Experimental group 2: 10 / 6.5 and 10 / 7.5 represent whey protein samples prepared by micronizing sample solutions at pH 6.5 / 7.5, respectively, which were prepared by keeping skim milk at 10°C for 24 hours, where the ratio of β-casein to whey protein was approximately 5:95.

[0091] Experimental group 3: 4 / 6.5 and 4 / 7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5 / 7.5, respectively, which were prepared by keeping skim milk at 4°C for 24 hours, where the ratio of β-casein to whey protein was approximately 10:90.

[0092] The experimental results are shown in Figure 5. Compared to the control sample WPI, all samples in the experimental group had higher viscosity. The lower the storage temperature of the skim milk, the higher the viscosity of the prepared 5% protein solution. When comparing the two pH levels, the viscosity of the protein solution was higher at pH 7.5. Example 6: Viscosity test of β-casein-rich whey protein samples

[0093] Experimental Objective: To determine the micronization effect on viscosity.

[0094] Experimental method: The viscosity of artificially simulated gastric and intestinal fluids was measured using an MCR301 rheometer and a cone plate.

[0095] Experimental Grouping:

[0096] Control group 1: WPC (Whey Protein Concentrate, Fonterra WPC392); WXR represents the competing product WheyRX.

[0097] Control Group 2: MW represents whey micelles;

[0098] Control Group 3: MWP6.5 and MWP7.5A represent undifferentiated whey protein WPC392 at pH 6.5 or 7.5, respectively;

[0099] Experimental group 1: BCNWP6.5 and BCNWP7.5 represent whey samples containing β-casein produced by membrane filtration when the pH was adjusted to 6.5 and 7.5, respectively (prepared according to the method of Step 1 of Example 1);

[0100] Experimental group 2: MBCNWP6.5 and MBCNWP7.5 represent β-casein-rich whey proteins undifferentiated at pH 6.5 or 7.5, respectively (prepared according to the method of steps 1-2 of Example 1);

[0101] The experimental results are shown in Figures 6 and 7. The viscosity of the undifferentiated experimental group 2 in gastric juice was significantly higher than that of the undifferentiated experimental group 1. In experimental group 2, the viscosity of the undifferentiated substance in gastric juice at pH 7.5 was significantly higher than that of the undifferentiated substance at pH 6.5. Example 7: In Vitro Digestion Experiment with Added Polysaccharide Raw Material - Viscosity Test

[0102] Experimental Objective: To determine the effect of polysaccharides on viscosity increase.

[0103] Experimental method: Polysaccharides were added at a ratio of 0.3% w / w, and the resulting mixture was uniformly mixed. The viscosity of artificially simulated gastric and intestinal fluids was detected using an MCR301 rheometer and a cone plate.

[0104] Experimental Grouping:

[0105] Control Group 1: WXR+ALG represents the commercial product WXR+ potassium alginate;

[0106] Control group 2: WXR+KCG represents the commercial product WXR+κ-carrageenan;

[0107] Experimental group 1: MBCNWP6.5+ALG represents finely differentiated β-casein-rich whey + potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1);

[0108] Experimental group 2: MBCNWP7.5+ALG represents finely differentiated β-casein-rich whey + potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1);

[0109] Experimental group 3: MBCNWP6.5+KCG represents finely differentiated β-casein-rich whey + κ-carrageenan at pH 6.5 (prepared according to steps 1-3 of Example 1);

[0110] Experimental group 4: MBCNWP7.5+KCG represents finely differentiated β-casein-rich whey + κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1);

[0111] The experimental results are shown in Figure 8. Experimental groups 2 and 4 formed complexes with polysaccharides at pH 7.5, which had a higher viscosity than that of the polysaccharides at pH 6.5 and the control group. Example 8: In Vitro Digestion Experiment with Added Polysaccharide Raw Material - Amino Acid Release

[0112] Experimental objective: To determine the two screened effects on the release rate of leucine.

[0113] Experimental method: Polysaccharides were added at a ratio of 0.3% w / w, and the resulting mixture was uniformly mixed. The target protein was enzymatically digested using the relevant digestive enzyme according to a standardized static in vitro digestion method, and the leucine content in the digested solution was detected using a Shimadzu LCMS 2010 EV system.

[0114] Experimental Grouping:

[0115] Control Group 1: WPC (Whey Protein Concentrate, Fonterra WPC392);

[0116] Experimental group 1: MBCNWP6.5 represents finely differentiated β-casein-rich whey at pH 6.5 (prepared according to steps 1-2 of Example 1);

[0117] Experimental group 2: MBCNWP7.5 represents finely differentiated β-casein-rich whey at pH 7.5 (prepared according to steps 1-2 of Example 1);

[0118] Experimental group 3: MBCNWP6.5+ALG represents finely differentiated β-casein-rich whey + potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1);

[0119] Experimental group 4: MBCNWP7.5+ALG represents finely differentiated β-casein-rich whey + potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1);

[0120] Experimental group 5: MBCNWP6.5+KCG represents finely differentiated β-casein-rich whey + κ-carrageenan at pH 6.5 (prepared according to steps 1-3 of Example 1);

[0121] Experimental group 6: MBCNWP7.5+KCG represents finely differentiated β-casein-rich whey + κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1);

[0122] Experimental results show that potassium alginate can reduce the rate of amino acid release from β-casein-rich undifferentiated whey protein during in vitro digestion (Fig. 9).

[0123] Although the addition of κ-carrageenan cannot significantly reduce the rate of leucine release from β-casein-rich undifferentiated whey protein in the intestinal tract (Fig. 10), considering that κ-carrageenan can cause a significant increase in the viscosity of gastric juice, it can be expected that whey protein may enter the intestine more slowly after supplementing with κ-carrageenan, which may help delay the release of free leucine in the small intestine in vivo. Example 9: Digestion Experiment - Analysis of Residual β-Lactoglobulin in Whey Protein

[0124] Experimental Objective: To compare the degree of digestion and determine the slow digestion ability.

[0125] Experimental method: A solution with a total protein content of 10% was prepared and in vitro digestion was performed. The residual amount of β-lactoglobulin before digestion, after gastric digestion, and after intestinal digestion was measured using size exclusion high-performance liquid chromatography (SCE-HPLC) and reverse-phase high-performance liquid chromatography (RP-HPLC).

[0126] Experimental Grouping:

[0127] Control group: WPI6.5 / 7.5 (Whey protein isolate, Fonterra WPI895, the same WPI in this test) represents undifferentiated whey protein isolate at pH 6.5 / 7.5.

[0128] Experimental Group 1: 15 / 6.5 and 15 / 7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5 / 7.5, respectively, which were prepared by maintaining skim milk at 15°C for 10 hours, where the ratio of β-casein to whey protein was approximately 4:96 (prepared according to the method of Step 1-2 of Example 1, with the temperature, cold treatment time, and pH parameters varied accordingly).

[0129] Experimental Group 2: 10 / 6.5 and 10 / 7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5 / 7.5, respectively, which were prepared by maintaining skim milk at 10°C for 10 hours, where the ratio of β-casein to whey protein was approximately 5:95 (prepared according to the method of Step 1-2 of Example 1, with the temperature, cold treatment time, and pH parameters varied accordingly).

[0130] Experimental Group 3: 4 / 6.5 and 4 / 7.5 were whey protein samples prepared by micronizing the sample solution at pH 6.5 / 7.5, respectively, and were prepared by maintaining skim milk at 4°C for 10 hours, wherein the ratio of β-casein to whey protein was approximately 10:90 (prepared according to the method of Step 1-2 of Example 1, with the temperature, cold treatment time, and pH parameters varied accordingly).

[0131] The experimental results are shown in Figure 11. As the temperature decreased, the ratio of β-casein to whey protein increased, and the ratio of undigested β-lactoglobulin in whey protein after in vitro digestion gradually increased. This means that the experimental group with a higher ratio of β-casein showed a slower digestion rate than the standard whey protein control group.

[0132] Compared to the residual amount of standard whey protein control groups (WPI / 6.5 and WPI / 7.5), a whey protein composition with a ratio of β-casein to whey protein of approximately 5:95 (strictly 4.6:95.4) or a composition with a higher ratio of β-casein to whey protein may have more residual β-lactoglobulin at the end of digestion than the control group. Therefore, the whey protein composition of the present invention having a mass ratio of β-casein to whey protein of 4.6:95.4 or higher may exhibit a slower digestion rate. Example 10: Digestion Experiment - Analysis of Degree of Hydrolysis 1

[0133] Experimental Objective: To compare the degree of hydrolysis and determine the slow digestion capacity.

[0134] Experimental method: The degree of hydrolysis of proteins digested in gastric juice and intestinal juice was tested using the method of J. Adler-Nissen (1979).

[0135] Experimental Grouping:

[0136] Control group 1: WXR + ALG-G / I represented commercial WXR + 0.3% potassium alginate, each digested by artificial simulated gastric / intestinal fluid.

[0137] Control group 2: WXR+KCG-G / I represented commercial WXR + 0.3% κ-carrageenan, each digested by artificial mock gastric / intestinal fluid.

[0138] Experimental group 1: MBCNWP6.5+ALG-G / I represents micronized β-casein-rich whey protein + 0.3% potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1), which was digested with artificial mock gastric juice and intestinal juice.

[0139] Experimental group 2: MBCNWP7.5+ALG-G / I represents micronized β-casein-rich whey protein + 0.3% potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1), which was digested with artificial simulated gastric and intestinal fluids.

[0140] Experimental group 3: MBCNWP6.5+KCG-G / I represents undifferentiated β-casein-rich whey protein + 0.3% κ-carrageenan at pH 6.5 (prepared according to the method of steps 1-3 of Example 1), which was digested with artificial mock gastric juice and intestinal juice.

[0141] Experimental group 4: MBCNWP7.5+KCG-G / I represents undifferentiated β-casein-rich whey protein + 0.3% κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1), which was digested with artificial mock gastric and intestinal fluids.

[0142] The experimental results are shown in Figure 12. The degree of hydrolysis in gastric fluid of experimental groups 1 and 2 was approximately 30%, while the degree of hydrolysis in gastric fluid of the control group with added ALG exceeded 35%. Additionally, the degree of hydrolysis in intestinal fluid of experimental groups 1 and 2 was approximately 40%, while the degree of hydrolysis in intestinal fluid of the control group with added ALG was close to 55%. The degree of hydrolysis in gastric fluid of samples in experimental groups 3 and 4 was also approximately 30%, while the degree of hydrolysis in gastric fluid of the control group with added KCG exceeded 35%. Furthermore, the degree of hydrolysis in intestinal fluid of experimental groups 3 and 4 was approximately 40%, while the degree of hydrolysis in intestinal fluid of the control group with added KCG was close to 50%. This indicates that the sustained-release effect was more pronounced in experimental groups 1, 2, 3, and 4. The degree of hydrolysis of the finely divided samples at pH 7.5 in experimental groups 1, 2, 3, and 4 was lower than that at pH 6.5, which means that the sustained-release effect of the finely divided samples at pH 7.5 is superior. Example 11: Digestion Experiment - Hydrolysis Degree 2 Analysis

[0143] Experimental Objective: To compare the degree of hydrolysis and determine the slow digestion capacity.

[0144] Experimental method: The degree of hydrolysis of proteins digested in gastric juice and intestinal juice was tested using the TNBS (trinitrobenzenesulfonic acid) method introduced by J. Adler-Nissen (1979).

[0145] Experimental Grouping:

[0146] Control Group 1: WPC (Whey Protein Concentrate, Fonterra WPC392);

[0147] Control Group 2: WXR (WheyRX, competitor product);

[0148] Experimental group 1: MBCNWP6.5+ALG represents micronized β-casein-rich whey protein + 0.3% potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1), which was broken down with an artificial mock digestive fluid.

[0149] Experimental group 2: MBCNWP7.5+ALG represents micronized β-casein-rich whey protein + 0.3% potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1), which was broken down with an artificial mock digestive fluid.

[0150] Experimental group 3: MBCNWP6.5+KCG represents undifferentiated β-casein-rich whey protein + 0.3% κ-carrageenan at pH 6.5 (prepared according to the method of steps 1-3 of Example 1), which was broken down with an artificial mock digestive fluid.

[0151] Experimental group 4: MBCNWP7.5+KCG represents undifferentiated β-casein-rich whey protein + 0.3% κ-carrageenan at pH 7.5 (prepared according to the method of steps 1-3 of Example 1), which was broken down with an artificial mock digestive fluid.

[0152] The experimental results are shown in Figure 13. The hydrolysis degree of samples in experimental groups 1, 2, 3, and 4 did not exceed 41%, while the hydrolysis degree of control group 1 exceeded 44% and the hydrolysis degree of control group 2 exceeded 42%, indicating that experimental groups 1, 2, 3, and 4 had a relatively prominent sustained-release effect. The degree of hydrolysis of the micronized samples in experimental groups 1, 2, 3, and 4 at pH 7.5 was lower than that at pH 6.5, which means that the sustained-release effect of the micronized samples at pH 7.5 is superior. Example 12: Digestion experiment after adding a sample to the formulation - Amino acid release

[0153] Experimental Objective: To determine the effect of different formulations on digestion rate.

[0154] Experimental method: The final product was prepared according to the formula in Table 1 below, the target protein was enzymatically digested using the relevant digestive enzyme according to a standardized static in vitro digestion method, and the leucine content in the digested solution was measured using a Shimadzu LCMS 2010 EV system. Powder (per 100g) P1 Solid Bar (per 60g) P2 ingredient Content (%) ingredient Content (%) Protein (MBCNWP7.5 / WXR) 28.1 Protein (MBCNWP7.5 / WXR) 46 κ-carrageenan 0.3 glucose syrup 27 Lactose 26.3 glycerin 17 Saccharides (Fructose 42% + Glucose) 26.6 Maltodextrin, DE ~ 17 3.5 fat (cream) 10 coconut oil 5 Vitamins Vitamin C Vitamin E (Tocopherol) Vitamin A Vitamin D 1 κ-carrageenan 0.3 Minerals: Calcium phosphate, Ferrous sulfate, Zinc sulfate, Magnesium sulfate 3 lecithin 0.5 lecithin 0.5 water 0.7 Water (contained in powder) 4-5

[0155] Experimental Grouping:

[0156] Control Group 1: P1+WXR represents the powder formulation P1 + the commercial product WXR, which slowly releases whey raw materials;

[0157] Control Group 2: P2+WXR represents a commercial product WXR that slowly releases the solid bar formulation P2 + whey raw material;

[0158] Experimental group 1: P1+MBCNWP7.5 represents powder formulation P1 + micronized β-casein-rich whey raw material at pH 7.5 + 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1);

[0159] Experimental group 2: P2+MBCNWP7.5 represents a solid bar formulation + micronized β-casein-rich whey raw material at pH 7.5 + 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1).

[0160] The experimental results are illustrated in Fig. 14. Different food formulations have a significant effect on the release rate of amino acids, and the digestion rate of the powder formulation is slower than that of the solid bar formulation; under the same food formulation, the whey protein composition of the present invention exhibited a leucine release effect similar to that of the WXR control sample. Example 13: Non-reduction (NR) and reduction (R) SDS-PAGE gel electrophoresis of different protein samples

[0161] Experimental Grouping:

[0162] 1. Milk;

[0163] 2. Whey protein isolated from milk of Group 1;

[0164] 3. β-casein-rich whey protein prepared from Group 2 whey protein (residue after ultrafiltration / diafiltration, total solids concentration 0.2%);

[0165] 4. Undifferentiated whey protein at pH 6.5 (identical to the whey protein in Group 2);

[0166] 5. Whey protein undifferentiated at pH 7.5 (identical to the whey protein in Group 2);

[0167] 6. Undifferentiated β-casein-rich whey protein at pH 6.5 (identical to the whey protein in Group 3, with a total solids concentration of 10%);

[0168] 7. Undifferentiated β-casein-rich whey protein at pH 7.5 (identical to the whey protein in Group 3, with a total solids concentration of 10%);

[0169] 8. β-casein-rich whey protein undifferentiated at pH 6.5 (same as Group 6, powder);

[0170] 9. β-casein-rich whey protein finely differentiated at pH 7.5 (same as Group 7, powder).

[0171] The results are shown in Fig. 15. Non-reducing (NR) gel electrophoresis shows only proteins that have not aggregated with other proteins, while reducing (R) gel electrophoresis shows all proteins as the aggregation reactions between proteins are destroyed. Lanes 4, 6, and 8 represent undifferentiated proteins at pH 6.5, and the bottom band (representing α-lactalbumin) in the non-reducing gel was nearly identical to Lane 3 of the undifferentiated control sample. Additionally, the second-to-bottom band (representing β-lactoglobulin) had almost disappeared, indicating that β-lactoglobulin aggregated almost completely after differentiation.

[0172] Lanes 5, 7, and 9 show undifferentiated protein at pH 7.5, where the bottom band became indistinct, and the second band was slightly stronger than the bands in lanes 4, 6, and 8, indicating that more α-lactalbumin and less β-lactoglobulin aggregated. Thus, different undifferentiation mechanisms of whey protein at two pH values ​​can be observed in this figure. Example 14: Viscosity test of whey protein with different polysaccharides added to artificially simulated digestive fluid

[0173] In artificial gastric juice (enzyme-free), the viscosity of a WPC392 (WP) protein solution with a protein concentration of 8% and 0.2 wt% or 0.3 wt% of a selected polysaccharide added was detected. ALG represents potassium alginate; CMC represents carboxymethyl cellulose; TGH represents tragacanth gum; XTH represents xanthan gum; Temp represents temperature; and GJ represents artificial gastric juice. The results are shown in Fig. 16.

[0174] In addition, the viscosity of a WPC392 protein solution with a protein concentration of 8% and 0.1-0.3 wt% of carrageenan added was detected in artificial gastric juice (no enzymes). Pure indicates 0.1 wt% of carrageenan. The results are shown in Fig. 17.

[0175] From Figures 16 and 17, it can be clearly seen that the viscosity of κ-carrageenan in the gastric juice of an in vitro digestion model was significantly higher compared to that of other tested polysaccharides, which was about 30-50 times higher than that of other polysaccharides. The viscosity of potassium alginate in the gastric juice of an in vitro digestion model was also significantly higher than that of other tested polysaccharides.

[0176] The foregoing is merely a preferred embodiment of the present invention, and those skilled in the art should note that several improvements and modifications may be made without departing from the principles of the present invention, and such improvements and modifications should be deemed to be included within the scope of protection of the present invention.

Claims

Claim 1 A whey protein composition comprising whey protein and β-casein, wherein the mass ratio of β-casein to the total mass of β-casein and whey protein is at least 4.5%; the whey protein composition is a micronized whey protein composition; said micronization is performed at a high temperature of 80 to 100°C and a high-speed shear of 6,000 to 20,000 rpm at a pH of 6.5 to 9.0; the whey protein composition further comprises a polysaccharide; said polysaccharide is selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, tragacanth gum, and mixtures thereof. Claim 2 A whey protein composition according to claim 1, wherein the mass ratio of β-casein to whey protein is 4.5:95.5 to 50:

50. Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 A method for preparing a whey protein composition comprising processing skimmed raw milk at a low temperature of 0 to 10°C for 10 to 24 hours, then performing filtration using a membrane of at least 20 to 200 nm, and concentrating the permeate by ultrafiltration to remove lactose to obtain a whey protein composition containing whey protein and β-casein; the method further comprises micronizing the whey protein composition; said micronizing is performed at a high temperature of 80 to 100°C and a high-speed shear of 6,000 to 20,000 rpm at a pH of 6.5 to 9.0; said method further comprises adding a polysaccharide; said polysaccharide is selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, tragacanth gum, and mixtures thereof. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 Whey protein composition prepared by the method according to paragraph 7. Claim 13 delete Claim 14 A whey protein product comprising a whey protein composition according to claim 1 or 2 or a whey protein composition according to claim 12, having slow digestion and / or slow release of amino acids. Claim 15 A Whey protein composition according to claim 1 or 2 or a whey protein composition according to claim 12, and a nutritional supplement comprising excipients and / or nutrients that may be added to food. Claim 16 A nutritional supplement according to claim 15, comprising a whey protein composition according to claim 1 or 2 or a whey protein composition according to claim 12, and a nutritional bar comprising glucose syrup, glycerin, maltodextrin, coconut oil, and lecithin. Claim 17 A nutritional supplement according to claim 15, wherein the whey protein composition according to claim 1 or 2 or the whey protein composition according to claim 12, and a powdered dairy product comprising lactose, fructose, glucose, cream, vitamins, minerals and lecithin. Claim 18 A muscle synthesis promoter comprising a whey protein composition according to claim 1 or 2 or a whey protein composition according to claim 12, and excipients and / or nutrients that may be added to food. Claim 19 A muscle synthesis promoter according to claim 18, wherein the whey protein composition according to claim 1 or 2 or the whey protein composition according to claim 12, and a nutrition bar comprising glucose syrup, glycerin, maltodextrin, coconut oil and lecithin. Claim 20 A muscle synthesis promoter according to claim 18, wherein the whey protein composition according to claim 1 or 2 or the whey protein composition according to claim 12, and a powdered dairy product comprising lactose, fructose, glucose, cream, vitamins, minerals and lecithin. Claim 21 A whey protein composition according to paragraph 1 or 2 or a whey protein composition according to paragraph 12, and a dairy product comprising excipients and / or nutrients that may be added to food. Claim 22 A dairy product according to claim 21, wherein the whey protein composition according to claim 1 or 2 or the whey protein composition according to claim 12, and a nutrition bar comprising glucose syrup, glycerin, maltodextrin, coconut oil and lecithin. Claim 23 A dairy product according to claim 21, wherein the whey protein composition according to claim 1 or 2 or the whey protein composition according to claim 12, and the powdered dairy product comprising lactose, fructose, glucose, cream, vitamins, minerals and lecithin.

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