Method for improving in-vitro antioxidant activity of pomegranate peel polyphenol based on microbial fermentation

Abstract

The invention discloses a method for improving in-vitro antioxidant activity of pomegranate peel polyphenol by utilizing microbial fermentation, and relates to the technical field of microbial fermentation. Pomegranate rind used in the invention is commercially available dry pomegranate rind. The microorganism used in the invention is commercially available saccharomyces cerevisiae, and the saccharomyces cerevisiae can decompose macromolecular substances in the pomegranate rind into small molecules, so that the extraction amount of polyphenol in the pomegranate rind is increased, and the antioxidant activity is further improved. The content of polyphenol and flavone and the in-vitro antioxidant activity in the fermentation process are measured, and the result shows that the maximum amount of polyphenol after fermentation is increased by 6.53% compared with that of non-fermentation, the content of flavone is increased by 13.38% compared with that of non-fermentation, and the antioxidant activity of DPPH is increased by 3.99%.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a method for fermenting pomegranate peel by utilizing a specific microbial strain to increase the polyphenol flavonoid content and in vitro antioxidant activity of the pomegranate peel. Background Art

[0002] Pomegranate peel is rich in components such as cellulose, pectin, and tannins, which limit the extraction efficiency of polyphenols and flavonoids. Microbial fermentation is a versatile and valuable biotechnology method. By utilizing enzymes produced by microorganisms, it can effectively degrade cellulose, pectin, and tannins, breaking down complex compounds into simpler and more easily absorbed forms, thereby increasing the extraction yield of polyphenols and flavonoids and enhancing their in vitro antioxidant activity. Furthermore, through microbial fermentation of pomegranate peel, waste materials such as pomegranate peel can be converted into high-value-added products, achieving resource recycling and having good environmental and economic benefits. Summary of the Invention

[0003] The present invention provides a method for enhancing the in vitro antioxidant activity of pomegranate peel polyphenols by using microbial fermentation. Saccharomyces cerevisiae is applied to the fermentation of pomegranate peel to increase the content of polyphenols and flavonoids in the pomegranate peel and their antioxidant activity.

[0004] To achieve the above object, the present invention provides the following scheme for fermenting pomegranate peel:

[0005] (1) Selecting Saccharomyces cerevisiae as the fermentation strain;

[0006] (2) Liquid fermentation of pomegranate peel was performed, and samples were taken every 12 h to determine the changes in polyphenols, flavonoids, and in vitro antioxidant activity during the fermentation process;

[0007] After yeast fermentation of pomegranate peel, it was found that the content of polyphenols and flavonoids increased to a certain extent, and the in vitro antioxidant activity also increased to a certain extent, indicating that yeast fermentation is beneficial to increasing the extraction amount of pomegranate peel polyphenols.

[0008] The present invention discloses the following technical effects:

[0009] The present invention ferments pomegranate peel with Saccharomyces cerevisiae, using the pomegranate peel as the main carbon and nitrogen source for yeast growth. Determination of polyphenol and flavonoid content reveals that both increase during the fermentation process, and that DPPH antioxidant activity also increases, indicating that microbial fermentation can effectively extract pomegranate peel polyphenols and enhance antioxidant activity. This method is simple, effectively reduces extraction costs, is suitable for large-scale industrial production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The changes in polyphenol content during the fermentation process.

[0011] Figure 2 The changes of flavonoid content during the fermentation process.

[0012] Figure 3 The changes in DPPH antioxidant activity during fermentation. DETAILED DESCRIPTION

[0013] The following are specific embodiments of the present invention. Methods not specifically described in the present invention are all conventional methods in the art.

[0014] Example 1: Fermentation of pomegranate peel

[0015] 1. Fermentation method: 4 g of pomegranate peel powder was added to 80 mL of saline solution (7.65 g / L NaNO3, 3.04 g / L KH2PO4, 1.52 g / L MgSO4, 1.52 g / L KCl, 2 g / L ammonium citrate, 20 g / L glucose), sterilized at 80°C for 10 min, then rapidly cooled, inoculated with the strain at an inoculum size of 3% (OD 600 =1.2) to explore the effect of fermentation time (0-72h) on fermentation results. The reaction was carried out at 28°C, and 5 mL of samples were taken every 12 hours. The samples were sterilized at 90°C for 10 minutes, centrifuged at 4000 rpm for 20 minutes, and the supernatant was diluted to 10 mL with distilled water and stored at 4°C until use.

[0016] 2. Fermentation strain: Saccharomyces cerevisiae, purchased from Angel Yeast Co., Ltd.

[0017] 3. Determine the polyphenol and flavonoid content and DPPH antioxidant activity in the fermentation supernatant.

[0018] 4. Determine polyphenol content using the folin-phenol method. Dilute 40 μL of sample to 2.4 mL with water, then add 200 μL of folin-phenol. Within 1-8 minutes, add 600 μL of 20% sodium carbonate and adjust the volume to 4 mL with 800 μL of water. Incubate at room temperature (22 ± 2°C) in the dark for 2 hours, then measure absorbance at 760 nm. Draw a standard curve using gallic acid (0-1600 μg / mL) as the standard. Polyphenol content is expressed as mg GAE (Gallic Acid Equivalent) / g DW (Dry Weight).

[0019] 5. Flavonoid Content Determination: Add 150 μL of 5% NaNO₂ to 2.5 mL of sample and let stand for 5 minutes. Then, add 1.5 mL of 10% Al(NO₃)₃ and let stand for 6 minutes. Then, add 2 mL of 4% NaOH and 200 μL of 70% ethanol and let stand for 10 minutes. Measure absorbance at 510 nm. A calibration curve is drawn using rutin (0-0.4 mg / mL) as the standard. Flavonoid content is expressed as mg RTE / g DW (RTE is Rutin Equivalent).

[0020] 6. DPPH Free Radical Scavenging Activity Assay: Prepare a 0.65 mM DPPH methanol solution and adjust its absorbance to 0.7 ± 0.02. Pipette 100 μL of this DPPH methanol solution and mix with 100 μL of sample or blank solution into a 96-well plate. Incubate in the dark for 30 min at room temperature. Measure the absorbance at 517 nm using a microplate reader. Trolox (10-30 μg / mL) is used as a standard. The antioxidant activity of the sample is expressed as mg of Trolox required to achieve the same elimination rate (mg TE / mg DW). The DPPH free radical scavenging activity is calculated using the following formula: Scavenging Rate (%) = [1-(Ai - Aj) / Ac] × 100%, where Ai is the absorbance of the sample plus DPPH reagent, Aj is the absorbance of the sample plus methanol, and Ac is the absorbance of the DPPH solution plus methanol.

Claims

1. A method for enhancing the in vitro antioxidant activity of pomegranate peel polyphenols by microbial fermentation, characterized in that: The steps include: (1) Selecting Saccharomyces cerevisiae as the fermentation strain; (2) The fermentation conditions were 28°C and the fermentation time was 0-72 hours. The changes in polyphenol and flavonoid contents and antioxidant activity were measured.

2. The method according to claim 1, characterized in that Saccharomyces cerevisiae was purchased from Angel Yeast Co., Ltd.

3. The method according to claim 1, characterized in that The polyphenol and flavonoid contents are determined by colorimetry.

4. The method according to claim 1, wherein The in vitro antioxidant activity was mainly determined by the DPPH free radical scavenging rate.

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