Screening method and application of fruit polyphenol oxidase inhibitor
By screening and optimizing organic acid inhibitors, the enzymatic browning problem catalyzed by polyphenol oxidase in fruit processing is solved, effective inhibition of fruit polyphenol oxidase is achieved, the fruit browning process is delayed, and the quality and efficiency in fruit processing is improved.
Patent Information
- Application Number
- CN202510034948.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-08
AI Technical Summary
During the fruit processing process, the problem of enzymatic browning catalyzed by polyphenol oxidase is difficult to effectively solve, affecting the quality and processing efficiency of fruits.
The optimal concentration, pH and temperature of organic acid inhibitors is determined to inhibit the activity of fruit polyphenol oxidase. Specific steps include extracting polyphenol oxidase, screening inhibitors, and optimizing pH and temperature conditions.
Effective inhibition of fruit polyphenol oxidase is achieved, delaying the browning process of fruits, and improving the quality and efficiency in the fruit processing process.
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Figure CN119769558A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of fine chemicals, and in particular relates to the screening of fruit polyphenol oxidase inhibitors and process optimization thereof. Background Art
[0002] With the improvement of modern living standards, people have an increasing demand for high-quality, nutritious fresh fruits. Therefore, the quality and safety requirements for fruits are getting higher and higher. However, during the fruit processing, enzymatic browning often occurs due to mechanical damage and cold damage, which affects the quality of the fruit and restricts the development of the fruit processing industry. Studies have shown that the enzymatic browning process of fruits is mainly related to polyphenol oxidase.
[0003] Polyphenol oxidase is widely present in vegetables, fruits and many other organisms. Polyphenol oxidase can catalyze the oxidation of phenolic substances to form melanin. In the presence of oxygen molecules, polyphenol oxidase hydroxylates monophenols, then oxidizes o-diphenols to dopaquinone, and finally forms melanin. This process is usually accompanied by a deepening of the color of the system, and eventually leads to browning of the system.
[0004] At present, physical or chemical methods are the most common treatment methods to inhibit the activity of polyphenol oxidase. The physical method is to inactivate polyphenol oxidase by heat treatment, which is a simple and efficient method in food processing. The chemical method is to inhibit the browning caused by polyphenol oxidase by browning inhibitors. In recent years, chemical inhibitors have been studied and used by a large number of scholars and have achieved good results in inhibiting enzymatic browning. Although many inhibitors have been discovered and proven to be able to effectively inhibit the activity of polyphenol oxidase, there are still great limitations in practical applications. Therefore, safe and efficient polyphenol oxidase inhibitors have become a current research hotspot. Summary of the invention
[0005] The invention aims to screen a fruit polyphenol oxidase inhibitor with good inhibitory effect, and provide the optimal concentration, pH and temperature of the most suitable inhibitor to inhibit the activity of fruit polyphenol oxidase, so as to provide a reference for comprehensive utilization in the production and processing of fruits after harvest.
[0006] The technical solution of the present invention is as follows:
[0007] (1) Extraction of fruit polyphenol oxidase: Select fruits of the same variety and maturity, peel and cut into pieces. Weigh the fruits and add them into phosphate buffer, beat them with a juicer, and then centrifuge to obtain the supernatant to obtain the crude polyphenol oxidase enzyme solution, which is stored in a 4°C refrigerator for later use.
[0008] (2) Screening of polyphenol oxidase inhibitors: Five organic acid solutions were prepared into inhibitors of different concentrations, and reacted with catechol in a water bath respectively. Then, the crude polyphenol oxidase enzyme solution was reacted with the above solutions in a water bath. After the reaction was completed, trichloroacetic acid solution was immediately added and mixed to terminate the reaction. The absorbance was then measured at a wavelength of 420 nm, and the inhibition rate was calculated using distilled water as a reference.
[0009] (3) Optimization of pH of the most suitable polyphenol oxidase inhibitor: Fruits were added to phosphate buffers of different pH values for juicing, and crude enzyme solutions were obtained after centrifugation. Organic acid solutions were reacted with catechol in a water bath, and then crude polyphenol oxidase solutions of different pH values were reacted with the above solutions in a water bath. After the reaction was completed, trichloroacetic acid solution was immediately added to mix and terminate the reaction, and then the absorbance was measured at a wavelength of 420 nm, with distilled water as a reference.
[0010] (4) Temperature optimization of the most suitable polyphenol oxidase inhibitor: The fruit was added to a phosphate buffer solution for juicing, and the crude enzyme solution was obtained after centrifugation. The organic acid solution was reacted with catechol in a water bath at different temperatures, and then the extracts with different pH values were reacted with the above solution in a water bath at different temperatures. After the reaction was completed, trichloroacetic acid solution was immediately added to mix and terminate the reaction, and then the absorbance was measured at a wavelength of 420 nm, with distilled water as a reference.
[0011] Preferably, the phosphate buffer solution in step 1 has a concentration of 0.01 mol / L and a pH of 6.8.
[0012] Preferably, in step 2, the concentration of organic acid inhibiting banana polyphenol oxidase is 0.2-1.0 g / L, and the concentration of organic acid inhibiting apple polyphenol oxidase is 0.02-0.1 g / L; in step 2, except for cinnamic acid being dissolved in anhydrous ethanol, the remaining acids are dissolved in deionized water; and in step 2, the water bath temperature is 30° C.
[0013] Preferably, the pH range of the phosphate buffer solution in step 3 is 6.0 to 8.0; and the water bath temperature in step 3 is 30°C.
[0014] Preferably, the concentration of the phosphate buffer in step 4 is 0.01 mol / L, and the pH is the optimal pH screened out in step 3; and the different temperature range in step 4 is 25-45°C. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 .The effect of different concentrations of inhibitors on the inhibition rate of banana PPO;
[0016] Figure 2 .The effect of different concentrations of inhibitors on the inhibition rate of apple PPO;
[0017] Figure 3 .The effect of tartaric acid on the inhibition rate of banana PPO at different pH;
[0018] Figure 4 .The effect of tartaric acid on the inhibition rate of apple PPO at different pH;
[0019] Figure 5 .The effect of tartaric acid on the inhibition rate of banana PPO at different temperatures;
[0020] Figure 6 .Effect of tartaric acid on the inhibition rate of apple PPO at different temperatures. DETAILED DESCRIPTION
[0021] Step 1: Extraction of fruit polyphenol oxidase
[0022] Select fruits of the same variety and maturity, peel and cut into pieces. Weigh 25g of fruit and add it to 100mL phosphate buffer (0.01mol / L, pH=6.8), and use a juicer to pulp. Then use a centrifuge to centrifuge the fruit juice at 4℃ and 10000r / min for 20min. The supernatant obtained is the crude polyphenol oxidase enzyme solution, which is stored in a 4℃ refrigerator for later use.
[0023] Step 2: Screening of polyphenol oxidase inhibitors
[0024] Citric acid, cinnamic acid, malic acid, tartaric acid and succinic acid solutions were prepared as inhibitors of different concentrations. The organic acid concentrations that inhibited banana polyphenol oxidase were 0.2, 0.4, 0.6, 0.8, and 1.0 g / L, and the organic acid concentrations that inhibited apple polyphenol oxidase were 0.02, 0.04, 0.06, 0.08, and 0.1 g / L. Cinnamic acid was dissolved in anhydrous ethanol, and the remaining acids were dissolved in deionized water. 1 mL of inhibitors of different concentrations and types were respectively mixed with 1 ml of catechol (0.1 mol / L) at 30 ° C for 10 min, and then 1 mL of polyphenol oxidase crude enzyme solution was reacted with the above solution in a 30 ° C water bath for 1 min. After the reaction was completed, 1 mL of trichloroacetic acid solution (10%, m / V) was immediately added to mix and terminate the reaction. The absorbance was then measured at a wavelength of 420 nm. With distilled water as a reference, the inhibition rate was calculated according to the formula:
[0025]
[0026] Where: A0 is the absorbance without adding inhibitor; A is the absorbance with adding inhibitor.
[0027] Step 3: pH optimization of the optimal polyphenol oxidase inhibitor
[0028] 25g of fruit was added to 100mL of phosphate buffer with different pH values (6.0, 6.5, 6.8, 7.0, 7.5, 8.0) to extract the juice, and the crude enzyme solution was obtained after centrifugation. 1mL of organic acid solution and 1mL of catechol (0.1mol / L) were placed in a 30℃ water bath for 10min, and then 1mL of crude polyphenol oxidase enzyme solution with different pH values was reacted with the above solution in a 30℃ water bath for 1min. After the reaction was completed, 1mL of trichloroacetic acid solution (10%, m / V) was immediately added to mix and terminate the reaction, and then the absorbance was measured at a wavelength of 420nm, and the inhibition rate was calculated with distilled water as a reference.
[0029] Step 4: Temperature optimization of the optimal polyphenol oxidase inhibitor
[0030] 25g of fruit was added to 100mL of phosphate buffer (0.01mol / L, optimal pH) to extract juice, and crude enzyme solution was obtained after centrifugation. 1mL of organic acid solution and 1mL of catechol (0.1mol / L) were placed in a water bath at different temperatures (25, 30, 35, 40, 45℃) for 10min, and then 1mL of crude polyphenol oxidase enzyme solution with different pH values was reacted with the above solution in a water bath at different temperatures (25, 30, 35, 40, 45℃) for 1min. After the reaction was completed, 1mL of trichloroacetic acid solution (10%, m / V) was immediately added to mix and terminate the reaction, and then the absorbance was measured at a wavelength of 420nm, and the inhibition rate was calculated with distilled water as a reference.
[0031] 1. Effects of different inhibitors on PPO activity
[0032] like Figure 1 and Figure 2 As shown: From the results, it can be seen that tartaric acid has a good inhibitory effect on banana PPO and apple PPO. The concentration of banana PPO inhibitor was selected as 0.6g / L, because the inhibition rate increased with the increase of inhibitor concentration, but when the concentration was higher than 0.6g / L, the increase was not obvious, so tartaric acid with a concentration of 0.6g / L was selected; tartaric acid with a concentration of 0.08g / L was selected for the apple PPO inhibitor concentration.
[0033] 2. Effect of different pH on PPO activity
[0034] like Figure 3 and Figure 4 As shown, it can be seen from the results that pH has an effect on the inhibition of banana PPO and apple PPO. The inhibition rate shows a trend of first increasing and then decreasing with the increase of pH, and the inhibition rate is the highest and the inhibition effect is the strongest at pH 6.8.
[0035] 3. Effect of different temperatures on PPO activity
[0036] like Figure 5 and Figure 6 As shown, it can be seen from the results that temperature has an effect on the inhibition of banana PPO and apple PPO. The inhibition rate shows a trend of first increasing and then decreasing with the increase of temperature, and the inhibition rate is the highest and the inhibition effect is the strongest at 30℃.
[0037] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily obtained by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. A method for screening fruit polyphenol oxidase inhibitors, characterized in that: At least the following steps are included: Step 1: Extraction of fruit polyphenol oxidase Select fruits of the same variety and the same maturity, peel them and cut them into pieces; weigh a certain weight of the fruits and add them into a phosphate buffer for pulping, wherein the weight-to-volume ratio of the fruits to the phosphate buffer is 1:4, and then separate them by centrifugation. The obtained supernatant is the crude polyphenol oxidase enzyme solution, which is stored in a 4°C refrigerator for later use; Step 2: Screening of polyphenol oxidase inhibitors Citric acid, cinnamic acid, malic acid, tartaric acid and succinic acid solutions were prepared as inhibitors of different concentrations; 1 mL of inhibitors of different concentrations and types were respectively bathed with 1 mL of catechol (0.1 mol / L) for 10 min, and then 1 mL of crude polyphenol oxidase prepared in step 1 was bathed with the above solutions for 1 min. After the reaction was completed, 1 mL of trichloroacetic acid solution (10%, m / V) was immediately added to mix and terminate the reaction, and then the absorbance was measured at a wavelength of 420 nm, and the inhibition rate was calculated using distilled water as a reference: Where: A0—absorbance without adding inhibitor; A—absorbance with adding inhibitor; Step 3: pH optimization of the optimal polyphenol oxidase inhibitor A certain amount of fruit is added to a certain volume of phosphate buffer with different pH values for juicing, the weight-to-volume ratio of the fruit to the phosphate buffer being 1:4, and a crude enzyme solution is obtained after centrifugation; 1 mL of the organic acid solution and 1 mL of catechol are bathed in water for 10 minutes, and the concentration of catechol is 0.1 mol / L; then 1 mL of the crude polyphenol oxidase solution with different pH values is bathed in water for reaction with the above solution for 1 minute, and immediately after the reaction is completed, 1 mL of trichloroacetic acid solution (10%, m / V) is added to mix and terminate the reaction, and then the absorbance is measured at a wavelength of 420 nm, with distilled water as a reference. Step 4: Temperature optimization of the optimal polyphenol oxidase inhibitor A certain weight of fruit was added to phosphate buffer for juicing, the weight-to-volume ratio of fruit to phosphate buffer was 1:4, and crude enzyme solution was obtained after centrifugation. 1 mL of organic acid solution and 1 mL of catechol were placed in a water bath at different temperatures for 10 min, and the concentration of catechol was 0.1 mol / L. Subsequently, 1 mL of crude polyphenol oxidase solution of different pH values was reacted with the above solution in a water bath at different temperatures for 1 min. After the reaction was completed, 1 mL of trichloroacetic acid solution (10%, m / V) was immediately added to mix and terminate the reaction, and then the absorbance was measured at a wavelength of 420 nm, with distilled water as a reference.
2. The method for fruit polyphenol oxidase inhibitor screening and process optimization according to claim 1, wherein: The concentration of the phosphate buffer described in step 1 is 0.01 mol / L, and the pH is 6.
8.
3. The method for fruit polyphenol oxidase inhibitor screening and process optimization according to claim 1, wherein: In step 2, the concentration of organic acid inhibiting banana polyphenol oxidase is 0.2-1.0 g / L, and the concentration of organic acid inhibiting apple polyphenol oxidase is 0.02-0.1 g / L; in step 2, except for cinnamic acid dissolved in anhydrous ethanol, the other acids are dissolved in deionized water; and in step 2, the water bath temperature is 30° C.
4. The method for fruit polyphenol oxidase inhibitor screening and process optimization according to claim 1, wherein: The pH range of the phosphate buffer in step 3 is 6.0 to 8.0; the water bath temperature in step 3 is 30°C.
5. The method for fruit polyphenol oxidase inhibitor screening and process optimization according to claim 1, wherein: The concentration of the phosphate buffer in step 4 is 0.01 mol / L, and the temperature range in step 4 is 25-45°C.