Combination of milk protein compounds for reducing astringent taste of coffee by combining with phenolic acid
By non-covalently binding with whey protein to form a stable complex, the problems of astringent taste and instability of coffee polyphenols are solved, and the effects of reducing astringent taste and improving bioavailability are achieved.
Patent Information
- Application Number
- CN202510023097.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-04
AI Technical Summary
Polyphenols in coffee, especially chlorogenic acid, contain unacceptable astringent flavors and are unstable during processing, affecting bioavailability.
By non-covalently binding with whey protein, a stable complex is formed, improving the structure and function of coffee polyphenols, thereby reducing its astringent taste and improving bioavailability.
It effectively reduces the astringent taste of coffee polyphenols and improves its bioaccessibility, providing a new way to improve the stability and taste of polyphenols.
Smart Images
Figure CN119744964A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food processing, and mainly relates to a combination of milk protein complexes capable of reducing the astringency of coffee by combining with phenolic acid. Background Art
[0002] Coffee is one of the most popular beverages in the world and is rich in polyphenols. Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in coffee and is formed by the condensation of caffeic acid carboxyl and quinic acid hydroxyl. CGA contains one carboxyl group and five active hydroxyl groups. The hydroxyl groups easily react with free radicals to generate hydrogen radicals that can scavenge hydroxyl radicals and superoxide anions, thus showing antioxidant capacity. In addition, CGA can also indirectly exert antioxidant effects by increasing the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Previous studies have shown that CGA can induce the activation of Nrf2 and heme oxygenase-1 (HO-1) and inhibit the phosphorylation of IKB and the subsequent nuclear translocation of nuclear factor κB. The two pathways have a synergistic effect, thereby alleviating the body's oxidative stress.
[0003] Chlorogenic acid, as the main phenolic compound in coffee, has an unacceptable astringency. Astringency is a complex feeling of shrinkage and dryness of the oral epithelium due to contact with astringent substances. After consumption, polyphenols interact with salivary proteins and salivary mucins, causing the oral epithelium to dry out and produce astringency. Studies have shown that adding milk to coffee can reduce its astringency. This may be because the protein in milk binds to the polyphenols in coffee, thereby reducing the chance of polyphenols binding to proteins and mucins in the saliva, thereby reducing the astringency of coffee. In addition to the obvious astringency, most polyphenols, including chlorogenic acid, are unstable to light and heat during processing and are easily oxidized to quinones under alkaline conditions, which greatly affects the bioavailability of polyphenols. Studies have shown that the combination of polyphenols and proteins can change the properties of proteins and improve the stability and bioavailability of polyphenols. Therefore, exploring the combination of protein and chlorogenic acid helps to reveal the mechanism by which milk protein reduces the astringency of coffee and improves the bioavailability of polyphenols.
[0004] Whey is a byproduct of cheese production, and whey protein accounts for 20% of the total raw milk protein. The three proteins with the highest content are β-lactoglobulin (β-Lg), α-lactalbumin (α-La) and glycomacropeptide (GMP), which account for about 60% of the total whey protein. β-Lg is a globular protein composed of 162 amino acid residues, and its secondary structure is mainly composed of β-folding and α-helix. α-La is a globular protein composed of 123 amino acid residues, which consists of α- and β-domains connected by a calcium binding loop. CMP is obtained by rennet cleaving the Phe105-Met106 bond of bovine κ-casein to obtain a hydrophilic C-terminal glycopeptide containing 64 amino acid residues (106-169), which exists in whey due to its good solubility.
[0005] The present invention improves the structure and function of the complex by non-covalently combining whey protein and coffee polyphenols. By exploring the functional properties of the complex after adding different whey proteins, the protein that most significantly reduces astringency is selected to ensure the stability of the complex while improving the astringency of polyphenols. Summary of the invention
[0006] The present invention utilizes the ability of polyphenols and proteins to change the spatial conformation of proteins by non-covalent binding, so that coffee polyphenols can be combined with different whey proteins to form a stable complex and improve the astringency of coffee polyphenols.
[0007] The present invention is achieved through the following technical solutions:
[0008] (1) Separation of whey protein: Acid whey powder was prepared into a protein solution with a concentration of 7%, 0.067 mol / L sodium citrate was added to the solution, the pH of the solution was adjusted to 3.48 with 3M citric acid, the solution was heated in a water bath at 45.4°C for 60 min, centrifuged (4000 g, 30 min), the supernatant was collected, the pH was adjusted to 6.7 with 4 mol / L NaOH, dialyzed for 48 h, and β-lactoglobulin (β-Lg) was obtained by freeze drying; the same amount of deionized water as that used to dissolve the protein was added to the precipitate, the pH was adjusted to 7.5 with 4 mol / L NaOH, the solution was heated in a water bath at 40°C for 30 min, centrifuged (4000 g, 30 min), the supernatant was collected, dialyzed for 48 h, and α-lactalbumin (α-La) was obtained by freeze drying; sweet whey powder was prepared into a protein solution with a concentration of 7.5. 5% protein solution, adjust the pH of the whey solution to 7.97 by adding NaOH, add calcium chloride to make its content in the solution 0.59g / L, heat the adjusted whey solution in a water bath at 90.78℃ for 30s, cool it quickly after the reaction, adjust the pH to 4.6 with 1mol / LHCl, centrifuge at 6000g for 15min, collect the supernatant, adjust the pH to 7.5 with 1mol / LNaOH, dialyze for 48h, and obtain casein glycomacropeptide (GMP) by freeze drying; (2) Preparation of protein polyphenol complex: The three proteins extracted in step (1) were dissolved in phosphate buffer solution (0.01M, pH7.2) to prepare a protein solution with a concentration of 10mg / mL, and after magnetic stirring at room temperature for 1h, the solution was completely hydrated in a refrigerator at 4℃ overnight. Chlorogenic acid (CGA) was added to the protein solution at a mass ratio of 10:0.25, 10:0.5, 10:1, 10:1, and 10:4, respectively, and mixed evenly. The complex was dialyzed in a dialysis bag (3 kDa) for 48 hours and then freeze-dried for further analysis. The protein sample without polyphenols was treated in the same way as a control; (3) the polyphenol binding rate of the complex in step (2) was determined by high performance liquid chromatography; (4) the Fourier transform infrared spectrum of the complex in step (2) was determined by potassium bromide tablet method; (5) simulated gastrointestinal digestion: the complex in step (2) was digested in vitro; 10 mL of a sample with a concentration of 10 mg / mL was taken, the sample was mixed with an equal volume of gastric juice, the pH was adjusted to 2.0, and digested in a 37°C incubator for 1 hour. After the digestion was completed, the pH was immediately adjusted to 7.0 to terminate the reaction; the gastric digestate and simulated intestinal juice were mixed in equal volumes, the pH was adjusted to 7.0, the temperature was 37°C, and after 2 hours of digestion, the pH was adjusted to 4-5 to inactivate the enzyme; the digested samples were collected for liquid chromatography analysis to determine the CGA content during the digestion process; (6) 70 mL of different sample solutions were taken, respectively, and placed in two electronic tongue special cups, placed on the electronic tongue sample stage, and measured using the Sample_Measurement program.
[0009] The purity of α-La is 72.28%, the purity of β-Lg is 83.61%, and the purity of GMP is 94.32%.
[0010] The optimal binding amount of CGA with α-La is 58.18 nmol / mg, the optimal binding amount with β-Lg is 124.30 nmol / mg, and the optimal binding amount with GMP is 41.77 nmol / mg.
[0011] The infrared absorption peaks of the protein all slightly red-shift in the amide A band, the amide I band and the amide II band, indicating that the secondary structure of the complex has changed in content compared with the individual proteins.
[0012] The bioaccessibility of the CGA is increased by 29.72% after being combined with α-La, by 30.43% after being combined with β-Lg, and by 16.81% after being combined with GMP.
[0013] The astringency of the CGA was improved after the three proteins were added. The effect of reducing astringency was most obvious when a mixture of α-La, β-Lg and GMP proteins in a ratio of 1:2:1 was added to the CGA.
[0014] Compared with the existing methods, the method for improving the structure and function of polyphenol protein complex proposed in this invention has the following characteristics:
[0015] (1) The preparation method of the CGA-protein complex of the present invention is simple, economical, mild in conditions, and easy to operate.
[0016] (2) The composite prepared by the present invention effectively improves the bioaccessibility of CGA after digestion and improves the astringency of CGA, providing a new idea for improving the astringency of polyphenols. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is the technical roadmap of the present invention;
[0018] Figure 2 is the polyphenol binding rate of the complex;
[0019] Figure 3 is the Fourier transform infrared spectrum of the complex;
[0020] Figure 4 is the bioaccessibility of the sample after digestion;
[0021] Figure 5 This is the electronic tongue result diagram of the sample. DETAILED DESCRIPTION
[0022] The specific embodiments are further described below in conjunction with the accompanying drawings.
[0023] A combination of milk protein complexes for reducing the astringency of coffee by combining with phenolic acid, characterized in that the method comprises the following steps: (1) separating whey protein: preparing acid whey powder into a protein solution with a concentration of 7%, adding 0.067 mol / L sodium citrate to the solution, adjusting the pH of the solution to 3.48 with 3M citric acid, heating the solution in a water bath at 45.4°C for 60 minutes and then centrifuging (4000g, 30 minutes), collecting the supernatant, adjusting the pH to 6.7 with 4 mol / L NaOH, dialyzing for 48 hours, and obtaining β-lactoglobulin (β-Lg) by freeze drying; adding an equal amount of deionized water to the precipitate when the protein was dissolved, and lysing with 4 mol / L NaOH; 1 / LNaOH was used to adjust the pH to 7.5, the solution was heated in a water bath at 40°C for 30 min and then centrifuged (4000g, 30min), the supernatant was collected, and after dialysis for 48h, α-lactalbumin (α-La) was obtained by freeze drying; the sweet whey powder was prepared into a protein solution with a concentration of 5%, the pH of the whey solution was adjusted to 7.97 by adding NaOH, calcium chloride was added to make its content in the solution 0.59g / L, the adjusted whey solution was heated in a water bath at 90.78°C for 30s, and the reaction was quickly cooled after completion, the pH was adjusted to 4.6 with 1mol / LHCl, and the solution was centrifuged at 6000g for 15min, the supernatant was collected, and the solution was purified by 1m ol / LNaOH was used to adjust the pH to 7.5. After dialysis for 48 hours, casein glycomacropeptide (GMP) was obtained by freeze drying. (2) Preparation of protein polyphenol complex: The three proteins extracted in step (1) were dissolved in phosphate buffer solution (0.01M, pH7.2) to prepare a protein solution with a concentration of 10 mg / mL. After magnetic stirring for 1 hour at room temperature, the solution was completely hydrated in a refrigerator at 4°C overnight. Chlorogenic acid (CGA) with a mass ratio of 10:0.25, 10:0.5, 10:1, 10:1, and 10:4 were added to the protein solution and mixed evenly. The complex was dialyzed in a dialysis bag (3 kDa) for 48 hours. The mixture was then freeze-dried for further analysis, and a protein sample without polyphenols was treated in the same manner as a control; (3) the polyphenol binding rate of the complex in step (2) was determined by high performance liquid chromatography; (4) the Fourier transform infrared spectrum of the complex in step (2) was determined by the potassium bromide tablet method; (5) simulated gastrointestinal digestion: the complex in step (2) was digested in vitro; 10 mL of a sample with a concentration of 10 mg / mL was taken, the sample was mixed with an equal volume of gastric juice, the pH was adjusted to 2.0, and digested in a 37°C incubator for 1 hour. After the digestion was completed, the pH was immediately adjusted to 7.0 to terminate the reaction; the gastric digestate and simulated intestinal juice were mixed in equal volumes, and the pH was adjusted to 7.0, temperature 37℃, after digestion for 2h, pH was adjusted to 4-5 to inactivate enzymes; the digested samples were collected for liquid chromatography analysis to determine the content of CGA during the digestion process; (6) 70mL of different sample solutions were taken and placed in two electronic tongue special cups respectively, placed on the electronic tongue sample table, and measured using the Sample_Measurement program.
[0024] The purity of α-La is 72.28%, the purity of β-Lg is 83.61%, and the purity of GMP is 94.32%.
[0025] The optimal binding amount of CGA with α-La is 58.18 nmol / mg, the optimal binding amount with β-Lg is 124.30 nmol / mg, and the optimal binding amount with GMP is 41.77 nmol / mg.
[0026] The infrared absorption peaks of the protein all slightly red-shift in the amide A band, the amide I band and the amide II band, indicating that the secondary structure of the complex has changed in content compared with the individual proteins.
[0027] The bioaccessibility of the CGA is increased by 29.72% after being combined with α-La, by 30.43% after being combined with β-Lg, and by 16.81% after being combined with GMP.
[0028] The astringency of the CGA was improved after the three proteins were added. The effect of reducing astringency was most obvious when a mixture of α-La, β-Lg and GMP proteins in a ratio of 1:2:1 was added to the CGA.
[0029] Example 1
[0030] (1) Preparation of protein-polyphenol non-covalent complexes: α-La was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare an α-La solution with a concentration of 10 mg / mL. After magnetic stirring at room temperature for 1 hour, the solution was completely hydrated in a refrigerator at 4°C overnight. CGA with a mass ratio of 10:0.25, 10:0.5, 10:1, 10:1, and 10:4 was added to the α-La solution and mixed evenly. The complex was dialyzed in a dialysis bag (3 kDa) for 48 hours and then freeze-dried for further analysis. The protein sample without polyphenols was treated in the same way as a control;
[0031] (2) Determination of the polyphenol binding rate of the complex by high performance liquid chromatography: The CGA standard sample was dissolved in methanol and prepared into different concentration gradients for drawing the standard curve. 0.5 mg of the freeze-dried sample was dissolved in 1 ml of methanol and the sample was passed through a 0.45 μm filter. Analyzed by Shimadzu LC-20AT. The results showed that the optimal binding amount of CGA and α-La was 58.18 nmol / mg;
[0032] (3) The Fourier transform infrared spectrum was measured by potassium bromide tablet method. The freeze-dried covalent complex was ground into powder, mixed with pre-dried potassium bromide at a ratio of 1:100, ground into powder, and pressed into tablets. The scanning conditions were set as follows: spectral range 4000-500cm -1 , scanning times 32 times, resolution 4cm -1 . Plot the graph with wavelength as the horizontal axis and absorbance as the vertical axis; α-La is at 1700-1500cm -1 There are two obvious absorption peaks in the region, at 1645.28 cm -1 and 1593.20cm of amide II band -1 There is also an obvious peak in the amide A band (3414.00 cm -1 ). After combining with CGA, the absorption peak intensity of α-La in the amide I band becomes stronger, and the absorption peaks of the amide II band and the amide A band shift;
[0033] (4) Determination of the bioaccessibility of the digested sample: Take 10 mL of a sample with a concentration of 10 mg / mL, mix the sample with an equal volume of gastric juice, adjust the pH to 2.0, and digest it in a 37°C incubator for 1 hour. After the digestion is completed, immediately adjust the pH to 7.0 to terminate the reaction; mix the gastric digest and simulated intestinal fluid in equal volumes, adjust the pH to 7.0, and the temperature to 37°C. After digestion for 2 hours, adjust the pH to 4-5 to inactivate the enzyme. Collect the digested samples for liquid chromatography analysis. The results show that the bioaccessibility is increased by 29.72% after being combined with α-La.
[0034] (5) Electronic tongue measurement results of the complex: 70 mL of each sample solution was taken and placed in two electronic tongue special cups respectively. The cups were placed on the electronic tongue sample table and measured using the Sample_Measurement program. The results showed that α-La could improve the astringency of CGA. The effect of reducing astringency was most obvious when a mixture of α-La, β-Lg and GMP protein in a ratio of 1:2:1 was added to CGA.
[0035] Example 2
[0036] (1) Preparation of protein-polyphenol non-covalent complexes: β-Lg was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare a β-Lg solution with a concentration of 10 mg / mL. After magnetic stirring at room temperature for 1 h, the solution was completely hydrated in a refrigerator at 4°C overnight. CGA with mass ratios of 10:0.25, 10:0.5, 10:1, 10:1, and 10:4 was added to the β-Lg solution and mixed evenly. The complex was dialyzed in a dialysis bag (3 kDa) for 48 hours and then freeze-dried for further analysis. The protein sample without polyphenols was treated in the same way as a control;
[0037] (2) Determination of polyphenol binding rate of the complex by high performance liquid chromatography: CGA standard sample was dissolved in methanol and prepared into different concentration gradients for drawing standard curve. 0.5 mg of freeze-dried sample was dissolved in 1 ml of methanol and the sample was passed through a 0.45 μm filter. Analyzed by Shimadzu LC-20AT. The results showed that the optimal binding amount of CGA and β-Lg was 124.30 nmol / mg;
[0038] (3) The Fourier transform infrared spectrum was measured by potassium bromide tablet method. The freeze-dried covalent complex was ground into powder, mixed with pre-dried potassium bromide at a ratio of 1:100, ground into powder, and pressed into tablets. The scanning conditions were set as follows: spectral range 4000-500cm -1 , scanning times 32 times, resolution 4cm -1 . Plot the graph with wavelength as the horizontal axis and absorbance as the vertical axis; β-Lg has an absorption peak in both the amide I band and the amide A band, which are located at 1645.28 cm -1 and 3402.43cm -1 After combining with CGA, the absorption peak of β-Lg in the amide I band red-shifted, and the peak shapes of the absorption peaks in the amide II band and the amide A band changed significantly;
[0039] (4) Determination of the bioaccessibility of the digested sample: Take 10 mL of a sample with a concentration of 10 mg / mL, mix the sample with an equal volume of gastric juice, adjust the pH to 2.0, and digest it in a 37°C incubator for 1 hour. After the digestion is completed, immediately adjust the pH to 7.0 to terminate the reaction; mix the gastric digest and simulated intestinal fluid in equal volumes, adjust the pH to 7.0, and the temperature to 37°C. After digestion for 2 hours, adjust the pH to 4-5 to inactivate the enzyme. Collect the digested samples for liquid chromatography analysis. The results show that the bioaccessibility is increased by 30.43% after complexing with β-Lg;
[0040] (5) Electronic tongue measurement results of the complex: 70 mL of each sample solution was taken and placed in two electronic tongue special cups respectively. The cups were placed on the electronic tongue sample table and measured using the Sample_Measurement program. The results showed that β-Lg could improve the astringency of CGA. The effect of reducing astringency was most obvious when a mixture of α-La, β-Lg and GMP protein in a ratio of 1:2:1 was added to CGA.
[0041] Example 3
[0042] (1) Preparation of protein-polyphenol non-covalent complexes: GMP was dissolved in phosphate buffer solution (0.01 M, pH 7.2) to prepare a GMP solution with a concentration of 10 mg / mL. After magnetic stirring at room temperature for 1 hour, the solution was completely hydrated in a refrigerator at 4°C overnight. CGA with a mass ratio of 10:0.25, 10:0.5, 10:1, 10:1, and 10:4 was added to the GMP solution and mixed evenly. The complex was dialyzed in a dialysis bag (3 kDa) for 48 hours and then freeze-dried for further analysis. The protein sample without polyphenols was treated in the same way as a control;
[0043] (2) Determination of polyphenol binding rate of the complex by high performance liquid chromatography: CGA standard sample was dissolved in methanol and prepared into different concentration gradients for drawing standard curve. 0.5 mg of freeze-dried sample was dissolved in 1 ml of methanol and the sample was passed through a 0.45 μm filter. Analyzed by Shimadzu LC-20AT. The results showed that the optimal binding amount of CGA and GMP was 41.77 nmol / mg;
[0044] (3) The Fourier transform infrared spectrum was measured by potassium bromide tablet method. The freeze-dried covalent complex was ground into powder, mixed with pre-dried potassium bromide at a ratio of 1:100, ground into powder, and pressed into tablets. The scanning conditions were set as follows: spectral range 4000-500cm -1 , scanning times 32 times, resolution 4cm -1 . Plot with wavelength as the horizontal axis and absorbance as the vertical axis; GMP is between 1700-1500cm -1 There are two obvious absorption peaks in the region, at 1637.57 cm -1 and 1593.20cm of amide II band -1 There is an obvious peak in amide A at 3412.08 cm -1 After binding with CGA, the absorption peaks of GMP in amide I and A bands changed significantly, and the absorption peak of amide II band red-shifted;
[0045] (4) Determination of the bioaccessibility of the digested sample: Take 10 mL of a sample with a concentration of 10 mg / mL, mix the sample with an equal volume of gastric juice, adjust the pH to 2.0, and digest it in a 37°C incubator for 1 hour. After the digestion is completed, immediately adjust the pH to 7.0 to terminate the reaction; mix the gastric digest and simulated intestinal fluid in equal volumes, adjust the pH to 7.0, and the temperature to 37°C. After digestion for 2 hours, adjust the pH to 4-5 to inactivate the enzyme. Collect the digested samples for liquid chromatography analysis. The results show that the bioaccessibility is increased by 16.81% after compounding with GMP;
[0046] (5) Electronic tongue measurement results of the complex: 70 mL of each sample solution was taken and placed in two electronic tongue special cups respectively. The cups were placed on the electronic tongue sample table and measured using the Sample_Measurement program. The results showed that GMP could improve the astringency of CGA. The effect of reducing astringency was most obvious when a mixture of α-La, β-Lg and GMP proteins in a ratio of 1:2:1 was added to CGA.
Claims
1. A combination of milk protein complexes for reducing coffee astringency by binding to phenolic acids, characterized in that: The method comprises the following steps: (1) separating whey protein: preparing acid whey powder into a protein solution with a concentration of 7%, adding 0.067 mol / L sodium citrate to the solution, adjusting the pH value of the solution to 3.48 with 3M citric acid, heating the solution in a water bath at 45.4°C for 60 minutes, centrifuging (4000g, 30 minutes), collecting the supernatant, adjusting the pH value to 6.7 with 4 mol / L NaOH, dialyzing for 48 hours, and obtaining beta-lactoglobulin (β-Lg) by freeze drying; adding an equal amount of deionized water to the precipitate when the protein is dissolved, adjusting the pH value to 7.5 with 4 mol / L NaOH, heating the solution in a water bath at 40°C for 30 minutes, centrifuging (4000g, 30 minutes), collecting the supernatant, dialyzing for 48 hours, and obtaining beta-lactoglobulin (β-Lg); After 48 hours of dialysis, α-lactalbumin (α-La) was obtained by freeze drying; sweet whey powder was prepared into a protein solution with a concentration of 5%, the pH of the whey solution was adjusted to 7.97 by adding NaOH, calcium chloride was added to make its content in the solution 0.59 g / L, the adjusted whey solution was heated in a water bath at 90.78°C for 30 seconds, and the reaction was quickly cooled after completion. The pH was adjusted to 4.6 with 1 mol / L HCl, centrifuged at 6000g for 15 minutes, the supernatant was collected, the pH was adjusted to 7.5 with 1 mol / L NaOH, and after 48 hours of dialysis, casein glycomacropeptide (GMP) was obtained by freeze drying; (2) Preparation of protein polyphenol complex: The three protein solutions obtained in step (1) were prepared by A protein solution with a concentration of 10 mg / mL was prepared in a phosphate buffer solution (0.01 M, pH 7.2). After magnetic stirring at room temperature for 1 hour, the solution was completely hydrated in a refrigerator at 4°C overnight. Chlorogenic acid (CGA) with a mass ratio of 10:0.25, 10:0.5, 10:1, 10:1, and 10:4 was added to the protein solution and mixed evenly. The complex was dialyzed in a dialysis bag (3 kDa) for 48 hours and then freeze-dried for further analysis. The protein sample without polyphenols was treated in the same way as a control; (3) The polyphenol binding rate of the complex in step (2) was determined by high performance liquid chromatography; (4) The Fourier transform infrared spectrum of the complex in step (2) was determined by potassium bromide tablet method; (5) Simulation Gastrointestinal digestion: digest the complex in step (2) in vitro; take 10 mL of a sample with a concentration of 10 mg / mL, mix the sample with an equal volume of gastric juice, adjust the pH to 2.0, and digest in a 37°C incubator for 1 hour. After digestion, immediately adjust the pH to 7.0 to terminate the reaction; mix equal volumes of gastric digesta and simulated intestinal fluid, adjust the pH to 7.0, and set the temperature to 37°C. After digestion for 2 hours, adjust the pH to 4-5 to inactivate the enzyme; collect the digested samples for liquid chromatography analysis to determine the CGA content during the digestion process; (6) take 70 mL of each different sample solution, put them into two electronic tongue special cups, place them on the electronic tongue sample table, and use the Sample_Measurement program for measurement.
2. A combination of milk protein complexes for reducing coffee astringency by combining with phenolic acids according to claim 1, characterized in that: The purity of α-La, the purity of β-Lg and the purity of GMP separated in step (1) are 72.28%, 83.61% and 94.32%, respectively.
3. The combination of a milk protein complex for reducing coffee astringency by combining with phenolic acid according to claim 1, characterized in that: The optimal binding amount of CGA described in step (3) with α-La is 58.18nmol / mg, the optimal binding amount with β-Lg is 124.30nmol / mg, and the optimal binding amount with GMP is 41.77nmol / mg.
4. The combination of a milk protein complex for reducing coffee astringency by combining with phenolic acid according to claim 1, characterized in that: The infrared absorption peaks of the protein described in step (4) all produce a slight red shift in the amide A band, the amide I band and the amide II band, indicating that the secondary structure of the complex has changed in content compared with the single protein.
5. The combination of a milk protein complex for reducing coffee astringency by combining with phenolic acid according to claim 1, characterized in that: The bioaccessibility of CGA in step (5) is increased by 29.72% after being combined with α-La, by 30.43% after being combined with β-Lg, and by 16.81% after being combined with GMP.
6. The combination of a milk protein complex for reducing coffee astringency by combining with phenolic acid according to claim 1, characterized in that: The astringency of the CGA described in step (6) was improved after the three proteins were added. The effect of reducing astringency was most obvious when a mixture of α-La, β-Lg and GMP proteins in a ratio of 1:2:1 was added to the CGA.