A whey protein enzymatic hydrolysate for promoting efficient digestion and absorption of branched-chain amino acids in the elderly, and a preparation method and application thereof
Whey protein hydrolysates were prepared by targeted enzymatic hydrolysis and alkaline heat treatment, which solved the problem of low whey protein digestion efficiency in the elderly population, and achieved efficient protein digestion and BCAA absorption. This method is suitable for nutritional intervention and sports nutrition supplementation in the elderly population.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2026-01-28
- Publication Date
- 2026-06-05
Smart Images
Figure CN122139849A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and functional food technology, specifically relating to a whey protein hydrolysate that promotes efficient digestion and absorption of branched-chain amino acids in the elderly, its preparation method, and its application. Background Technology
[0002] Sarcopenia is an age-related syndrome characterized by a progressive decline in skeletal muscle mass, muscle strength, and physical function. With the accelerating aging of the global population, sarcopenia has become a significant public health issue. The "Expert Consensus on the Diagnosis and Treatment of Sarcopenia in the Elderly in China (2021)" indicates that the prevalence of sarcopenia among elderly people in the community ranges from 8.9% to 38.8%. Nutritional intervention is a core strategy for the prevention and management of sarcopenia, with supplementation with high-quality protein, especially protein sources rich in branched-chain amino acids (BCAAs), playing a crucial role in stimulating muscle protein synthesis and improving muscle function. Whey protein, due to its complete amino acid profile and high BCAA content, is widely considered an ideal substrate for muscle nutrition.
[0003] While some progress has been made in the development of whey protein-related products, several technological limitations remain. One major limitation is the insufficient consideration of the unique gastrointestinal physiology of the elderly. Aging-related decline in digestive function, reduced intestinal mucosal surface area, and insufficient digestive enzyme secretion significantly impact protein digestion and absorption efficiency. However, most current products are not specifically designed for the gastrointestinal system of this population. For example, Chinese patent application CN120585070A discloses a whey protein peptide-based nutritional food for the elderly, focusing on its formulation and preparation, but failing to establish a systematic evaluation of protein digestibility and absorption under simulated elderly gastrointestinal conditions, making it difficult to guarantee its actual bioavailability. Secondly, existing solutions generally neglect the holistic nature of the "digestion-absorption" process as a continuous physiological process, resulting in a fragmented evaluation system. Protein bioavailability begins with gastrointestinal digestion and ends with intestinal absorption, both jointly determining its final nutritional efficacy. However, current research largely focuses on absorption endpoint indicators, lacking consideration of the digestion stage. Chinese patent application CN120436331A primarily relies on the final apparent absorption rate for efficacy evaluation, without thoroughly examining the content of absorbable portions during digestion. Its products may suffer from incomplete digestion, resulting in insufficient usable substrate. Even with a acceptable apparent absorption rate, efficient delivery of BCAAs is difficult to achieve. Furthermore, existing processes fail to balance digestive characteristics and functional integrity in terms of molecular weight control. Intact whey protein, due to its large molecular structure, is difficult to digest efficiently in the gastrointestinal environment of the elderly; while deep enzymatic hydrolysis strategies can improve solubility, they may damage the BCAA structure or lead to an increased proportion of free amino acids, as described in Chinese patent application CN120501165A. Current technologies have not yet achieved a balance between improving digestive efficiency and maximizing the preservation of BCAA functional integrity.
[0004] Therefore, by coupling targeted enzymatic hydrolysis with alkaline-thermal induction treatment, whey protein hydrolysates with small particle size and good dispersibility are formed. These hydrolysates can achieve efficient digestion in the gastrointestinal environment and improve the absorption efficiency of BCAAs in the intestine, thus achieving a dual improvement in digestion and absorption. This provides an important technical premise and methodological basis for developing nutritional intervention programs to prevent sarcopenia in the elderly. Summary of the Invention
[0005] The main objective of this application is to provide a method for preparing whey protein hydrolysate that promotes efficient digestion and absorption of branched-chain amino acids in the elderly and has good dispersibility of small-sized particles, aiming to solve the problem in the prior art that it is difficult to simultaneously improve both protein digestibility and BCAA absorption.
[0006] This invention is achieved through the following technical solution: A method for preparing whey protein hydrolysate that promotes efficient digestion and absorption of branched-chain amino acids in the elderly includes the following steps: Whey protein was mixed with water, and protease was added for targeted enzymatic hydrolysis, controlling the degree of hydrolysis to 5%-15%. After the reaction, the enzyme was inactivated to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was then subjected to alkaline-thermal induction treatment under the following conditions: pH was adjusted to 11.0-13.0, and the mixture was stirred at 65-95°C for 0.5-1.5 h, followed by pH adjustment to 6.5-7.5. Finally, the whey protein hydrolysate was obtained through post-treatment.
[0007] Furthermore, the mass ratio of whey protein to water is 1:10 to 1:50.
[0008] Furthermore, the protease is at least one of trypsin, alkaline protease, and subtilisin.
[0009] Furthermore, the amount of protease added is 0.1% to 2.0% of the whey protein content.
[0010] Furthermore, the enzymatic hydrolysis reaction is carried out at a pH of 7.0-9.0, a temperature of 45-60℃, and a reaction time of 4-8 h.
[0011] Furthermore, the post-processing includes one or more operations such as centrifugation, filtration, concentration, and drying.
[0012] Furthermore, the protein digestibility of the whey protein hydrolysate in an in vitro simulated elderly digestion model was significantly higher than that of unhydrolyzed whey protein.
[0013] A whey protein hydrolysate prepared by the method described in any of the preceding claims, which promotes efficient digestion and absorption of branched-chain amino acids in the elderly.
[0014] Application of a whey protein hydrolysate as described above in the preparation of functional foods that promote the absorption and utilization of branched-chain amino acids.
[0015] The application of a whey protein hydrolysate as described above in the preparation of nutritional formulations for the prevention or improvement of sarcopenia in the elderly.
[0016] Application of a whey protein hydrolysate as described above in the preparation of sports nutrition supplements.
[0017] The whey protein hydrolysate prepared by the above method has a degree of hydrolysis controlled at 10%, and has a high retention rate of branched-chain amino acids. The in vitro protein digestibility and the BCAA absorption rate in the Caco-2 cell model are significantly improved compared with whey protein.
[0018] The present invention has the following advantages over the prior art: This application provides a method for preparing whey protein peptides suitable for the gastrointestinal characteristics of the elderly, based on the targeted enzymatic hydrolysis coupled with alkaline heat treatment using trypsin, alkaline protease, and Bacillus subtilis protease. By precisely controlling the types, ratios, and degrees of hydrolysis of enzymes, and through efficient alkaline heat treatment, whey protein peptides with both high BCAA retention and high absorption properties are prepared. It can be seen that this application effectively improves protein digestibility while maximally preserving the structural integrity and functional activity of BCAAs, forming whey protein hydrolysates with small particle size and uniform dispersion. This provides a reliable raw material and technical route for developing highly efficient nutritional formulations suitable for elderly individuals with sarcopenia. Attached Figure Description
[0019] Figure 1 This is a graph showing the molecular weight results of whey protein and enzymatic hydrolysis products involved in the embodiments of this application; Figure 2 This is a graph showing the in vitro protein digestibility results of whey protein and enzymatic hydrolysates involved in the embodiments of this application in two digestion models: adults and the elderly. Figure 3 This is a graph showing the BCAA absorption rate of whey protein and its enzymatic hydrolysis products involved in the embodiments of this application. Detailed Implementation
[0020] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0021] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.
[0022] The whey protein and protease used in the examples and comparative examples are all commercially available reagents.
[0023] Examples 1-3 (1) Enzymatic hydrolysis Weigh 10 g of whey protein isolate, add deionized water to prepare a 4% (w / w) protein solution, hydrate overnight, and maintain at 50℃, pH 8.0 ± 0.2, and stir at 160 rpm; add trypsin (≥ 2500 U / mg, source leaf S31655) at an enzyme / substrate ratio of 0.5% (w / w).
[0024] (2) Enzyme inactivation After enzymatic hydrolysis, the hydrolysate was placed at 100℃ for 10 min to inactivate the enzyme and then cooled to room temperature. It was then centrifuged at 8000 × g for 10 min, and the supernatant was collected. The hydrolysate was freeze-dried to obtain the whey protein hydrolysate, denoted as Try-1.
[0025] (3) Alkali heat treatment The enzymatic hydrolysis supernatant was heated to 65℃ or 95℃ and kept at that temperature for 30 min. The pH of the mixture was then adjusted to 12.0, and the reaction was stirred at room temperature for 1 h. Finally, the pH of the mixture was adjusted to 7.0. The reaction at 65℃ is designated Try-2, and the reaction at 95℃ is designated Try-3.
[0026] Examples 4-6 Whey protein hydrolysates were prepared according to the method in Example 1, except that the enzyme was replaced with alkaline protease (2.4 AU / g, enzyme dosage 0.5% w / w (2.4 AU / g protein), purchased from Novozymes AG, Copenhagen, Denmark). The degree of hydrolysis was measured after enzyme inactivation to obtain the whey protein hydrolysates. Samples were taken 7 h after enzyme inactivation, and the degree of hydrolysis was measured after DH = 10%, denoted as Alc-1. The supernatant was heated to 65°C or 95°C and incubated for 30 min; then the pH of the mixture was adjusted to 12.0, and the reaction was stirred at room temperature for 1 h; finally, the pH of the mixture was adjusted to 7.0. The hydrolysate reacting at 65°C was denoted as Alc-2, and the hydrolysate reacting at 95°C was denoted as Alc-3.
[0027] Examples 7-9 Whey protein hydrolysates were prepared according to the method in Example 1, except that the enzyme was replaced with subtilisin (7-15 units / mg solid, 0.5% w / w, purchased from Sigma-Aldrich). The degree of hydrolysis was measured after enzyme inactivation to obtain the whey protein hydrolysates. Samples were taken 6 h after enzyme inactivation, and the degree of hydrolysis was measured after enzyme inactivation; DH = 10%, denoted as Sub-1. The supernatant was heated to 65°C or 95°C and incubated for 30 min; then the pH of the mixture was adjusted to 12.0, and the reaction was stirred at room temperature for 1 h; finally, the pH of the mixture was adjusted to 7.0. The reaction at 65°C was denoted as Sub-2, and the reaction at 95°C was denoted as Sub-3.
[0028] Comparative Example 1 Undigested whey protein Experimental Example 1 The particle size of whey protein and its enzymatic hydrolysates can be analyzed using DLS. The particle size of the hydrolysates prepared under different conditions was determined. The samples were diluted with deionized water to 0.5 mg / mL and placed in cuvettes to ensure uniform suspension of the digested sample particles in the solution. The laser angle was set to 173°. Subsequently, the particle size and potential of the samples were measured using a Mastersizer laser scattering analyzer.
[0029] The particle size distribution and potential of the whey protein enzymatic hydrolysates are shown in Table 1. The results indicate that compared to the untreated whey protein's particle size of 1168.73 nm and PDI of 0.74, the particle size of all enzymatically hydrolyzed samples was significantly reduced to 136.20–273.97 nm, and the PDI decreased to 0.20–0.41. The trypsin-treated group (Try-3) had the smallest particle size (136.20 nm) and the most uniform distribution (PDI = 0.29), while the subtilisin group (Sub-1) had the highest absolute potential (-31.67 mV). These results demonstrate that enzymatic hydrolysis coupled with alkaline heat treatment can effectively disrupt the aggregate structure of whey protein, reduce particle size, and improve dispersion uniformity. Trypsin showed the best performance in preparing small, uniform particles, while subtilisin treatment helped enhance system stability, providing a basis for the targeted regulation of whey protein functional properties.
[0030] Table 1 ; Experiment Example 2 Molecular weight determination method: An Ultimate 3000 high-performance liquid chromatograph (Thermo Fisher, MA, USA) equipped with a TSKgel G2000 SWXL column (7.8 × 300 mm, 5 μm particle size) was used at a wavelength of 220 nm. The mobile phase was 0.1% trifluoroacetic acid acetonitrile eluted at isodense density at a flow rate of 1 mL / min. Molecular weight was calculated from retention time.
[0031] By determining the molecular weight distribution of the enzymatic hydrolysis products, such as... Figure 1 As shown, the experimental results indicate that with increasing alkaline heat treatment temperature, the proportion of <1 kDa components increased significantly, with Alc-1 to Alc-3 increasing from 34.35% to 43.52%, Try-1 to Try-3 from 32.55% to 45.35%, and Sub-1 to Sub-3 from 31.16% to 46.91%. Conversely, the proportion of >5 kDa components decreased significantly, with Alc-1 to Alc-3 decreasing from 36.84% to 19.16%, Try-1 to Try-3 from 25.05% to 13.87%, and Sub-1 to Sub-3 from 24.62% to 13.67%. Furthermore, under 95℃ treatment conditions, the <1 kDa component of the Bacillus subtilis proteome (Sub-3) had the highest proportion (46.91%), indicating its greatest advantage in the generation of small peptides. The results above indicate that alkaline heat treatment can effectively enhance enzymatic hydrolysis efficiency and promote the conversion of macromolecular components into small molecule peptides, and trypsin has a more prominent ability to generate low molecular weight peptides under high temperature conditions.
[0032] Experimental Example 3 According to GB 5009.124-2016 standard, the total amino acid content was determined using an A300 amino acid automated analyzer (Bodenheim, Germany): the sample was hydrolyzed at 110℃ for 24 h. After drying in an oven (60℃, 2 h), the sample was dissolved in 1 mL of buffer solution and filtered through a 0.22 μm membrane filter. The hydrolysate was derivatized with phthalaldehyde-mercaptoethanol, separated by cation exchange chromatography in the amino acid analyzer, and then quantitatively analyzed using a UV / fluorescence detector.
[0033] The amino acid composition was determined and is shown in Table 2. The experimental results showed that after alkaline heat treatment at 95℃, the total amino acid content of the trypsin group (Try-3) and the alkaline protease group (Alc-3) reached 93.84 g / 100 g and 93.92 g / 100 g, respectively, which remained stable compared with the untreated WPI (92.79 g / 100 g), while the content of the subtilis protease group (Sub-3) decreased significantly to 71.91 g / 100 g. At the same time, the retention rate of branched-chain amino acids reached 21.38 g / 100 g in the Try-3 group and 21.69 g / 100 g in the Alc-3 group, which was significantly higher than 17.14 g / 100 g in the Sub-3 group. In addition, the changes in the content of essential amino acids showed the same trend, with the Try-3 and Alc-3 groups retaining 44.44 g / 100 g and 44.28 g / 100 g, respectively, while the Sub-3 group only retained 32.43 g / 100 g. The results above indicate that trypsin and alkaline protease can effectively maintain the integrity of amino acid composition under 95℃ treatment conditions, while subtilisin leads to significant loss of nutrients, suggesting that the first two enzymes are more advantageous in preserving the nutritional value of whey protein.
[0034] Table 2 ; Experiment Example 4 (1) Adult in vitro simulated digestion: The test samples (Examples 1-9 and Comparative Example 1) were prepared into a 10% (w / w) solution, preheated in a 37°C water bath for 30 min, and approximately 4 mL of SGF was added and mixed. The pH of the solution was adjusted to 3.0 using 6 M HCl. An appropriate amount of 0.3 M CaCl2 was added, and pepsin was added at a ratio of 2% (enzyme to substrate). The digestion was carried out in a 37°C water bath for 2 h. Then, 4 mL of SIF was added and the pH of the solution was adjusted to 7.0 to enter the intestinal digestion stage. An appropriate amount of 10 mM bile salt solution and 0.3 M CaCl2 solution were added to the digestion system, and pancreatic enzyme was added at a ratio of 2% (enzyme to substrate). The digestion was carried out in a 37°C water bath shaker for 2 h. After digestion, the digestive solution was cooled in an ice-water bath to room temperature and the pH was adjusted to 7.0 to form the adult in vitro digestive solutions of Examples 1-9 and Comparative Example 1, respectively.
[0035] (2) In vitro simulated digestion in the elderly: the ratio of enzyme to substrate added to pepsin was changed to 1.2%, the pH of the solution was adjusted to 3.7 using 6 M HCl, the ratio of enzyme to substrate added to trypsin was changed to 1.6%, the concentration of bile salts was changed to 6.7 mM, and the remaining operations were the same as those for adult in vitro simulated digestion, thus forming the in vitro digestive solutions of the elderly in Examples 1-6 and Comparative Example 1, respectively.
[0036] (3) In vitro digestibility determination: Take 10 mL of digestion solution, add an equal volume of 24% trichloroacetic acid to precipitate the protein, centrifuge at 10000 g for 15 min, take the supernatant, and determine the protein content of the digestible portion in the supernatant according to the Kjeldahl method in GB 5009.5-2010. The solution without added digestion solution is used as a blank. The total protein content of the sample is determined by the Kjeldahl method. The formula for calculating the in vitro protein digestibility is: ; like Figure 2 As shown, the digestibility of whey protein and its intestinal phase proteins after alkaline heat treatment was determined using in vitro simulated digestion models in two population groups. The results of the in vitro simulated digestion experiments showed that in the adult digestion model, the Try-3 group had the highest digestibility (90.50%), while the Alc-2, Alc-3, and Sub-3 groups also reached 85.03%–88.96%, all significantly higher than the untreated WPI group (77.07%–79.84%). In the elderly digestion model, the digestibility of the Sub-3 group further increased to 90.82%, the Try-3 group reached 90.83%, while the WPI group only reached 72.72%–73.28%. Meanwhile, with increasing treatment temperature, the digestibility of each enzymatic hydrolysis group showed an increasing trend in both digestion models, with the Try-3 and Sub-3 groups treated at 95℃ exhibiting the best performance. These results indicate that enzymatic hydrolysis combined with alkaline heat treatment can significantly improve the digestibility of whey protein in a simulated digestive tract environment, and the enzymatic hydrolysates obtained from subtilisin and trypsin treated at 95℃ exhibit good digestibility characteristics in an elderly population digestion model. The reason why enzymatic hydrolysis may improve BCAA absorption is that proteins are mainly absorbed in the intestine in the form of amino acids and peptides after trypsin hydrolysis, while the decomposition effect of pepsin in the stomach is limited; targeted enzymatic pretreatment degrades large protein molecules into more easily absorbed small peptides in advance, thereby potentially improving the bioavailability of BCAAs. The results show that enzymatic hydrolysis treatment not only significantly enhances the overall protein digestibility of whey protein and reduces age-related differences in digestion, but also provides a reference for developing whey protein peptides with high BCAA absorption efficiency.
[0037] Experimental Example 5 (1) Cell culture inoculation: at 37℃, 5% CO2, and 90% relative humidity, at a height of 75 cm² 2 In tissue culture flasks, Caco-2 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 1% antibiotic solution and 1% non-essential amino acids) to the logarithmic growth phase (the medium was changed every 2-3 days). When the Caco-2 cells reached 80%-90% confluence, they were passaged using trypsin-EDTA solution. Cells from passages 10 to 20 were selected for subsequent cell uptake experiments.
[0038] (2) Cell plating and culture: Caco-2 cells were plated at a density of 1 × 10⁻⁶ cells / cells. 5 cells / cm 2 Cells were seeded at a density suitable for 12-well plates. Caco-2 cells were cultured in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and placed in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity, with the medium changed every 2 days. When the Caco-2 cells had filled all the wells, they were treated with in vitro digestion solutions of Examples 1-6 and Comparative Example 1 at a concentration of 1 mg / mL for 4 h.
[0039] (3) The samples were measured according to the Beyotime BCAA kit instructions. The BCAA absorption rate was calculated using the following formula: ; Based on a comprehensive consideration of BCAA retention rate and protein digestibility, a series of trypsin and alkaline protease samples were selected for cell absorption verification experiments. Figure 3 As shown, the BCAA absorption efficiency of whey protein raw materials and their products after different enzymatic hydrolysis-alkali heat treatments was evaluated using the Caco-2 cell model. The results showed that the BCAA absorption rate of whey protein raw materials (WPI) was 1.71%, while the absorption rate was significantly improved after enzymatic hydrolysis-alkali heat treatment. The trypsin 95℃ treatment group (Try-3) achieved the highest absorption rate of 3.65%, approximately 1.13 times higher than WPI; the alkaline protease 95℃ treatment group (Alc-3) achieved 2.80%, approximately 0.64 times higher. The overall absorption rate improvement of the enzymatically hydrolyzed samples ranged from 0.15 to 1.13 times. Alkaline heat treatment effectively enhanced the ability of whey protein hydrolysates to promote BCAA intestinal absorption, and trypsin showed the greatest advantage in improving BCAA absorption efficiency under high-temperature treatment conditions.
[0040] Therefore, the directional enzymatic hydrolysis preparation method provided by this invention is simple, highly controllable, and suitable for industrial continuous production. It can obtain whey protein hydrolysates with high BCAA retention, high digestibility, and high absorption rate at a low production cost. At the same time, the enzymatic hydrolysis process is green and efficient, making it particularly suitable for the design and development of muscle recovery nutritional preparations for the elderly, thus broadening the application prospects of whey protein in the fields of precision nutrition and elderly food.
[0041] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for preparing whey protein hydrolysate that promotes efficient digestion and absorption of branched-chain amino acids in the elderly, characterized in that, Includes the following steps: Whey protein was mixed with water, and protease was added for targeted enzymatic hydrolysis, controlling the degree of hydrolysis to 5%-15%. After the reaction, the enzyme was inactivated to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was then subjected to alkaline-thermal induction treatment under the following conditions: pH was adjusted to 11.0-13.0, and the mixture was stirred at 65-95°C for 0.5-1.5 h, followed by pH adjustment to 6.5-7.
5. Finally, the whey protein hydrolysate was obtained through post-treatment.
2. The preparation method according to claim 1, characterized in that, The mass ratio of whey protein to water is 1:10 to 1:
50.
3. The preparation method according to claim 1, characterized in that, The protease is at least one of trypsin, alkaline protease, and subtilisin.
4. The preparation method according to claim 1, characterized in that, The amount of protease added is 0.1% - 2.0% of the whey protein content.
5. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out at a pH of 7.0-9.0, a temperature of 45-60℃, and a reaction time of 4-8 hours.
6. The preparation method according to claim 1, characterized in that, The post-processing includes one or more of the following operations: centrifugation, filtration, concentration, and drying.
7. A whey protein hydrolysate prepared by the method according to any one of claims 1-6, which promotes efficient digestion and absorption of branched-chain amino acids in the elderly.
8. The application of the whey protein hydrolysate as described in claim 7 in the preparation of functional foods that promote the absorption and utilization of branched-chain amino acids.
9. The use of the whey protein hydrolysate as described in claim 7 in the preparation of nutritional formulations for the prevention or improvement of sarcopenia in the elderly.
10. The use of the whey protein hydrolysate as described in claim 7 in the preparation of sports nutrition supplements.