Phenolic acid-hydrolyzed casein complex, method for preparing the same, and use thereof

Abstract

The present application belongs to the technical field of food processing, and relates to a phenolic acid-hydrolyzed casein protein complex, a preparation method and application thereof. Specifically, the present application obtains a high-antioxidant-activity component with a molecular weight of <3KDa by hydrolyzing casein protein with alkaline protease; and the hydrolyzed casein protein is mixed with a phenolic acid solution to form a complex. In vitro simulation of gastrointestinal digestion tests show that the polyphenol content and total antioxidant capacity of the complex are significantly higher than those of the phenolic acid group alone, and the digestion stability of the phenolic acid is improved. Animal tests show that complex intervention can shorten the defecation time, improve the gastrointestinal propulsion rate, and the digestion-promoting effect is better than that of the single component, and is equivalent to that of the positive drug mosapride; among them, the coffee acid and p-coumaric acid-hydrolyzed casein protein complex has the best effect, with a synergistic index CI <1, which confirms the synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and relates to phenolic acid-hydrolyzed casein complex, its preparation method and application. Background Technology

[0002] Indigestion is a common clinical syndrome in which patients have no organic lesions and experience symptoms such as upper abdominal pain, postprandial fullness, or early satiety. Because medical equipment cannot detect any organic lesions, patients often do not receive proper treatment, and most patients with indigestion suffer greatly from it.

[0003] Currently, the main methods for improving indigestion include acid suppression therapy, prokinetic drug therapy, and psychological and behavioral therapy. However, acid suppression therapy can only improve indigestion caused by excessive gastric acid secretion, limiting its application scenarios; while common prokinetic drugs can achieve certain effects in the early stages, their long-term efficacy is poor, and they have drawbacks such as high relapse rates and many side effects; psychological and behavioral therapies have limited feasibility and do not achieve ideal results.

[0004] In recent years, the search for novel drugs to improve indigestion from natural plants or traditional Chinese medicine has been a hot topic in related research. Studies have shown that flavonoids in polyphenols can promote gastrointestinal motility and improve indigestion to some extent, while the functional effects of phenolic acids in polyphenols on indigestion have not been reported in detail. Phenolic acids in polyphenols include caffeic acid, ferulic acid, p-coumaric acid, gallic acid, and syringic acid, which are widely distributed in fruits, vegetables, and grains. They have been shown to have various biological activities such as antioxidant, anti-inflammatory, immunomodulatory, and anticancer effects, and have the potential to improve indigestion. However, due to the generally poor water solubility and stability of phenolic acid polyphenols, their bioavailability is low, which greatly limits their application in the food, pharmaceutical, and cosmetic industries.

[0005] Casein, as an important nutrient, possesses various functional properties. Numerous studies have shown that combining casein with polyphenols can significantly enhance the stability and bioactivity of polyphenols. Due to its structural characteristics, casein is not easily digested; however, hydrolysis of casein can impart characteristics such as high digestibility, low allergenicity, and antioxidant properties. Currently, there are no reports of phenolic acid polyphenols combined with hydrolyzed casein, and the function of this complex in improving indigestion remains unexplained. Summary of the Invention

[0006] To provide a wider range of potential therapeutic drugs for improving indigestion, this application provides the following technical solutions: The first aspect of the present invention is to provide a phenolic acid-hydrolyzed casein complex, the complex being formed by the non-covalent bonding of phenolic acid and hydrolyzed casein, wherein the hydrolyzed casein is a component of casein with a molecular weight of less than 3 kDa obtained by protease hydrolysis and ultrafiltration. Furthermore, the protease is an alkaline protease. Further, the phenolic acid is selected from one or more of caffeic acid, p-coumaric acid, gallic acid, syringic acid, or ferulic acid; preferably, the phenolic acid is selected from caffeic acid and / or p-coumaric acid; Further, the molar ratio of phenolic acid to hydrolyzed casein is 1 to 3:1; preferably, the molar ratio is 1:1.

[0007] A second aspect of the present invention is to provide a method for preparing the phenolic acid-hydrolyzed casein complex described in the first aspect, comprising the following steps: hydrolyzing casein to obtain hydrolyzed casein, and mixing the hydrolyzed casein with a phenolic acid solution.

[0008] Furthermore, the preparation method is performed under conditions where the pH value is 6.5 to 7.5; Furthermore, the preparation method is carried out at a temperature of 25-45°C; Furthermore, in the preparation method, after mixing, the mixture is kept at a certain temperature for 0.5 to 2 hours, preferably 1 hour. Further, the preparation of the hydrolyzed casein includes: dispersing casein in water, adding protease for hydrolysis, inactivating the enzyme after hydrolysis, centrifuging and filtering, and then collecting the permeate using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain a hydrolyzed casein component with a molecular weight of less than 3 kDa. Furthermore, the protease is an alkaline protease, the enzyme-to-substrate ratio is 1:100 w / w, the hydrolysis time is 2-5 hours, the optimal pH is adjusted according to the enzyme properties, and the enzyme is inactivated by heating after hydrolysis.

[0009] Furthermore, the concentration of the hydrolyzed casein is 0.3~0.5 mg / ml, the concentration of the phenolic acid solution is adjusted according to the molar ratio, and the pH value is adjusted using HCl and NaOH.

[0010] A third aspect of the present invention provides the use of the phenolic acid-hydrolyzed casein complex according to the first aspect in the preparation of a medicament for improving indigestion; Furthermore, the improvement of indigestion includes promoting gastrointestinal motility, shortening defecation time, and increasing gastrointestinal propulsion rate; Furthermore, the drug is suitable for patients with functional dyspepsia, and the dosage of the compound is 800-1200 mg / kg / Day.

[0011] The beneficial effects of this invention include: 1) In this invention, casein is first hydrolyzed with alkaline protease (enzyme:substrate 1:100 w / w) for 3 h, then inactivated, centrifuged, filtered, and ultrafiltered to obtain a high antioxidant active component with a molecular weight <3 kDa; then, the hydrolyzed casein (0.4 mg / ml) is mixed with phenolic acid solution at a ratio of 1:1 and kept at pH 7 and 25-45℃ for 1 h to form a complex.

[0012] 2) Characterization results showed that the complex was formed through a static quenching mechanism (binding sites n≈1, molar ratio 1:1), with hydrogen bonds and van der Waals forces being the main interactions. In vitro simulated gastrointestinal digestion experiments showed that the complex had significantly higher polyphenol content and total antioxidant capacity than the phenolic acid group alone, thus improving the digestibility and stability of phenolic acids.

[0013] 3) Animal experiments used a mouse model of indigestion induced by loperamide. Complex intervention (1000 mg / kg, containing 100 mg of phenolic acid) shortened defecation time and increased gastrointestinal propulsion, demonstrating better digestive effects than the individual components and comparable to the positive control drug mosapride. Among the components, caffeic acid- and p-coumaric acid-hydrolyzed casein complexes showed the best effect, with a synergistic index (CI) < 1, confirming a synergistic effect.

[0014] In summary, the complex of the present invention has antioxidant and digestive-promoting functions, and can be used to prepare drugs or food supplements to improve functional dyspepsia, with broad application prospects. Attached Figure Description

[0015] Figure 1 Antioxidant activity of casein hydrolysates from different proteases: A: Total antioxidant activity T-AOC determined by FRAP method; B: DPPH free radical scavenging rate; C: ABTS free radical scavenging rate. Figure 2 Antioxidant activity of casein hydrolysates from different proteases after ultrafiltration: A: Total antioxidant activity T-AOC determined by FRAP method; B: DPPH free radical scavenging rate; C: ABTS free radical scavenging rate. Figure 3 Fluorescence intensity of caffeic acid and hydrolyzed casein complexed at different temperatures: A: 25℃, B: 35℃, C: 45℃; Figure 4 The Stern-Volmer equation (A) and the double logarithmic equation (B) are used to calculate the complex of caffeic acid and hydrolyzed casein, respectively. Figure 5 A: Polyphenol content of caffeic acid-hydrolyzed casein complex group in in vitro simulated gastrointestinal digestion; B: Total antioxidant capacity T-AOC (FRAP method). Figure 6 Fluorescence intensity of coumaric acid and hydrolyzed casein complexed at different temperatures: A: 25℃, B: 35℃, C: 45℃; Figure 7 The Stern-Volmer equation (A) and the double logarithmic equation (B) are used to calculate the complex of coumaric acid and hydrolyzed casein, respectively. Figure 8 A: Polyphenol content of caffeic acid-hydrolyzed casein complex group in in vitro simulated gastrointestinal digestion; B: Total antioxidant capacity T-AOC (FRAP method). Figure 9 Phenolic acid-hydrolyzed casein complex promotes digestion; A: defecation time; B: gastrointestinal propulsion rate. Figure 10 Defecation time in the caffeic acid-hydrolyzed casein (A) and p-coumaric acid-hydrolyzed casein (B) intervention groups; Figure 11 Gastrointestinal propulsion rate in the caffeic acid-hydrolyzed casein (A) and coumaric acid-hydrolyzed casein (B) intervention groups. Detailed Implementation

[0016] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0017] Example 1: Preparation of hydrolyzed casein 1. Hydrolysis and ultrafiltration of casein Based on the characteristics of antioxidant peptides (high content of hydrophobic residues, hydrophobic or aromatic amino acids at the C-terminus or N-terminus, mostly including 5-16 amino acids), five hydrolases with cleavage sites consistent with the characteristics of antioxidant peptides were selected from the three major classes of enzymes: animal proteases: trypsin; plant proteases: bromelain, fig protease; microbial proteases: alkaline protease, flavor protease.

[0018] Casein powder was dispersed in water at 2% w / w, stirred at room temperature for 1 h, and then stored at 4°C overnight. Enzyme pre-incubation was performed for 10 min, followed by addition to the casein solution at an enzyme-to-substrate ratio of 1:100 w / w. The pH was adjusted to the optimal level according to the enzyme properties. After hydrolysis for 3 h, the mixture was heated at 100°C for 10 min to inactivate the enzyme, and then centrifuged at 12000 rpm for 10 min at 4°C. The hydrolyzed solution was centrifuged at 12000 rpm for 10 min at 4°C and filtered through a 0.45 μm aqueous filter membrane. Ultrafiltration was performed using ultrafiltration membranes with different molecular weight cutoff sizes (3, 10, 50, 100 kDa), and the permeate and retentate were collected at each filtration stage.

[0019] 2. Detection indicators The antioxidant activity of the solutions after hydrolysis of five proteases and hydrolyzed casein of different molecular weights was determined by FRAP, DPPH and ABTS methods, respectively. The kits were provided by Solarbio.

[0020] 3. Results like Figure 1 As shown in AC, the casein hydrolysate exhibits the highest antioxidant activity after being hydrolyzed by alkaline protease; therefore, alkaline protease was selected for subsequent experiments.

[0021] like Figure 2 As shown in AC, the casein alkaline protease hydrolysate was divided into five groups according to molecular weight after ultrafiltration (<3KDa, 3-10KDa, 10-50KDa, 50-100KDa, >100KDa). Among them, the <3KDa component had the highest antioxidant activity, so the <3KDa component was selected for subsequent experiments.

[0022] Example 2: Complexation and Characterization of Phenolic Acid-Hydrolyzed Casein 1. Phenolic acid-hydrolyzed casein complex Hydrolyzed casein (<3 kDa) was diluted to 0.4 mg / ml with PBS to prepare a series of phenolic acid solutions of different concentrations. The hydrolyzed casein solution and the phenolic acid solution were thoroughly mixed at a 1:1 ratio, and the pH of the system was adjusted to 7 with HCl and NaOH. The mixture was incubated at different temperatures (25℃, 35℃ and 45℃) for 1 h to obtain the phenolic acid-hydrolyzed casein complex.

[0023] 2. In vitro simulated gastrointestinal digestion test of phenolic acid-hydrolyzed casein The samples were tested using in vitro gastrointestinal models during the gastric and small intestinal phases.

[0024] Simulated gastric digestion: 1 mL of sample was mixed with 1 mL of simulated gastric juice (purchased from FEIMOBIO, sterile, containing pepsin). The mixture was then adjusted to pH 2.5 with HCl solution and continuously rotated at 90 rpm at 37°C for 2 hours.

[0025] Simulated intestinal digestion: 1 mL of gastric phase sample was transferred to a sterile enzyme-free centrifuge tube. After adjusting the pH of the mixture to 6.8, it was mixed with 1 mL of simulated intestinal fluid (purchased from FEIMOBIO, sterile, containing trypsin) and continuously rotated at 90 rpm at 37°C for 2 hours.

[0026] 3. Detection indicators 3.1 Fluorescence spectroscopy determination: The instrument was set to an excitation wavelength of 280 nm, an emission wavelength of 290-450 nm, a slit width of 5 nm, and a scan rate of 600 nm / min. PBS solution was used as a blank control.

[0027] The fluorescence quenching mechanism of protein-small molecule interactions can be divided into dynamic or static types. The following conclusions can be drawn from the Stern-Volmer equation: F0 / F=1+K q τ0[Q]=1+K SV [Q] Where F and F0 represent the fluorescence intensity of proteins with and without phenolic acid, respectively; [Q] represents the concentration of the quencher; K SV It is the quenching constant; K q τ is the bimolecular quenching rate constant; τ0 is the lifetime of the fluorophore in the absence of a quencher, and the average lifetime of biomacromolecules is about 10⁻⁸ s.

[0028] For static quenching, combined with the constant (K) A The number of binding sites (n) can be calculated from a double logarithmic equation: log(F0-F) / F=logK A +nlog[Q] To further investigate the thermodynamic parameters involved in the interaction between phenolic acids and hydrolyzed casein, the Van't Hoff equation was introduced to calculate the enthalpy change (ΔH), entropy change (ΔS), and free energy change (ΔG).

[0029] lnK A =-ΔH / RT+ΔS / R ΔG=ΔH-TΔS Where R represents the gas constant (8.314 J·mol⁻¹) −1 ·K −1 ), T represents absolute temperature.

[0030] 3.2 Determination of total polyphenol content: The sample solution was appropriately diluted with PBS. Then, 2.5 mL of phenol reagent (0.2 M) was added to 0.5 mL of the sample solution, followed by 3 mL of Na₂CO₃ solution (7.5%, w / v). After reacting in the dark for 2 h, the absorbance at 760 nm was measured using a UV-Vis spectrophotometer. A standard curve was generated using standard phenolic acid solutions (0–0.2 mg / mL), with PBS used as a blank control.

[0031] 4. Results 4.1 Construction and characterization of caffeic acid-hydrolyzed casein like Figure 3 As shown, hydrolyzed casein was subjected to different temperatures: 25℃ ( Figure 3 A) 35℃ Figure 3 B) and 45℃ Figure 3 The strongest fluorescence absorption value was observed at 300 nm for caffeic acid (C). As the concentration of caffeic acid increased, the fluorescence intensity gradually decreased, demonstrating that caffeic acid and hydrolyzed casein can recombine. This is based on the Stern-Volmer equation (…). Figure 4 A) and double logarithmic equations ( Figure 4 B) Drawing Figure 4 The statistical results are shown in Table 1. The Kq values ​​of caffeic acid and hydrolyzed casein are greater than the maximum diffusion collision quenching constant of 2.0 × 10⁻⁶. 10 The L / (mol·s) value indicates that the quenching mechanism is static quenching. The number of binding sites (n) is approximately 1, indicating that caffeic acid and hydrolyzed casein can form a complex in a 1:1 molar ratio. The Ka value increases with increasing temperature, indicating that the reaction is endothermic, but the overall result shows an exothermic reaction, with the exothermic reaction dominating. This may be related to the high content and specific structure of the individual phenolic compounds that may induce the exothermic reaction. Thermodynamic parameters can be used to determine the binding properties; ΔH<0 and ΔS<0 for the caffeic acid and hydrolyzed casein complex indicate that the main forces are hydrogen bonds and van der Waals forces.

[0032] like Figure 5 As shown in Figure A, after in vitro simulated gastrointestinal digestion, the polyphenol content of the caffeic acid-hydrolyzed casein complex group was significantly higher than that of the caffeic acid alone group. Simultaneously, the total antioxidant capacity of the complex group was significantly higher during the initial digestion stage, gastric digestion, and intestinal digestion. Figure 5 B) Significantly higher than the caffeic acid alone group and the hydrolyzed casein group. This indicates that the combination of caffeic acid and hydrolyzed casein significantly improves the stability of caffeic acid during in vitro digestion and increases the antioxidant capacity of the complex after gastrointestinal digestion.

[0033] Table 1 4.2 Construction and characterization of coumaric acid-hydrolyzed casein like Figure 6 As shown, hydrolyzed casein was subjected to different temperatures: 25℃ ( Figure 6 A) 35℃ Figure 6 B) and 45℃ Figure 6 C) exhibits the strongest fluorescence absorption at 300 nm. As the concentration of p-coumaric acid increases, the fluorescence intensity gradually decreases, demonstrating that p-coumaric acid and hydrolyzed casein can recombine. According to the Stern-Volmer equation (… Figure 7 A) and double logarithmic equations ( Figure 7 B) The graph was plotted, and the statistical results are shown in Table 2. The Kq values ​​for coumaric acid and hydrolyzed casein are greater than the maximum diffusion collisional quenching constant of 2.0 × 10⁻⁶. 10 The L / (mol·s) value indicates that the quenching mechanism is static quenching. The number of binding sites (n) is approximately 1, indicating that coumaric acid and hydrolyzed casein can form a complex in a 1:1 molar ratio. The Ka value decreases with increasing temperature, indicating that the reaction is exothermic. The ΔH < 0 and ΔS < 0 values ​​for the coumaric acid and hydrolyzed casein complex indicate that the main forces are hydrogen bonds and van der Waals forces.

[0034] Table 2 like Figure 8 As shown, after in vitro simulated gastrointestinal digestion, the polyphenol content of the coumaric acid-hydrolyzed casein complex group was significantly higher than that of the coumaric acid-only group. Figure 8 A), while the total antioxidant capacity of the complex group during the initial digestion stage, gastric digestion, and intestinal digestion stage (A), Figure 8 B) Significantly higher than the groups containing only p-coumaric acid and hydrolyzed casein. This indicates that the combination of p-coumaric acid and hydrolyzed casein significantly improves the stability of p-coumaric acid during in vitro digestion and increases the antioxidant capacity of the complex after gastrointestinal digestion.

[0035] Example 3: Verification of the digestive-promoting function of phenolic acid-hydrolyzed casein 1. Animal experiment grouping and dosage design Sixty healthy, 6-week-old male SPF-grade C57BL / 6J mice, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., were randomly divided into 10 groups, including a blank control group, a model group, a caffeic acid group, a p-coumaric acid group, a hydrolyzed casein group, a caffeic acid-hydrolyzed casein complex group, a p-coumaric acid-hydrolyzed casein complex group, a gallic acid-hydrolyzed casein complex group, a syringic acid-hydrolyzed casein complex group, and a positive control group.

[0036] After 7 days of adaptation, mice were administered loperamide hydrochloride by gavage to establish a model. The loperamide dosage for modeling was 10 mg / kg / day, while the blank control group was administered an equal volume of PBS by gavage. Simultaneously, each example group underwent intervention, with the complex administered daily by gavage at a dose of 1000 mg / kg (calculated at a 1:1 molar ratio; the mass ratio of hydrolyzed casein to phenol was 10:1; mass spectrometry analysis of hydrolyzed casein showed a molecular weight of approximately 1600, while the molecular weight of phenol was smaller, approximately 160; the mass ratio was approximately 10:1 at the same molar ratio; based on the molar ratio of phenolic acid to hydrolyzed casein, each 1000 mg of hydrolyzed casein contained 100 mg of phenolic acid). The blank control and model groups were administered an equal volume of PBS by gavage, while the positive control group was administered 10 mg / kg of mosapride solution by gavage. Modeling and intervention were performed concurrently and continuously by gavage for 14 days.

[0037] 2. Detection indicators 2.1 Fourteen days after gavage, a charcoal propulsion experiment was conducted to record the time of the first black stool in mice. 2.2 Determination of gastrointestinal propulsion rate: Before the experiment, mice were fed but not watered for 16 hours. On the day of the experiment, each group was given 0.4 mL of carbon powder indicator by gavage. The mice were sacrificed 30 minutes later, and the gastrointestinal propulsion rate of the mice was measured.

[0038] Gastrointestinal propulsion rate = (Length of carbon powder indicator propelled (cm) / Total small intestine length (cm)) × 100% 2. Results like Figure 9 As shown in Figure A, compared with the blank control group, the defecation time of the model group was significantly increased, indicating that loperamide modeling caused indigestion in mice. The intervention of each complex could shorten the defecation time of mice and reach the level of positive drugs, indicating that the phenolic acid-hydrolyzed casein complex has a certain digestive function, among which caffeic acid-hydrolyzed casein and p-coumaric acid-hydrolyzed casein have the best digestive effect.

[0039] like Figure 9 As shown in Figure B, compared with the blank control group, the gastrointestinal propulsion rate of the model group was significantly decreased. Caffeic acid-hydrolyzed casein and p-coumaric acid-hydrolyzed casein significantly improved the gastrointestinal propulsion rate of mice, reaching the level of positive control drugs, indicating good digestive promotion function. While gallic acid-hydrolyzed casein complex and syringic acid-hydrolyzed casein complex interventions also improved the gastrointestinal propulsion rate of mice, there was no significant difference compared with the model group, indicating limited digestive promotion function. In conclusion, caffeic acid-hydrolyzed casein and p-coumaric acid-hydrolyzed casein showed the best digestive promotion effects.

[0040] The two groups with the best digestive effects were subjected to complex synergistic analysis, such as... Figure 10As shown in AB, the defecation time in the caffeic acid-hydrolyzed casein and coumaric acid-hydrolyzed casein intervention groups was significantly shorter than that in the hydrolyzed casein and phenolic acid groups alone. Figure 11 As shown in Figures AB, the gastrointestinal propulsion rates of the complex intervention groups were significantly higher than those of the hydrolyzed casein and phenolic acid groups alone. Specifically, the gastrointestinal propulsion rate of the caffeic acid-hydrolyzed casein group was approximately 39% higher than that of the caffeic acid group and approximately 34% higher than that of the hydrolyzed casein group. Furthermore, the gastrointestinal propulsion rate of the p-coumaric acid-hydrolyzed casein group was approximately 21% higher than that of the p-coumaric acid group and approximately 19% higher than that of the hydrolyzed casein group. This indicates that the digestive-promoting ability of the combination of phenolic acids and hydrolyzed casein is superior to that of either substance alone.

[0041] The synergistic effect of the two combined drug combinations was calculated using the Loewe equivalent line method. The calculation method is as follows: Plot the single drug doses DA and DB at any effect point on the X and Y axes of the coordinate system. Connecting the two points yields the linear equation: x / DA + x / DB = 1. When the two doses dA and dB are used together, the synergistic index CI = dA / DA + dB / DB is used to achieve the effect of DA or DB alone. When CI < 1, it is a synergistic effect; CI = 1, it is an additive effect; and CI > 1, it is an antagonistic effect.

[0042] The calculation results are shown in the table below. The CI values ​​of the caffeic acid + hydrolyzed casein (Tables 3 and 5) and p-coumaric acid + hydrolyzed casein (Tables 4 and 6) combinations are all less than 1, indicating that they are synergistic effects.

[0043] Table 3. Calculation of the synergistic effect of caffeic acid-hydrolyzed casein on defecation time. caffeic acid 96.8 100 82.13 / Hydrolyzed casein 102.3 1000 777.13 / Caffeic acid-hydrolyzed casein 79.5 / / 0.78 Table 4. Calculation of the synergistic effect of coumaric acid-hydrolyzed casein on defecation time. p-coumaric acid 98.4 100 77.92 / Hydrolyzed casein 102.3 1000 749.46 / p-Coumaric acid-hydrolyzed casein 76.67 / / 0.75 Table 5. Calculation of the synergistic effect of caffeic acid-hydrolyzed casein on gastrointestinal propulsion rate caffeic acid 60.37 100 72.19 / Hydrolyzed casein 62.5 1000 747.34 / Caffeic acid-hydrolyzed casein 83.63 / / 0.75 Table 6. Calculation of the synergistic effect of coumaric acid-hydrolyzed casein on gastrointestinal propulsion rate p-coumaric acid 61.33 100 82.33 / Hydrolyzed casein 62.50 1000 823.33 / p-Coumaric acid-hydrolyzed casein 74.49 / / 0.82 The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A phenolic acid-hydrolyzed casein complex, characterized in that, The complex is formed by the non-covalent bonding of phenolic acid and hydrolyzed casein, wherein the hydrolyzed casein is a component with a molecular weight of less than 3 kDa obtained by protease hydrolysis and ultrafiltration of casein; the protease is an alkaline protease; and the phenolic acid is selected from caffeic acid and / or p-coumaric acid. The molar ratio of phenolic acid to hydrolyzed casein is 1~3:

1.

2. The method for preparing the phenolic acid-hydrolyzed casein complex according to claim 1, characterized in that, Includes the following steps: Casein is hydrolyzed to obtain hydrolyzed casein, which is then mixed with a phenolic acid solution.

3. The preparation method according to claim 2, characterized in that, The preparation of the hydrolyzed casein includes: dispersing casein in water, adding protease for hydrolysis, inactivating the enzyme after hydrolysis, centrifuging and filtering, and then collecting the permeate using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain a hydrolyzed casein component with a molecular weight of less than 3 kDa.

4. The preparation method according to claim 3, characterized in that, The protease is an alkaline protease with an enzyme-to-substrate ratio of 1:100 w / w. The hydrolysis time is 2-5 hours, and the optimal pH is adjusted according to the enzyme properties. The enzyme is inactivated by heating after hydrolysis.

5. The method according to claim 4, characterized in that, The concentration of the hydrolyzed casein is 0.3~0.5 mg / ml, the concentration of the phenolic acid solution is adjusted according to the molar ratio, and the pH value is adjusted using HCl and NaOH.

6. The use of the phenolic acid-hydrolyzed casein complex of claim 1 in the preparation of a drug for improving indigestion.

7. The application according to claim 6, characterized in that, The improvement of indigestion includes promoting gastrointestinal motility, shortening defecation time, and increasing gastrointestinal propulsion rate.

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