Method for adjusting flavor and digestion characteristics of peptide by utilizing casein peptide and polyphenol self-assembly

Abstract

The invention belongs to the technical field of functional food processing, and particularly relates to a method for regulating peptide flavor and digesting characteristics by utilizing casein peptide and polyphenol self-assembly. According to the research, the casein peptide with good GABA-T inhibitory activity is taken as a raw material, and ferulic acid is added to be self-assembled with the casein peptide, so that the bitter taste of the casein peptide is reduced, and the GABA-T inhibitory activity of the casein peptide is improved in an in-vitro digestion stage. Compared with a traditional zymolyte debitterizing method, the method has the advantages that the biological activity of peptide is reserved, no toxic chemical reagent is introduced or added, and the method is safer, more efficient, more environmentally friendly and more sustainable. The method can be widely applied to functional food or health care products.

Description

Technical Field

[0001] The present invention relates to the technical field of functional food processing, and specifically to a method for regulating the flavor and digestive characteristics of peptides by self-assembly of casein peptides and polyphenols. Background Art

[0002] With the evolution of the global food technology landscape, consumers' interest in functional foods has increased significantly. These foods not only have conventional nutritional value but also are beneficial to health and disease prevention. As a potential source of multifunctional foods, bioactive peptides have been favored by the industry due to their significant health benefits. Milk casein is rich in tryptophan (Trp) and proline (Pro), and can relieve anxiety and insomnia symptoms through Trp metabolism and the HPA axis, making it an excellent material for preparing anti-anxiety peptides. However, the functional peptides produced by casein hydrolysis exhibit strong bitterness, which is difficult for consumers to accept and affects the application of casein hydrolysates.

[0003] Currently, there have been numerous studies on the debittering methods of enzyme hydrolysates at home and abroad, such as separation methods, masking methods, and exopeptidase digestion methods, etc. However, many of these methods will result in the loss of the bioactive sites of peptides, so they are not suitable for functional food applications. In addition, peptides will face chemical degradation during gastrointestinal digestion, resulting in reduced bioactivity and limited application. Therefore, it is necessary to develop methods for masking the bitterness of peptides and improving the bioactivity of peptides. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for regulating the flavor and digestive characteristics of peptides by self-assembly of casein peptides and polyphenols. In this study, casein peptides with good GABA-T inhibitory activity were used as raw materials, and ferulic acid was added to self-assemble with them to reduce the bitterness of casein peptides and ensure the improvement of the GABA-T inhibitory activity of casein peptides during the in vitro digestion stage.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] (1) A method for regulating the flavor and digestive characteristics of peptides by self-assembly of casein peptides and polyphenols, the specific steps are as follows: (1) Hydrolyze casein with alkaline protease. After hydrolysis, heat the enzyme solution to inactivate the enzyme, and then cool it to room temperature; centrifuge to take the supernatant and lyophilize it into powder for subsequent experiments; (2) Uniformly mix the lyophilized casein peptides in step (1) with ferulic acid, and the concentration of ferulic acid is 0.3, 0.6, 0.9, 1.2 mM, and then dialyze to obtain a complex; (3) Use an electronic tongue for analysis to measure the bitterness intensity of casein peptides and the complex; (4) During the simulated gastrointestinal tract stage, measure the GABA-T inhibitory activity of casein peptides and the complex.

[0007] In step (1), the protease for hydrolyzing casein is alkaline protease;

[0008] In step (1), the casein concentration is 5%, the hydrolysis time is 150 min, and the enzyme-substrate ratio is 4800 U / g.

[0009] In step (2), the casein peptide concentration is 40 mg / mL.

[0010] In step (2), the optimal concentration of ferulic acid is 1.2 mM.

[0011] In step (2), the dialysis time is 48 h.

[0012] In step (4), the GABA-T detection method is as follows: Disconnect the spine of the mouse to put it to death, immediately take the brain and store it in liquid nitrogen until the test determination; grind the brain tissue into a brain homogenate in a buffer solution composed of 20% glycerol, 0.10 mmol / L glutathione, 0.10 mmol / L PLP, 1 mmol / L disodium EDTA, 0.13% Triton-X100, 0.01 mmol / L K 2 HPO 4 , and pH 6.8, centrifuge at 10000 g at 4°C for 20 min, and collect the supernatant. Add 180 μL of incubation medium (100 mmol / L potassium pyrophosphate, 5 mmol / L α-ketoglutaric acid, 4 mmol / L nicotinamide adenine dinucleotide (NAD), 3.50 mmol / L 2-mercaptoethanol, 0.01 mmol / L pyridoxal 5-phosphate (PLP), pH 8.6), 20 μL of tissue homogenate and 10 μL of sample solution to each well of a 96-well plate; pre-incubate the plate at 37°C for 15 min, then add 50 mL of 115 mmol / L GABA to initiate the reaction, and measure the production of NADH at 340 nm at 37°C for 15 min using an enzyme-labeled instrument; repeat all tests three times; calculate according to the following formula:

[0013]

[0014] T is the test (incubation solution, GABA, homogenate and sample), T0 is the test blank (water, homogenate and sample), A is the control (incubation solution, homogenate, water, GABA), and A0 is the control blank (water, brain homogenate).

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. The present invention uses self-assembly to process casein peptides, which can simultaneously achieve the effects of improving the in vitro digestion GABA-T inhibitory activity and reducing its bitterness intensity, providing a new idea.

[0017] 2. The method of the present invention does not introduce or add any toxic chemical reagents. It only utilizes the self-assembly of ferulic acid and casein peptide to form a complex. The process is safe, efficient, green, environmentally friendly and sustainable. The obtained product can be directly applied to foods or health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the process flow chart of the present invention;

[0019] Figure 2 is the turbidity diagram of casein peptide and the complex;

[0020] Figure 3 is the particle size and PDI diagram of casein peptide and the complex;

[0021] Figure 4 is the surface hydrophobicity diagram of casein peptide and the complex;

[0022] Figure 5 is the electronic tongue diagram of casein peptide and the complex;

[0023] Figure 6 is the in vitro digestion GABA-T inhibitory activity diagram of casein peptide and the complex. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The following further describes the specific embodiments in conjunction with the drawings.

[0025] A method for regulating the flavor and digestion characteristics of peptides by self-assembly of casein peptide and polyphenol, characterized in that the method comprises the following steps:

[0026] Example 1

[0027] (1) The casein concentration is 5%, the enzyme-substrate ratio is 4800 U / g, alkaline protease is selected, and casein is hydrolyzed for 150 min. After hydrolysis, the enzyme is inactivated in a water bath at 100 °C for 10 min. After inactivation, it is cooled to room temperature, the pH is adjusted to 4.6 with glacial acetic acid, and then centrifuged at 8000 g for 10 min. After centrifugation, the supernatant is taken and freeze-dried into powder for subsequent experiments;

[0028] (2) Using the method of controlling variables, the sample prepared in step (1) is formulated into 40 mg / mL, 0.3 mM of ferulic acid is added thereto and uniformly mixed to form a complex, and the complex is dialyzed for 48 h to remove free ferulic acid;

[0029] (3) The complex prepared in step (2) is diluted 10 times, and the turbidity is measured. As Figure 2 shown, the turbidity of the complex prepared with 0.3 mM of ferulic acid added is 0.18, and the turbidity is reduced by 42.7% compared with the casein peptide without added ferulic acid;

[0030] (4) Dilute the complex prepared in step (2) 80 times and measure the particle size and PDI. As Figure 3 shown, the particle size and PDI of the complex prepared with 0.3% ferulic acid addition are 823.3 nm and 0.53 respectively, which are significantly lower than those of the casein peptide without ferulic acid addition;

[0031] (5) Dilute the complex prepared in step (2) 80 times and measure the surface hydrophobicity. As Figure 4 shown, the surface hydrophobicity of the complex prepared with 0.3% ferulic acid addition is 12.34, which is significantly lower than that of the casein peptide without ferulic acid addition. This indicates that when ferulic acid is added, the microenvironment of hydrophobic amino acids in casein peptide changes.

[0032] Example 2

[0033] (1) The casein concentration is 5%, the enzyme-substrate ratio is 4800 U / g, select alkaline protease, hydrolyze casein for 150 min. After hydrolysis, inactivate the enzyme in a water bath at 100 °C for 10 min. After inactivating the enzyme, cool to room temperature, adjust the pH to 4.6 with glacial acetic acid, then centrifuge at 8000 g for 10 min. After centrifugation, take the supernatant and freeze-dry it into powder for subsequent experiments;

[0034] (2) Using the method of controlling variables, prepare the sample prepared in step (1) into 40 mg / mL, add 0.6 mM ferulic acid to it and mix evenly to form a complex, and dialyze the complex for 48 h to remove free ferulic acid;

[0035] (3) Dilute the complex prepared in step (2) 10 times and measure the turbidity. As Figure 2 shown, the turbidity of the complex prepared with 0.6 mM ferulic acid addition is 0.19, which is 41.2% lower than that of the casein peptide without ferulic acid addition;

[0036] (4) Dilute the complex prepared in step (2) 80 times and measure the particle size and PDI. As Figure 3 shown, the particle size and PDI of the complex prepared with 0.6 mM ferulic acid addition are 591.2 nm and 0.44 respectively, which are significantly lower than those of the casein peptide without ferulic acid addition;

[0037] (5) Dilute the complex prepared in step (2) 80 times and measure the surface hydrophobicity. As Figure 4 shown, the surface hydrophobicity of the complex prepared with 0.6 mM ferulic acid addition is 12.34, which is significantly lower than that of the casein peptide without ferulic acid addition.

[0038] Example 3

[0039] (1) With a casein concentration of 5%, an enzyme-substrate ratio of 4800 U / g, select alkaline protease, hydrolyze casein for 150 min. After hydrolysis, inactivate the enzyme in a water bath at 100 °C for 10 min. After inactivating the enzyme, cool to room temperature, adjust the pH to 4.6 with glacial acetic acid, then centrifuge at 8000 g for 10 min. After centrifugation, take the supernatant and lyophilize it into powder for subsequent experiments;

[0040] (2) Using the method of controlling variables, prepare the sample prepared in step (1) into 40 mg / mL, add 0.9 mM of ferulic acid to it and mix evenly to form a complex, and dialyze the complex for 48 h to remove free ferulic acid;

[0041] (3) Dilute the complex prepared in step (2) by 10 times and measure the turbidity. As Figure 2 shown, the turbidity of the complex prepared with 0.9 mM of ferulic acid added is 0.18, which is 42.7% lower than that of the casein peptide without added ferulic acid;

[0042] (4) Dilute the complex prepared in step (2) by 80 times and measure the particle size and PDI. As Figure 3 shown, the particle size and PDI of the complex prepared with 0.9 mM of ferulic acid added are 545.67 nm and 0.356 respectively, which are significantly lower than those of the casein peptide without added ferulic acid;

[0043] (5) Dilute the complex prepared in step (2) by 80 times and measure the surface hydrophobicity. As Figure 4 shown, the surface hydrophobicity of the complex prepared with 0.9 mM of ferulic acid added is 11.29, which is significantly lower than that of the casein peptide without added ferulic acid.

[0044] Example 4

[0045] (1) With a casein concentration of 5%, an enzyme-substrate ratio of 4800 U / g, select alkaline protease, hydrolyze casein for 150 min. After hydrolysis, inactivate the enzyme in a water bath at 100 °C for 10 min. After inactivating the enzyme, cool to room temperature, adjust the pH to 4.6 with glacial acetic acid, then centrifuge at 8000 g for 10 min. After centrifugation, take the supernatant and lyophilize it into powder for subsequent experiments;

[0046] (2) Using the method of controlling variables, prepare the sample prepared in step (1) into 40 mg / mL, add 1.2 mM of ferulic acid to it and mix evenly to form a complex, and dialyze the complex for 48 h to remove free ferulic acid;

[0047] (3) Dilute the complex prepared in step (2) by 10 times and measure the turbidity. As Figure 2As shown, the turbidity of the complex prepared with 1.2 mM of ferulic acid added was 0.17, a 57.1% reduction in turbidity compared to the casein peptide without ferulic acid added;

[0048] (4) Dilute the complex prepared in step (2) by 80 times and measure the particle size and PDI. As Figure 3 shown, the particle size and PDI of the complex prepared with 1.2 mM of ferulic acid added were 542.1 nm and 0.23 respectively, showing a significant decrease in particle size and PDI compared to the casein peptide without ferulic acid added;

[0049] (5) Dilute the complex prepared in step (2) by 80 times and measure the surface hydrophobicity. As Figure 4 shown, the surface hydrophobicity of the complex prepared with 1.2 mM of ferulic acid added was 10.34, showing a significant decrease in surface hydrophobicity compared to the casein peptide without ferulic acid added;

[0050] (6) Measure the bitterness intensity of the complex prepared in step (2). As Figure 5 shown, when the ferulic acid addition amount was 1.2 mM, the bitterness intensity decreased by 30.2% compared to the casein peptide without ferulic acid added, indicating that adding ferulic acid can inhibit the bitterness of casein peptide;

[0051] (7) Measure the in vitro digestion GABA-T inhibitory activity of the complex prepared in step (2). As Figure 6 shown, when the ferulic acid addition amount was 1.2 mM, compared to the casein peptide without ferulic acid added, the GABA-T inhibition rate at the end of the intestine increased significantly, indicating that after adding ferulic acid, the digestion GABA-T inhibitory activity of casein peptide can be improved.

Claims

1. A method for regulating peptide flavor and digestion properties by self-assembly of casein peptides and polyphenols, characterized in that: The following steps are involved: (1) hydrolyzing casein with alkaline protease, heating the hydrolyzate to inactivate the enzyme, and then cooling it to room temperature; centrifuging to obtain the supernatant, freeze-drying it into powder, and using it in subsequent experiments; (2) uniformly mixing the freeze-dried casein peptides in step (1) with ferulic acid, wherein the concentration of ferulic acid is 0.3, 0.6, 0.9, and 1.2 mM, and then dialyzing to obtain a complex; (3) using an electronic tongue to analyze and determine the bitterness intensity of the casein peptides and the complex; and (4) determining the GABA-T inhibitory activity of the casein peptides and the complex in a simulated gastrointestinal stage.

2. The hydrolysis method according to claim 1, characterized in that In the above (1), the enzyme is alkaline protease.

3. The hydrolysis method according to claim 1, characterized in that In the above (1), the casein concentration is 5% and the enzyme-substrate ratio is 4800 U / g.

4. The method for preparing the composite according to claim 1, characterized in that In the above (2), the casein peptide concentration is 40 mg / mL.

5. The method for preparing the composite according to claim 1, characterized in that In the above (2): the optimal concentration of ferulic acid is 1.2 mM.

6. The method for preparing the composite according to claim 1, characterized in that In the (2) above, the dialysis time is 48 hours.

7. The GABA-T detection method according to claim 1, characterized in that In the above (4): the mice were killed by spinal fracture, and the brains were immediately taken out and stored in liquid nitrogen until the test was measured; the brain tissue was ground into a buffer solution consisting of 20% glycerol, 0.10mmol / L glutathione, 0.10mmol / L PLP, 1mmol / L disodium EDTA, 0.13% Triton-X100, 0.01mmol / L K2HPO4, pH 6.8 to prepare a brain homogenate, and the mixture was centrifuged at 10000×g for 20min at 4°C to collect the supernatant; 180μL of incubation medium (100mmol / L potassium pyrophosphate, 5mmol / L α-ketoglutaric acid, 4mmol / L nicotinamide adenine dinucleotide (NAD), 3.50mmol / L 2-mercaptoethanol, 0.01mmol / L pyridoxal 5-phosphate (PLP), pH 8.6), 20μL tissue homogenate and 10μL sample solution; the plate was pre-incubated at 37℃ for 15min, then 50μL 115mmol / L GABA was added to start the reaction, and the NADH production within 15min at 37℃ 340nm was measured by microplate reader; all experiments were repeated three times; calculated according to the following formula: T is the test (incubation solution, GABA, homogenate and sample), T0 is the test blank (water, homogenate and sample), A is the control (incubation solution, homogenate, water, GABA), and A0 is the control blank (water, homogenate).

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