BLUEBERRY FRESH-KEEPING AGENT, PREPARATION METHOD AND APPLICATIONS THEREOF

Abstract

The present disclosure provides a blueberry fresh-keeping agent, preparation method and applications thereof, the fresh-keeping agent consisting of 0.03%-0.5% of a Galla chinensis extract, 0.5%-1.0% of ascorbic acid and 1.0%-2.0% of chitosan, and pH is 2.0-6.0. The fresh-keeping agent provided by the present disclosure may significantly inhibit blueberry Botrytis cinerea, effectively alleviate post-harvest blueberry membrane lipid peroxidation, maintain blueberry cell membrane completeness, and extend storage time of blueberries from 20 days in conventional cooling to 50 days, blueberry decay rate is reduced within 10%, and weight loss rate is controlled within 5%, achieving relatively long-term, high-quality fresh-keeping storage of blueberries. The blueberry fresh-keeping agent of the present disclosure is safe to use, and blueberries treated have no toxic and harmful substances, and have the characteristics of high efficiency, safety and environment-friendly.

Claims

1. A blueberry fresh-keeping agent, wherein the blueberry fresh-keeping agent is consisted of 0.03%-0.5% of a Galla chinensis extract, 0.5%-1.0% of ascorbic acid and 1.0%-2.0% of chitosan.

2. The blueberry fresh-keeping agent according to claim 1, wherein the blueberry fresh-keeping agent has a pH of 2.0-6.0.

3. The blueberry fresh-keeping agent according to claim 1, wherein when preparation of the fresh-keeping agent, a ratio of solid to liquid during ethanol extraction of Galla chinensis is 1:10-1:20 (W/V), and an ultrasonic treatment time is 0.5-2 hours, a vacuum rotational evaporation temperature is no more than 60 C.

4. The blueberry fresh-keeping agent according to claim 1, wherein when preparation of the fresh-keeping agent, Tween 80 of 0.5% to 2% is added for solubilization during the dissolving.

5. A method for preparing the blueberry fresh-keeping agent of claim 1, wherein the method is carried out according to the following steps: (1) preparing the Galla chinensis extract: removing internal eggs of high-quality dried Galla chinensis, then pulverizing using a high-speed powder machine, and ultrasonically extracting with 75% ethanol at room temperature, centrifuging and collecting a supernatant, and rotationally evaporating the supernatant to dryness, a crude extract is obtained; redissolving the crude extract with water, extracting with ethyl acetate and collecting an ethyl acetate layer, evaporating the ethyl acetate layer to dryness, the Galla chinensis extract is obtained; (2) separately adding the Galla chinensis extract, the ascorbic acid and the chitosan by weight percentage to distilled water for dissolving; (3) after a mixed solution is thoroughly stirred and mixed, pH is adjusted to 2.0-6.0, the blueberry fresh-keeping agent is obtained.

6. A method for treating blueberries with the blueberry fresh-keeping agent according to claim 1, comprising the following steps: (1) rinsing blueberries freshly picked with water, draining for use; (2) soaking the rinsed blueberries in the blueberry fresh-keeping agent, taking out, drying and placing in a plastic box with air holes; (3) pre-cooling the blueberries treated with the blueberry fresh-keeping agent in a pre-cooling storage and storing at a low temperature.

7. The method according to claim 6, wherein a time for soaking with the blueberry fresh-keeping agent is 1-2 min, a temperature of the pre-cooling storage is 1 C.-5 C., a pre-cooling time is 2-4 hours, and the temperature of the storing is 1 C.-5 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a diagram of inhibition rate of the Galla chinensis extract against Botrytis cinerea.

[0023] FIG. 2 is a diagram of the effect of pH on antibacterial stability of the Galla chinensis extract.

[0024] FIG. 3 is a diagram of the effect of temperature on antibacterial stability of the Galla chinensis extract.

[0025] FIG. 4 is a diagram of the effect of irradiation time on antibacterial stability of the Galla chinensis extract.

[0026] FIG. 5 is a diagram of the effect of the fresh-keeping agent on decay rate and weight loss rate of blueberry fruit, where FIG. (A) shows the decay rate of blueberry fruit, and FIG. (B) shows the weight loss rate of blueberry fruit.

[0027] FIG. 6 is a diagram of the effect of the fresh-keeping agent on blueberry fruit membrane permeability and membrane lipid peroxidation, wherein FIG. (A) shows the effect of the fresh-keeping agent on membrane permeability, and FIG. (B) shows the effect of the fresh-keeping agent on membrane lipid peroxidation.

[0028] FIG. 7 is a diagram of the effect of the fresh-keeping agent treatment on the activity of enzymes related to blueberry disease resistance, wherein FIG. (A) shows the effect of the fresh-keeping agent treatment on the activity of phenylalanine ammonia-lyase, and FIG. (B) shows the effect of the fresh-keeping agent treatment on the activity of polyphenol oxidase.

DESCRIPTION OF EMBODIMENTS

[0029] The contents of the present disclosure will be more specifically described below with reference to embodiments. It is to be understood that the present disclosure is not limited to the embodiments described below, and that any form of modifications and/or changes made to the present disclosure are intended to fall within the protection scope of the present disclosure.

[0030] In the present disclosure, unless otherwise specified, all parts and percentages are units of weight, and all devices and raw materials are commercially available or commonly used in the industry. Unless otherwise specified, the methods employed in examples are general techniques in the art.

[0031] Galla chinensis used in examples was picked from Zhangjiajie, Hunan; blueberry species was Legacy, and they were picked from the Senzhilan planting base in Anji County, Hangzhou City, Zhejiang Province.

Example 1 Inhibition of the Galla chinensis Extract, Chitosan and Ascorbic Acid Against Botrytis cinerea

[0032] 1. Preparation of the Galla chinensis Extract

[0033] Removing internal eggs of high-quality dried Galla chinensis and then pulverizing using a high-speed powder machine, and ultrasonically extracting with 75% (V/V) ethanol, solid-liquid ratio of 1:15 (W/V) at room temperature (power 300 W) for 1 h, centrifuging for 10 min and collecting a supernatant, and rotationally evaporating the supernatant to dryness at 50 C., a crude extract is obtained; redissolving the crude extract with water, extracting with ethyl acetate and collecting an ethyl acetate layer, and evaporating the ethyl acetate layer to dryness, the Galla chinensis extract is obtained.

[0034] 2. A Inhibition Test of the Galla chinensis Extract, Chitosan and Ascorbic Acid Against Botrytis cinerea

[0035] Test species and reagents: Botrytis cinerea was isolated from the picked blueberries in the laboratory, and chitosan and ascorbic acid were purchased commercially.

[0036] Medium and culture conditions: a PDA medium was used, and cultured at 28 C. for 6 days.

[0037] Antibacterial effect test: after Botrytis cinerea was activated with the PDA medium, lawn of Botrytis cinerea was picked up and suspended with sterile water to obtain a bacterial suspension. Scraps of filter paper with a diameter of 1 cm were made from a highly absorbent filter paper by using a puncher, and the scraps of filter paper were subjected to dry heat sterilization and soaked in the Galla chinensis extract, a chitosan solution, and an ascorbic acid solution, respectively, and dried for use after 4 hours soaking. The solid PDA medium was melted, poured into a culture dish, cooled and solidified, then 0.1 mL of the bacterial suspension was added and uniformly coated, and then the scraps of filter paper soaked with the above solutions were attached to the bacteria-containing culture dish, and a scrap of filter paper impregnated with sterile water was used as a control. The culture dish treated above was cultured at 28 C. for 6 days, and the diameters of inhibition zones were measured.

TABLE-US-00001 TABLE 1 Antibacterial effect of the Galla chinensis extract, chitosan, ascorbic acid and gallic acid reagents inhibition zone diameter/mm Galla chinensis extract 18.30 chitosan 0.00 ascorbic acid 0.00 gallic acid 0.00

[0038] The above results show that the Galla chinensis extract has a strong inhibitory effect on Botrytis cinerea, while chitosan and ascorbic acid have no inhibitory effect on Botrytis cinerea.

[0039] In order to determine active substances that have antibacterial effects in the Galla chinensis extract, antibacterial effect of gallic acid (commercially purchased), one of the main components in Galla chinensis, on Botrytis cinerea was studied. The result showed that gallic acid had no inhibitory effect on Botrytis cinerea, indicating that the substance that has antibacterial effect in the Galla chinensis extract was not gallic acid.

Example 2 Antibacterial Effect of the Galla chinensis Extract in Different Concentrations

[0040] The Galla chinensis extract was dissolved in 10% of Tween80 to fully emulsify, a Galla chinensis suspension was obtained. When the melted PDA medium was cooled to about 50 C., to which a certain volume of the Galla chinensis suspension was added, and the mixture was subjected to fully shaking and mixing, then poured the mixture onto plates, such that final concentrations of the Galla chinensis extract in the plates were 37.5 L/mL, 75 L/mL, 150 L/mL, 300 L/mL, and 600 L/mL. A plate to which no Galla chinensis extract was added in the medium was used as a control. After culturing for 7 days, a bacteria block with a diameter of 6 mm was taken from a pathogenic bacterial plate (which can be obtained by: a solid PDA medium was melted, poured into a culture dish, cooled and solidified, then 0.1 mL of the bacterial suspension was added and uniformly coated on the medium, then the pathogenic bacterial plate was cultured) and placed on the center of each PDA plate (90 mm in diameter) using a sterile puncher, which were cultured in a biochemical incubator at 28 C. for 6 days to observe antibacterial effect. The diameters of bacterial colonies were measured using a cross method and inhibition rate was calculated. There were three plates for each concentration gradient and the test was repeated for 3 times. Inhibition rate (%)=(control colony diameter-treated colony diameter)/(control colony diameter6 mm)100. The minimum antibacterial agent concentration with inhibition rate of 100% was its minimum inhibitory concentration (MIC). As can be seen from FIG. 1, the MIC of the Galla chinensis extract against Botrytis cinerea was 300 L/mL.

Example 3 Effect of pH on Antibacterial Effect of the Galla chinensis Extract

[0041] pH values of the Galla chinensis extract were adjusted to 2, 4, 6, 8, 10, and 12 using acetic acid, and the inhibitory effects of Galla chinensis extracts with different pH values on Botrytis cinerea were compared, and the experimental procedure was described in Example 1.

[0042] It could be seen from FIG. 2 that pH had a great influence on the antibacterial activity of the Galla chinensis extract. With the increase of pH value, diameters of inhibition zones for the Galla chinensis extract gradually decreased. The Galla chinensis extract had a strong antibacterial activity under acidic condition, while in alkaline condition, the Galla chinensis extract substantially loss its antibacterial activity. That was because part carboxyl groups and phenolic hydroxyl groups of some compounds in the Galla chinensis extract will have different degrees of dissociation with the increase of pH value in an external environment, which will leading to destroy of the original structure of the compounds. Therefore, the acidic condition is more conducive to the antibacterial effect of the Galla chinensis extract.

[0043] It was reported that gallnut tannin was a polyphenolic compound that was easily hydrolyzed, which may be hydrolyzed under acidic and basic conditions to produce intermediates such as gallic acid and penta galloy glucose (-PGG). It is possible that the mutual transformation between these intermediates causes the contents of gallnut tannin, gallic acid and -PGG showing corresponding changes under different pH conditions.

[0044] In combination with the experimental result of Example 1, we speculated that the substance that has antibacterial effect in the Galla chinensis extract was not gallic acid, may be gallnut tannin, or it is a result of combination of multiple components and multiple factors, what component exactly playing an antibacterial role and the antibacterial mechanism thereof needs to be further studied.

Example 4 Effect of Temperature on Antibacterial Effect of a Galla chinensis Extract

[0045] Antibacterial activity of the Galla chinensis extract at different temperatures was measured by using the diameters of inhibition zones of Botrytis cinerea as detection objects. It can be seen from FIG. 3 that the overall trend of antibacterial activity of the Galla Chinensis extract decreased as the increase of temperature, but only decreased by 26.32%, the Galla chinensis extract partially degraded as the increase of temperature, but due to a short treatment time, the overall downward trend was not obvious. High-temperature treatment in a short time has a certain effect on activity of the Galla chinensis extract, and high-temperature treatment in a long time would lead to the degradation, structural change of active substances in the Galla chinensis extract, thereby causing the decrease of antibacterial activity. In order to ensure antibacterial activity of the Galla chinensis extract, an extraction temperature or use temperature should be lower than 60 C.

Example 5 Effect of Irradiation on Antibacterial Effect of the Galla chinensis Extract

[0046] The effect of irradiation time on antibacterial effect of the Galla chinensis extract was shown in FIG. 4. As the increase of irradiation time, the diameters of inhibition zones showed a decrease trend, especially after 3 days of continuous irradiation, the diameters of inhibition zones were decreased more obviously. This was due to the Galla chinensis extract includes tannins, gallic acid and the like, and there was a decrease in absorbance of these substances under uninterrupted irradiation, resulting in a decrease in stability, thus antibacterial effect was affected. Thus, the Galla chinensis extract should be prepared when it will be used, so as to avoid long-term exposure.

Example 6 Inhibition of a Fresh-Keeping Agent Containing the Galla chinensis Extract as a Main Component Against Botrytis cinerea

[0047] 1. Preparation of the Fresh-Keeping Agent

[0048] The Galla chinensis extract, ascorbic acid, and chitosan, by weight percentage, were dissolved in distilled water, solubilized with 2% of Tween 80, stirred and mixed, and pH was adjusted to 4.0 with acetic acid.

[0049] 2. Antibacterial Experiments of Fresh-Keeping Agents with Different Weight Percentages

[0050] The prepared fresh-keeping agents with different weight percentages were subjected to Botrytis cinerea antibacterial experiment, and the Galla chinensis extract was used as a control. The results were shown in the following table:

TABLE-US-00002 TABLE 2 Antibacterial effect of fresh-keeping agents with different percentages composition of the inhibition zone fresh-keeping diameter/ No. agent mm 1 Galla chinensis extract 0.03% 16.5 ascorbic acid 0 chitosan 0 2 Galla chinensis extract 0.03% 21.8 ascorbic acid 0.5% Chitosan 1.0% 3 Galla chinensis extract 0.03% 23.5 ascorbic acid 1.0% Chitosan 2.0% 4 Galla chinensis extract 0.1% 25.7 ascorbic acid 0.5% Chitosan 1.0% 5 Galla chinensis extract 0.1% 27.2 ascorbic acid 0.8% Chitosan 1.5% 6 Galla chinensis extract 0.5% 29.8 ascorbic acid 1.0% Chitosan 2% 7 Galla chinensis extract 0.8% 30.4 ascorbic acid 1.2% Chitosan 2.5%

[0051] It can be seen from the results that the combination of ascorbic acid, chitosan and the Galla chinensis extract significantly increased antibacterial activity of the Galla chinensis extract. Gallnut tannin had strong reducibility property, and the addition of ascorbic acid has a protected effect to the Gallnut tannin. Chitosan was a good film-forming agent, which may form a relatively dense protective film on a fruit surface, and block most of the air so as to inhibit respiratory metabolism of the fruit. The Galla chinensis extract was cooperated with the antioxidant and the film-forming agent, and produced significant antiseptic and antibacterial abilities against Botrytis cinerea, thereby achieving antisepsis and fresh-keeping effect of blueberries.

Example 7 Method for Fresh-Keeping Blueberries with the Blueberry Fresh-Keeping Agent

[0052] A method for applying a blueberry fresh-keeping agent to blueberry fresh-keeping, which was carried out as follows.

[0053] 1. Preparation of the Galla chinensis extract: removing internal eggs of high-quality dried Galla chinensis and then pulverizing using a high-speed powder machine, and ultrasonically extracting with 75% (V/V) ethanol, solid-liquid ratio of 1:15 (W/V) at room temperature (power 300 W) for 1 h, centrifuging for 10 min and collecting a supernatant, and rotationally evaporating the supernatant to dryness at 50 C., a crude extract is obtained; redissolving the crude extract with water, extracting with ethyl acetate and collecting an ethyl acetate layer, and evaporating the ethyl acetate layer to dryness, the Galla chinensis extract is obtained.

[0054] 2. Preparation of a blueberry green fresh-keeping agent: the Galla chinensis extract 0.1%, ascorbic acid 0.5%, chitosan 1.0%, balanced with distilled water, which were solubilized with 0.5% of Tween80, stirred and mixed, pH value was adjusted to 6.0 with acetic acid.

[0055] 3. Treatment method: fresh blueberries, which were relatively consistent in maturity (89 mature), had uniform size, no mechanical damage, no pests and diseases, were selected, rinsed with water, drained, soaked in the fresh-keeping agent solution for 1 min, taken out and dried, then placed in a plastic box with air holes for storage. After the fresh blueberries were pre-cooled for 3 h in a cold storage at 4 C., they were stored at 4 C. for cooling. The mildew and decay condition of fruit was check every 10 days until the end of storage.

[0056] Further, taking a storage method without using the fresh-keeping agent as a control, and the treatment method was as follows.

[0057] Fresh blueberries, which were relatively consistent in maturity (89 mature), had uniform size, no mechanical damage, no pests and diseases, were selected, rinsed with water, drained, then placed in a plastic box with air holes for storage. After the fresh blueberries were pre-cooled for 3 h in a cold storage at 4 C., they were stored at 4 C. for cooling. The mildew and decay condition of fruit was check every 10 days until the end of storage.

[0058] 4. Blueberry fruits in the treated group and the control were tested according to the following methods. The sampling time was 0 d, 10 d, 20 d, 30 d, 40 d, 50 d, 60 d and 70 d, where:

[0059] (1) fruit decay rate measurement: fruit decay rate (%)=number of decayed fruits/total number of fruits100%, the decayed fruit refers to that at least one place on the surface of the fruit was juice leaking, softening or decayed.

[0060] (2) weight loss rate: it was measured using a weighing method, using initial weight of a sample to minus weight weighed each time during storage to obtain a difference, a ratio of the difference to the initial weight of the sample was the weight loss rate, expressed in %.

[0061] (3) pulp membrane permeability measurement: the pulp membrane permeability was studied by measuring relative conductivity. 1 mm thick blueberry slices were cut, mixed and taken 20 slices out. The 20 slices were totally 3.0 g, and placed in a 25 mL tube, 25.0 mL of deionized water was added therein, after shaking for 30 min on a shaker, solution conductivity P.sub.1 was measured using a conductivity meter. After the measurement of conductivity, the solution was boiled for 10 min, cooled to room temperature, water was added to an original scale, and solution conductivity P.sub.2 was measured using the conductivity meter; deionized water conductivity P.sub.0 was measured. The relative conductivity was calculated as follows, representing the pulp membrane permeability.

P=(P.sub.1P.sub.0)/(P.sub.2P.sub.0)100%

[0062] (4) malondialdehyde content (MDA) measurement: the effect of the fresh-keeping agent on blueberry membrane lipid peroxidation was characterized by the change of MDA content. 1 g of a blueberry frozen sample was weighed and placed in a 10 mL centrifuge tube, 5 mL 100 g/L of a TCA solution was added therein to obtain a mixture, centrifuging the mixture at 10 000 r/min for 20 min at 4 C., and a supernatant was collected. 2.0 mL of the supernatant (2.0 mL 100 g/L TCA solution was added in a blank control) was taken, to which 2.0 mL 0.67% of a thiobarbituric acid solution was added, mixed and boiled in a boiling water bath for 20 min, and absorbance values at wavelengths of 450, 532 and 600 nm were measured, respectively.

[0063] (5) pulp phenylalanine ammonia-lyase (PAL) activity measurement: 3 mL of a reaction solution includes 1.5 mmol/L of ascorbic acid 1 mL, 0.1875 mmol/L of EDTA-Na 1.6 mL, 1 mmol/L of H.sub.2O.sub.2 0.3 mL and an enzyme solution 0.1 mL. Starting with the addition of H.sub.2O.sub.2, the changes of light absorption value at 290 nm were recorded within 1 min. A change of 0.01 per minute indicates an enzymatic unit (U).

[0064] (6) pulp polyphenol oxidase (PPO) activity measurement: a reaction system included 0.1 mol of catechol 2.9 mL, a supernatant enzyme solution 0.1 mL, which were mixed, placed in a UV spectrophotometer within 15 s, and measured the changes of absorbance at 420 nm within 3 min, using a reaction solution without a substrate of catechol as a control. A change of 0.01 per minute indicates an enzymatic unit (U).

[0065] The results of the measurements were shown in FIGS. 3, 4 and 5. It can be seen that with the prolongation of storage time, the decay rate and weight loss rate of blueberry fruit gradually increased, and the quality gradually decreased. The method of the present disclosure may reduce the fruit decay rate and weight loss rate well, when storage for 30 days, the fruit decay rate in general storage methods reached to 13.26%, while the fruit in the present method had not decayed, and when storage time was prolonged to 50 days, the fruit decay rate in general storage methods reached to 23.00%, while the decay rate using the present disclosure may reduce within 10%. At the same time, the present method may well inhibit the water loss during blueberry storage, resulting in the weight loss rate of blueberries was controlled within 5%. The fresh-keeping agent and fresh-keeping method of the present disclosure may effectively alleviate post-harvest blueberry membrane lipid peroxidation and maintain blueberry cell membrane completeness. In addition, the present disclosure may also significantly (P<0.05) improve the activity of phenylalanine ammonia-lyase and polyphenol oxidase related to self-resistance to disease of post-harvest blueberry, and effectively improve blueberry disease resistance and prolong storage time. In summary, applying the fresh-keeping agent according to the present disclosure may significantly inhibit decay and water loss of post-harvest blueberries, alleviate membrane lipid peroxidation, improve blueberry disease resistance, and maintain storage quality of blueberries.

Example 8 Method for Fresh-Keeping Blueberries with the Blueberry Fresh-Keeping Agent

[0066] A method for applying the blueberry fresh-keeping agent for blueberry fresh-keeping, which was carried out as follows.

[0067] 1. Preparation of the Galla chinensis extract: removing internal eggs of high-quality dried Galla chinensis and then pulverizing using a high-speed powder machine, and ultrasonically extracting with 75% (V/V) ethanol, solid-liquid ratio of 1:20 (W/V) at room temperature (power 300 W) for 2 h, centrifuging for 10 min and collecting a supernatant, and rotationally evaporating the supernatant to dryness at 45 C., a crude extract is obtained; redissolving the crude extract with water, extracting with ethyl acetate and collecting an ethyl acetate layer, and evaporating the ethyl acetate layer to dryness, the Galla chinensis extract is obtained.

[0068] 2. Preparation of the blueberry green fresh-keeping agent: the Galla chinensis extract 0.03%, ascorbic acid 1.0%, chitosan 2.0%, balanced with distilled water, which were solubilized with 1.0% Tween80, stirred and mixed, pH value was adjusted to 2.0 with acetic acid.

[0069] 3. Treatment method: fresh blueberries, which were relatively consistent in maturity (89 mature), had uniform size, no mechanical damage, no pests and diseases, were selected, rinsed with water, drained, soaked in the fresh-keeping agent solution for 2 min, taken out and dried, then placed in a plastic box with air holes for storage. After the fresh blueberries were pre-cooled for 2 h in a cold storage at 1 C., they were stored at 1 C. for cooling. The mildew and decay condition of fruit was check every 10 days until the end of storage.

[0070] The technical effects tested through experimental in this example are the same to that achieved in the Example 7, and will not be repeated here.

Example 9 Method for Fresh-Keeping Blueberries with the Blueberry Fresh-Keeping Agent

[0071] A method for applying the blueberry fresh-keeping agent for blueberry fresh-keeping, which was carried out as follows.

[0072] 1. Preparation of a Galla chinensis extract: removing internal eggs of high-quality dried Galla chinensis and then pulverizing using a high-speed powder machine, and ultrasonically extracting with 75% (V/V) ethanol, solid-liquid ratio of 1:10 (W/V) at room temperature (power 300 W) for 0.5 h, centrifuging for 10 min and collecting a supernatant, and rotationally evaporating the supernatant to dryness at 60 C., a crude extract is obtained; redissolving the crude extract with water, extracting with ethyl acetate and collecting an ethyl acetate layer, and evaporating the ethyl acetate layer to dryness, the Galla chinensis extract is obtained.

[0073] 2. Preparation of the blueberry green fresh-keeping agent: the Galla chinensis extract 0.5%, ascorbic acid 1.0%, chitosan 2.0%, balanced with distilled water, which were solubilized with 2.0% Tween80, stirred and mixed, pH was adjusted to 4.0 with acetic acid.

[0074] 3. Treatment method: fresh blueberries, which were relatively consistent in maturity (89 mature), had uniform size, no mechanical damage, no pests and diseases, were selected, rinsed with water, drained, soaked in a fresh-keeping agent solution for 1.5 min, taken out and dried, then placed in a plastic box with air holes for storage. After the fresh blueberries were pre-cooled for 4 h in a cold storage at 5 C., they were stored at 5 C. for cooling. The mildew and decay condition of fruit was check every 10 days until the end of storage.

[0075] The technical effects tested through experimental in this example are the same to that achieved in the Example 7, and will not be repeated here.

Example 10 Method for Fresh-Keeping Grapes and Black Plums with the Blueberry Fresh-Keeping Agent

[0076] A method for applying the blueberry fresh-keeping agent for fresh-keeping of grapes and black plums, which was carried out as follows.

[0077] 1. Preparation of a Galla chinensis extract: removing internal eggs of high-quality dried Galla chinensis and then pulverizing using a high-speed powder machine, and ultrasonically extracting with 75% (V/V) ethanol, solid-liquid ratio of 1:10 (W/V) at room temperature (power 300 W) for 0.5 h, centrifuging for 10 min and collecting a supernatant, and rotationally evaporating the supernatant to dryness at 60 C., a crude extract is obtained; redissolving the crude extract with water, extracting with ethyl acetate and collecting an ethyl acetate layer, and evaporating the ethyl acetate layer to dryness, the Galla chinensis extract is obtained.

[0078] 2. Preparation of a blueberry green fresh-keeping agent: the Galla chinensis extract 0.5%, ascorbic acid 1.0%, chitosan 2.0%, balanced with distilled water, which were solubilized with 2.0% Tween80, stirred and mixed, pH value was adjusted to 4.0 with acetic acid.

[0079] 3. Treatment method: fresh grapes and black plums, which were relatively consistent in maturity (89 mature), had uniform size, no mechanical damage, no pests and diseases, were selected, rinsed with water, drained, soaked in a fresh-keeping agent solution for 1.5 min, taken out and dried, then placed in a plastic box with air holes for storage. After the fresh blueberries were pre-cooled for 3 h in a cold storage at 4 C., they were stored at 4 C. for cooling. The mildew and decay condition of fruit was check every 10 days until the end of storage. Taking a storage method without using the fresh-keeping agent as a control.

[0080] With the prolongation of storage time, the decay rate and weight loss rate of grapes and black plums gradually increased, and the quality gradually decreased. When storage for 30 days, the decay rate of grape fruit reached to 20.36%, the decay rate of black plum fruit reached to 10.57% in the treatment group, and pulp thereof became softening. The water loss of the two fruits was obvious, and electrical conductivity was not significantly lower than that of the control groups, that is, the membrane lipid peroxidation of grapes and black plums was not effectively alleviated. The above results indicated that the fresh-keeping agent has no obvious fresh-keeping effect to grapes and black plums, and specific reasons need further analysis.

[0081] In combination with the above examples, the fresh-keeping agent according to the present disclosure may significantly inhibit decay and water loss of the post-harvest blueberries, alleviate blueberry membrane lipid peroxidation, and significantly improve the activity of phenylalanine ammonia-lyase and polyphenol oxidase related to self-resistance to disease of post-harvest blueberry, so as to enhance blueberry disease resistance, and effectively maintain storage quality of the blueberries.