Preparation method and application of chlorella pyrenoidosa polypeptide-polyphenol composite antioxidant

Abstract

The invention discloses a preparation method and application of a chlorella pyrenoidosa polypeptide-polyphenol composite antioxidant. The preparation method is characterized by comprising the following steps: a) extracting protein from chlorella pyrenoidosa by using an alkali-solution and acid-isolation method; b) performing enzymolysis on the protein obtained in the step a) by using alkaline protease to obtain polypeptide; and c) adding polyphenol into the polypeptide, and mixing to obtain the composite antioxidant with higher antioxidant activity. Wherein the final concentration of the chlorella pyrenoidosa polypeptide is 1mg / mL, and the final concentration of the gallic acid is 1, 2, 3, 4 and 5 mu g / mL; the final concentration of the chlorogenic acid is 5, 10, 15, 20, 25, 30 and 35 mu g / mL; the final concentration of EGCG is 2, 4, 6, 8, 10 [mu] g / mL. The invention is beneficial to the development and application of the chlorella pyrenoidosa polypeptide-polyphenol composite antioxidant in the fields of food, medicines and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method and application of a polypeptide-polyphenol complex antioxidant, in particular to a preparation method and application of a Chlorella pyrenoidosa polypeptide-polyphenol complex antioxidant. BACKGROUND

[0002] In recent years, food-derived antioxidant peptides have attracted much attention from researchers due to their safety and wide sources. These antioxidant peptides are mainly derived from plant proteins, animal proteins, and microbial proteins. Chlorella pyrenoidosa is a new type of protein source with great development potential. It has a fast growth rate, high protein content (more than 50% of dry weight), and is certified as a safe food raw material. Antioxidant peptides are contained in Chlorella pyrenoidosa protease hydrolysate, and can produce higher antioxidant activity when combined with polyphenols. Therefore, the preparation of Chlorella pyrenoidosa polypeptide-polyphenol complex antioxidant is crucial for expanding the application of Chlorella pyrenoidosa as a dietary antioxidant. SUMMARY

[0003] The present application provides a preparation method and application of a Chlorella pyrenoidosa polypeptide-polyphenol complex antioxidant, which can promote the application of Chlorella pyrenoidosa polypeptide as a dietary antioxidant.

[0004] The purpose of the present application is achieved by a preparation method and application of a Chlorella pyrenoidosa polypeptide-polyphenol complex antioxidant, which comprises the following steps: a) extracting proteins from Chlorella pyrenoidosa using an alkali dissolution and acid precipitation method; b) subjecting the proteins obtained in step a) to enzymatic hydrolysis using alkaline protease to obtain polypeptides; and c) adding polyphenols to the polypeptides, and mixing to obtain a complex antioxidant with higher antioxidant activity.

[0005] In a preferred embodiment of the present application, the final concentration of Chlorella pyrenoidosa polypeptide is 1 mg / mL, the final concentration of gallic acid is 1, 2, 3, 4, 5 μg / mL, the final concentration of chlorogenic acid is 5, 10, 15, 20, 25, 30, 35 μg / mL, and the final concentration of EGCG is 2, 4, 6, 8, 10 μg / mL. The Chlorella pyrenoidosa polypeptide is mixed with the polyphenol solution, the pH is adjusted to 7, and the mixture is incubated in a 100 r / min shaker for 2 h in the dark to obtain a polypeptide-polyphenol complex.

[0006] The present application has the following advantages:

[0007] The preparation method and application of a Chlorella pyrenoidosa polypeptide-polyphenol complex antioxidant of the present application use a simple enzymatic hydrolysis method to obtain polypeptides, and through a polyphenol addition method, a better antioxidant effect is achieved, which is conducive to the development and application of Chlorella pyrenoidosa polypeptide-polyphenol complex antioxidant in the fields of food and medicine. Attached image description:

[0008] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0009] Figure 1 It refers to the scavenging activity of polypeptides, gallic acid, and their complexes against ABTS free radicals.

[0010] Figure 2 It refers to the scavenging activity of peptides, chlorogenic acid, and their complexes against ABTS free radicals.

[0011] Figure 3 It refers to the scavenging activity of peptides, EGCG, and their complexes against ABTS free radicals. Detailed implementation method:

[0012] The following is a detailed description of the method of the present invention used to improve the antioxidant activity of Chlorella protein peptides, including the following steps:

[0013] a) Extraction of protein from Chlorella pulmonale using the alkali-soluble acid precipitation method: Chlorella pulmonale powder with broken cell walls was mixed with 95% ethanol at a ratio of 1:10 (g / mL) for defatting. The mixture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was retained and evaporated to dryness. The defatted Chlorella pulmonale powder was mixed with 0.3M NaCl solution at a ratio of 1:10 (g / mL), the pH was adjusted to 10 using NaOH, and the mixture was centrifuged at 5000 rpm for 20 min. The supernatant was collected, and the pH was adjusted to 4.2 using HCl. The mixture was centrifuged at 5000 rpm for 20 min to obtain the protein precipitate, which was then decolorized using 95% ethanol.

[0014] b) The protein obtained in step a) was enzymatically hydrolyzed using alkaline protease to obtain peptides: 1.945 g of protein was weighed and dissolved in 500 mL of Na₂HPO₄ (pH 7.0), the pH was adjusted to 6.5, and 1.67 mL of alkaline protease solution (7000 U / mg) was added. The mixture was hydrolyzed at 60°C for 3 h. After hydrolysis, the enzyme was inactivated in a 95°C water bath for 10 min. The hydrolysate was centrifuged at 4°C and 6000 rpm for 20 min to obtain the supernatant. The supernatant was then freeze-dried to obtain peptide powder for later use.

[0015] c) Adding polyphenols to peptides and mixing them yields a complex antioxidant with higher antioxidant activity: Equal volumes of peptides (2 mg / mL) and polyphenol solutions of different concentrations (gallic acid: 2, 4, 6, 8, 10 μg / mL, chlorogenic acid: 10, 15, 20, 25, 30, 40, 50, 60, 70 μg / mL, EGCG: 4, 8, 12, 16, 20 μg / mL) were mixed, the pH was adjusted to 7.0, and the mixture was reacted in a shaker at 100 r / min under closed conditions in the dark for 2 h to obtain a complex containing polyphenols of different concentrations.

[0016] The prepared peptide-polyphenol complex was tested for ABTS free radical scavenging activity. The results showed that the activity of the composite antioxidant was higher than that of the peptide and polyphenol alone.

[0017] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing and applying a protein-nucleated Chlorella polypeptide-polyphenol composite antioxidant, characterized in that... The process includes the following steps: a) extracting proteins from Chlorella pulvinata using an alkaline dissolution and acid precipitation method; b) enzymatically hydrolyzing the proteins obtained in step a) using an alkaline protease to obtain polypeptides; c) adding polyphenols to the polypeptides and mixing them to obtain a compound antioxidant with higher antioxidant activity.

2. The method for preparing the protein-nucleated Chlorella polypeptide-polyphenol composite antioxidant according to claim 1, characterized in that, Chlorella vulgaris was dissolved at pH 10, and the protein was precipitated at pH 4.

2. The protein was then enzymatically hydrolyzed for 3 h at pH 6.5, E / S 3%, and 60 °C using alkaline protease. The resulting polypeptide (2 mg / mL) was mixed with equal volumes of polyphenol solutions of different concentrations (gallic acid: 2, 4, 6, 8, 10 μg / mL; chlorogenic acid: 10, 15, 20, 25, 30, 40, 50, 60, 70 μg / mL; EGCG: 4, 8, 12, 16, 20 μg / mL). The pH was adjusted to 7.0, and the mixture was reacted for 2 h in a closed, light-protected environment at 100 r / min.

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