Changes in antioxidant activity during enzyme fermentation

Chapter 4 Changes in Antioxidant Activity during Enzyme Fermentation

Fruits are rich in nutrients and come in many varieties. The production of substances during fermentation is complex and varied, and there are many studies on their antioxidant activity. People are always exploring and discovering, looking for foods that are beneficial to human health, so enzymes have become a hot topic in recent years. The traditional enzyme production process is to obtain enzymes by natural fermentation of fruits and vegetables. Based on previous research, this article adds 4 beneficial fermentation bacteria. This bacteria itself also exists in the enzyme. After the additional addition of bacteria, the number and type of microorganisms in the fermentation process are changed. What effect will this change have on its antioxidant activity? In the previous chapter, we took apples as an example for a detailed explanation. The comparison of the antioxidant activity of the experimental group and the control group at different enzyme concentrations showed that the antioxidant activity of the enzyme in the experimental group was greater than that of the control group. The intensity of antioxidant activity also reflects the reliability of the enzyme in a certain way.

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This chapter adds common fruits pears and citrus to the original experiment, and tracks and detects the changes in their antioxidant activity during fermentation. On the basis of reducing costs, through experiments, we hope to more comprehensively reflect the biological activity of enzymes after adding bacteria, and provide a certain theoretical basis and data support for the production of microbial enzymes by artificial inoculation of strains.

 

 

 

4.1 Materials and methods

4.1.1 Materials

(1) Experimental materials

Wash fresh apples, pears and citrus fruits with sterile water under sterile conditions, dry them naturally on a sterile operating table, peel them and slice them for later use. Add white sugar and fruits to a sterilized glass jar at a mass ratio of 1:1. Activate the bacteria required for the experiment and inoculate them into the enzyme according to the optimal scheme (this operation step is omitted for the control group), seal it and place it in a cool and dry place. Take samples at different time periods of fermentation at room temperature to obtain all the enzyme liquid containing fruit pulp, and filter it. Take the supernatant as the experimental sample, and use it to measure relevant data after centrifuging it at 10,000 rpm in a high-speed centrifuge for 15 minutes. It is worth noting that in the process of making enzymes, in order to avoid unnecessary contamination, all our operations are carried out under sterile conditions.

(2) Main instruments

The instruments required for the experiment are the same as 3.1.1 (2).

 

4.1.2 Methods

(1) Changes in total phenol content during fermentation

450μL of sample solution was added with distilled water to make the volume 46mL, and then 1mL of Folin-phenol reagent was added. Mix well and react for 3min. Then 3mL of 20% NaCO3 was added. The mixture was shaken in a constant temperature water bath at 25℃ for 2h. Distilled water was used as blank control. The absorbance A was measured at 760nm by spectrophotometer. Three replicates were performed for each treatment. The total phenol content was calculated according to the standard curve equation.

(2) Changes in reducing power during fermentation

Add 450μL of sample to 2.5mL of phosphate buffer with a concentration of 0.2mol/L and a pH value of 6.6, then add 2.5mL of potassium ferricyanide (w/v) with a mass concentration of 1%, react at 50℃ for 30 minutes, add 2.5mL of trichloroacetic acid (w/v) with a mass concentration of 10%, centrifuge at 3000rpm for 10 minutes, take out and immediately draw 2.5mL of supernatant into a volumetric flask, add 2.5mL of distilled water and 0.5mL of ferric chloride (w/v) with a mass concentration of 0.1%. Use distilled water as a blank control, and measure the absorbance A at a wavelength of 700nm with a spectrophotometer. Perform 3 replicates for each treatment. The strength of reducing power is determined based on the absorbance value.

 

(3) Changes in superoxide anion radical scavenging ability during fermentation
Place 4.5 mL of 0.05 mol/L pH 8.2 Tris-HCl buffer in a constant temperature water bath and adjust the temperature to 25 ℃. After 20 min, add 1 mL of enzyme sample solution and 0.4 mL of pyrogallol solution with a molar concentration of 25 mmol/L. Mix well and place in a 25 ℃ water bath for 5 min. Add 1.0 mL of 8 mol/L HCl to terminate the reaction. Use Tris-HCl buffer as a reference and use a spectrophotometer to measure the absorbance A at 299 nm to calculate the scavenging rate. The blank control group uses 1 mL of solvent instead of sample. The superoxide anion radical scavenging rate is calculated according to formula 4.1: Superoxide anion radical scavenging rate (%) = (A1-A2)/A1×100 (4.1) Where: A1 is the absorbance of the blank control group; A2 is the absorbance of the enzyme sample solution of the experimental group.

 

(4) Changes in hydroxyl radical scavenging ability during fermentation

 

Add water to 450μL sample solution to 2mL, then add it to 1.4mL of hydrogen peroxide with a molar mass concentration of 6mmol/L, then add 0.6mL of sodium salicylate with a molar mass concentration of 20mmol/L and 2mL of ferrous sulfate with a molar mass concentration of 1.5mmol/L, and heat in a constant temperature water bath at 37℃ for 1h. Zero with distilled water, and measure the absorbance A at a wavelength of 562nm with a spectrophotometer. Three replicates were performed for each treatment. The hydroxyl radical scavenging rate was calculated according to formula 4.2: Hydroxyl radical scavenging rate (%) = [(A1-A2)/A1] × 100 (4.2) Where: A1 is the average absorbance of the blank; A2 is the average absorbance of the sample solution.

 

(5) Changes in DPPH free radical scavenging ability during fermentation
Accurately transfer 2 mL of enzyme sample solution to a 10 mL volumetric flask, then add 2 mL of 80% DPPH ethanol aqueous solution to the volumetric flask, with a molar mass concentration of 2×10-4 mol/L. Mix well and let stand at room temperature for 30 min. Take 80% ethanol solution as reference and measure the absorbance of the sample at a wavelength of 517 nm, which is recorded as A1. Mix 2 mL of DPPH solution and 2 mL of 80% ethanol solution, and measure their absorbance at the same wavelength, which is recorded as A0. Mix 2 mL of enzyme solution and 2 mL of 80% ethanol solution, and measure their absorbance at the same wavelength, which is recorded as A2. Three replicates were performed for each treatment. Calculate the DPPH free radical scavenging rate according to formula 4.3:
DPPH free radical scavenging rate (%) = [1-(A1-A2)/A0] × 100 (4.3) Where: A1 is the average absorbance of 2 mL DPPH 80% ethanol aqueous solution and 2 mL sample solution; A2 is the absorbance of 2 mL enzyme solution and 2 mL 80% ethanol mixed solution; A0 is the absorbance of 2 mL DPPH solution and 2 mL 80% ethanol mixed solution.

(6) Changes in ABTS free radical scavenging ability during fermentation
8 μL sample solution was supplemented to 10 μL with phosphate buffer (5 mmol/L pH 7.4) and then mixed evenly with 10 mL of potassium sulfate and ABTS mixed solution to react at a reaction temperature of 30 °C and a reaction time of 5 min. Zero the solution with distilled water and measure the absorbance A at a wavelength of 734 nm using a spectrophotometer. Three replicates were performed for each treatment. The ABTS free radical scavenging rate was calculated according to formula 4.4:
ABTS free radical scavenging rate (%) = [(A1-A2)/A1] × 100 (4.4) Where: A1 is the absorbance of the blank control group; A2 is the absorbance of the sample experimental group.