Application of natural inducer in fruit preservation

Abstract

The invention discloses application of a natural inducer in fruit preservation, and particularly relates to the technical field of fruit preservation. According to the application of the natural inducer in fruit preservation, the natural inducer is used as an active ingredient to inhibit growth of botrytis cinerea on fruits and induce immune systems of the fruits to enhance active oxygen metabolism; the natural inducer is theanine; the fruits are kiwi fruits. When the natural inducer provided by the invention is applied to fruit preservation, a relatively good preservation effect can be exerted on kiwi fruits.

Description

Technical Field

[0001] The invention relates to the technical field of fruit preservation, in particular to the application of a natural inducer in fruit preservation. Background Art

[0002] Kiwifruit is a nutritious fruit with a unique taste and is loved by consumers. However, during storage and transportation, kiwifruit is easily infected by pathogens, which leads to fruit rot and quality deterioration, resulting in economic losses. Botrytis cinerea is one of the main pathogens causing postharvest diseases of kiwifruit. Although traditional chemical fungicides can control the occurrence of diseases to a certain extent, there are residual problems and potential risks to the environment and human health. Therefore, finding a safe and effective alternative method has become the focus of current research.

[0003] L-theanine also has a strong antioxidant effect, which can help fruits and vegetables resist the damage of free radicals, reduce the oxidative damage of reactive oxygen species (ROS) to membrane lipids, and delay the aging and decay of fruits and vegetables, thereby extending their shelf life. In addition, L-theanine can also maintain the nutrients in fruits and vegetables, such as vitamin C, which helps to maintain the nutritional value of fruits and vegetables. It can also inhibit the activity of some enzymes in fruits and vegetables, delay the browning and softening process of fruits and vegetables, and help maintain the taste and texture of fruits and vegetables. However, there are few studies on the application of theanine as an exogenous substance in the prevention and control of kiwifruit postharvest diseases.

[0004] Therefore, it is necessary to provide an application of a natural inducer in fruit preservation to solve the above technical problems. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an application of a natural inducer in fruit preservation, which can exert a good preservation effect on kiwifruit.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The invention discloses an application of a natural inducer in fruit preservation, wherein the natural inducer is used as an active ingredient to inhibit the growth of gray mold on the fruit and induce the immune system of the fruit to enhance active oxygen metabolism; the natural inducer is theanine; and the fruit is kiwi fruit.

[0007] Preferably, the active oxygen metabolism includes the hydrogen peroxide content and the superoxide anion free radical generation rate, and the hydrogen peroxide content decreases and the superoxide anion free radical generation rate increases under theanine treatment.

[0008] Preferably, the active oxygen metabolism further comprises antioxidant enzymes, and the antioxidant enzymes comprise catalase, superoxide dismutase, and peroxidase, and the activities of catalase, superoxide dismutase, and peroxidase are all increased under the treatment of theanine.

[0009] Preferably, the concentration of theanine is 1-20 mmol L -1 In the range of 10 mmol L -1 Theanine has the best effect in inhibiting the growth of Botrytis cinerea hyphae.

[0010] Preferably, theanine is used to regulate the active oxygen metabolism of kiwifruit at a concentration of 1-10 mmol L -1 In the range of 10 mmol L -1 Theanine has the best effect in regulating the metabolism of active oxygen in kiwi fruit.

[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) Inhibition of Botrytis cinerea growth: Theanine treatment can significantly inhibit the growth of Botrytis cinerea, especially at 10 mmol L -1 The antibacterial effect is best at the concentration.

[0012] (2) Reduce the decay rate and lesion diameter of kiwifruit: Theanine treatment can effectively reduce the decay rate and lesion diameter of kiwifruit during storage. -1 The concentration of theanine treatment had the most significant effect.

[0013] (3) Delaying fruit softening: Theanine treatment can delay the softening of kiwifruit, especially at 10 mmol L -1 At the highest concentration, the effect of delaying fruit softening is most significant.

[0014] (4) Reducing the accumulation of reactive oxygen species: Theanine treatment can significantly reduce the content of hydrogen peroxide (H2O2) and superoxide anion free radicals (O2· - ) production rate, reducing the oxidative damage to kiwifruit caused by the massive accumulation of reactive oxygen species.

[0015] (5) Enhance the activity of antioxidant enzymes: Theanine treatment can enhance the activity of superoxide dismutase (SOD) and peroxidase (POD) antioxidant enzymes in kiwifruit, thereby improving the antioxidant capacity of the fruit.

[0016] (6) Prolonging the storage life: Through the above mechanism, theanine treatment helps to extend the storage life of kiwifruit and maintain the quality and freshness of the fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The mycelial growth under different concentrations of theanine; Figure 2 It is a bar graph of plaque diameter under different concentrations of theanine; Figure 3 The induction effect of different concentrations of theanine on kiwifruit gray mold; Figure 4 The effect of different concentrations of theanine on the diameter of kiwifruit lesions; Figure 5 The effect of different concentrations of theanine on the decay index of kiwifruit; Figure 6 The effect of different concentrations of theanine on the firmness of kiwifruit fruit; Figure 7 This is the changing trend of hydrogen peroxide content in kiwifruit during storage; Figure 8 The changing trend of superoxide anion radical generation rate during storage of kiwifruit; Fig. 9 The change trend of catalase activity during storage of kiwifruit; Fig.10 The change trend of superoxide dismutase activity in kiwifruit during storage; Fig.11 This is the changing trend of peroxidase activity in kiwifruit during storage. DETAILED DESCRIPTION

[0018] The present invention is further described below, and the embodiments of the present invention include but are not limited to the following embodiments.

[0019] The invention discloses an application of natural inducers in fruit preservation, using theanine as an active ingredient to inhibit the growth of gray mold on kiwifruit and induce the immune system of the fruit to enhance active oxygen metabolism; active oxygen metabolism includes hydrogen peroxide content, superoxide anion free radical generation rate and antioxidant enzymes, and the hydrogen peroxide content is reduced and the superoxide anion free radical generation rate is increased under theanine treatment, and the antioxidant enzymes include catalase, superoxide dismutase and peroxidase, and the catalase activity, superoxide dismutase activity and peroxidase activity are all improved under theanine treatment.

[0020] The following is an experiment on the effect of theanine on the preservation of kiwi fruit.

[0021] Experimental materials and strains: Kiwifruits with no pests, no mechanical damage, and uniform size were selected for use in the experiment. The gray mold (B. cinerea) was obtained from the strains reserved by the research group. B. cinerea was grown on potato dextrose agar (PDA) medium (containing 20 g L -1 Glucose, 20 g L -1 Agar and 200 g L -1 potato extract) and incubated at 28 °C.

[0022] In vitro antifungal activity assay: The bacterial cake was heated to 40 °C with theanine concentrations of 0, 2.5, 5, 10, and 20.0 mmol L -1 The treated group was cultured in PDA medium, and the control group was treated with sterile water. Both were cultured at 28 °C for 5 days. The colony diameter was measured by the cross method every 24 hours. Three parallel lines were taken for each group, and the test was repeated three times. The specific results are shown in Figure 2. Figure 1 As shown. Figure 1 and Figure 2 It can be seen that at 2.5, 5, 10, and 20.0 mmol L -1 Theanine at all concentrations had an inhibitory effect on the growth of Botrytis cinerea, but 10 mmol L -1 Under the condition of high concentration of theanine, the colony area is the smallest and the ability to inhibit the growth of Botrytis cinerea is the strongest.

[0023] Selection of optimal concentration of theanine: Kiwifruit were randomly distributed in five different concentration treatment groups, each containing 108 fruits, and repeated three times. The kiwifruit were then immersed in 1% (v / v) sodium hypochlorite for 1 min and then washed three times with sterile deionized water. After air drying, the fruits were immersed in different concentrations of theanine solution (0, 1, 2.5, 5, 10 mmol L -1 ) for 10 min. Then, the air-dried kiwifruit was injected with a sterile needle (5 mm deep) at the equator of the wound. The kiwifruit was stored in a plastic box for 12 days at a temperature of 20 ± 1 °C and a relative humidity of 85-90%.

[0024] Specifically: Theanine formulation: (1) Weigh 3.484 g of theanine and dissolve it in 2 L of water to obtain 10 mM theanine. (2) Weigh 2 L of water and dissolve it in 2 L of theanine aqueous solution in (1) to obtain 5 mM theanine; (3) Weigh 2 L of water and dissolve it in 2 L of theanine aqueous solution in (2) to obtain 2.5 mM theanine; (4) Weigh 3 L of water and dissolve it in 2 L of theanine aqueous solution in (3) to obtain 1 mM theanine.

[0025] Preparation of spore suspension: 1. Use a sterile syringe to slowly add 10 mL of sterile saline containing 0.05% Tween 80 to the PDA plate with good growth of Botrytis cinerea, and use an applicator to evenly scrape the mycelium to wash off the spores; 2. Filter the eluate through a cell strainer into a 50 mL centrifuge tube to obtain the spore stock solution, and count it using a hemocytometer; 3. Perform gradient dilution of the stock solution, i.e., take 0.5 mL of the stock solution into a 10 mL centrifuge tube, add 4.5 mL of sterile water, and so on, to adjust the spore suspension to 10. 6 cfm -1 stand-by; 4. Prepare 4 mL of Botrytis cinerea spore suspension.

[0026] Fruit processing: 1. Fruit selection: Select kiwifruit that are about 90% ripe and slightly larger, ensuring that the fruit size, maturity, and defects are uniform; 2. Sterilization: Soak the kiwifruit in 1% sodium hypochlorite for 1 minute to disinfect the surface of the fruit and reduce field microbial contamination; 3. Rinse and air-dry: Hold the kiwi fruit in your hands and rinse it three times in sterile distilled water (30 seconds each time), place it on sterile newspapers prepared in advance, and dry it at room temperature.

[0027] 4. Add preservative: Place kiwifruit in 0, 1, 2.5, 5, 10 mmol L -1 The kiwifruits were immersed in theanine solution with a concentration of 10 min and then placed on sterile paper to air dry naturally. Each treatment group had 3 kiwifruits and the treatment was repeated 3 times. 5. Make wounds: Cut off the tip of the 10 μL gun tip and use the gun tip to make two wounds with a depth of 5 mm and a width of 3 mm in an area below the equatorial plane of each kiwi fruit. Immediately use sterile tweezers to hold sterile gauze to absorb the kiwi juice oozing out of the wounds; 6. Inoculate Botrytis cinerea: Use a 20 μL pipette to add 10 μL of 10 6 cfm -1 A spore suspension was injected into each wound site.

[0028] Kiwifruit Storage: The fruits were placed in a sealed white box that was disinfected with alcohol cotton in advance (the inside and outside of the white box should be wiped with alcohol 1 day in advance, and then covered with plastic wrap sprayed with alcohol). Each treatment group was repeated 3 times, and each repeated group was represented by ABC, such as: CKA (control group A); CKB (control group B); CKC (control group C). Sterile plastic foam was placed on the bottom of the box to prevent the kiwifruit from moving. The disease test was carried out at 20 ℃ and 85-90% relative humidity and stored for 12 days. Samples were collected every 3 days (the collection results are shown in Figure 3 ), subsequently frozen in liquid nitrogen and finally stored at -80 °C for further biochemical examination.

[0029] The lesion diameter (LD), decay index (DS) and hardness of kiwifruit in the late storage period were tested. The test method is as follows. The test results are as follows: Figure 4-6 shown.

[0030] Lesion diameter: Based on the average length and width of kiwifruit lesions in orthogonal directions, the result is expressed in millimeters (mm).

[0031] Through Figure 3-4 It can be seen that in the later stage of storage, at 1, 2.5, 5, and 10 mmol L -1 The lesions on kiwifruit under the treatment of theanine concentration were smaller than those in the control group, but -1 The diameter of the lesions on kiwifruit fruits treated with theanine at the highest concentration was the smallest.

[0032] Rot index: According to the proportion of the rotten area to the whole fruit, the degree of rot can be divided into 4 levels: Level 0, no rot; Level 1, 1-25%; Level 2, 26%-50%; Level 3, 51%-100%. Calculation formula: Rot index = Σ[(rotten level × number of fruits at that level) / (highest rotten level × total number of fruits)].

[0033] Through Figure 5 It can be seen that in the later stage of storage, at 1, 2.5, 5, and 10 mmol L -1 The rot degree of kiwifruit under the treatment of theanine concentration was less than that of the control group, but 10 mmol L -1 The degree of decay of kiwifruit fruit treated with high concentration of theanine was the lowest.

[0034] Hardness: Texture profile analysis (TPA) was performed using a cylindrical probe with a diameter of 5 mm (P / 5 probe). Each time, the same position of 9 kiwifruits was selected for testing, with a downward pressure distance of 5 mm. The probe descending speed before the test, the test speed, and the probe return speed after the test were 3 mm s -1 , 1 mm s-1 and 1 mm s -1 , and the result is expressed in g.

[0035] Through Figure 6 It can be seen that in the later stage of storage, at 1, 2.5, 5, and 10 mmol L -1 The hardness of kiwifruit fruits under the treatment of theanine concentration was greater than that of the control group, but 10 mmol L -1 The kiwifruit fruit hardness was the highest under the treatment of theanine concentration.

[0036] When fruits are infected by pathogens, they stimulate the rapid defense response of the immune system and produce a lot of reactive oxygen species (ROS). The increase of ROS is the cause of local cell death and activation of the ROS scavenging system in fruits. The main ROS in fruits and vegetables include hydrogen peroxide (H2O2), superoxide anion free radical (O2 - ), including H2O2 content, O2· - The generation rate can reflect the accumulation degree of reactive oxygen species.

[0037] Hydrogen peroxide (H2O2) content, superoxide anion (O2 - ) Determination of production rate: Determination of H2O2 content: Weigh 0.3 g of sample and grind it into a homogenate in 0.75 mL of pre-cooled acetone solution, then centrifuge it at 4 °C and 12000 × g for 20 min. Take 0.15 mL of supernatant, add 0.015 mL of 10% (v / v) titanium tetrachloride-hydrochloric acid solution and 0.03 mL of concentrated ammonia water, mix well, react for 5 min, and centrifuge again at 12000 × g and 4 °C for 15 min. Discard the supernatant and keep the precipitate for later use. Use pre-cooled acetone to repeatedly wash the remaining precipitate obtained by centrifugation until the precipitate is colorless. Then add 0.45 mL of 2 mmol L -1 Sulfuric acid, after complete mixing and dissolution, measure the absorbance at 412 nm. According to the absorbance of the solution, find the corresponding amount of hydrogen peroxide from the standard curve, and calculate the content of hydrogen peroxide per gram of fruit and vegetable tissue (fresh weight). The result is expressed in μmol g -1 express.

[0038] O2 - Production rate determination method: weigh 0.3 g sample, add 0.75 mL extract and 2% polyvinyl pyrrolidone (PVP), grind and homogenize, centrifuge at 4 °C, 12000 × g for 20 min. After centrifugation, take 1 mL of supernatant and 1 mL of 0.05 mol L -1, pH 7.8 PBS buffer, and 1 mL 1 mmol L -1 The hydroxylamine hydrochloride solution was mixed evenly and then reacted in a 25 °C water bath for 60 min. Then 1.0 mL of 17 mmol L -1 p-Aminobenzenesulfonic acid solution and 1.0 mL 7 mmol L -1 α-naphthylamine solution, react again in a 25 ℃ water bath environment for 20 min. The control group uses distilled water instead of the supernatant as a reference blank for zero adjustment. Immediately after the water bath, the absorbance value of the colorimetric solution at a wavelength of 530 nm is measured. According to the difference in absorbance between the colorimetric solution in the sample tube and the colorimetric solution in the control tube, the corresponding amount of superoxide anion substance is found from the standard curve, and the amount of superoxide anion substance produced per gram of fruit and vegetable tissue (fresh weight) per minute is used as O2· - The production rate is expressed in nmol g -1 min -1 express.

[0039] like Figure 7 The figure shows the changing trend of H2O2 content in kiwifruit during storage. The H2O2 content generally showed an upward trend during storage. Among them, the H2O2 content under theanine and control group treatments began to show significant differences on the 6th day (P<0.05), and its content was 10.41% lower than that of the control group. Until the 12th day of storage, the H2O2 content under theanine treatment was still significantly reduced (P<0.05), and its content was 11.74% lower than that of the control group. From the above, it can be seen that the treatment of theanine significantly inhibited the accumulation of H2O2 in kiwifruit during storage, thereby reducing the oxidative damage to fruits and vegetables caused by the large accumulation of reactive oxygen.

[0040] like Figure 8 The figure shows the O2· - The changing trend of the production rate. - The production rate increased with the extension of storage time during storage. - There was no significant difference in the production rate between the two groups on the 3rd day (P<0.05). - The production rate increased rapidly under the treatment of theanine. On the 6th day, a significant difference began to appear between the control group and the treatment group (P<0.05), and the content was 29.84% higher than that of the control group. Until the 12th day of storage, there was still a significant difference between the control group and the treatment group (P<0.05), and the content was 13.66% higher than that of the control group.

[0041] The metabolic balance of active oxygen in plants is regulated by a series of antioxidant enzymes, among which key enzymes include catalase (SOD), superoxide dismutase (CAT), and peroxidase (POD). These antioxidant enzymes can reduce oxidative damage to fruits and vegetables and reduce fruit decay.

[0042] Determination of SOD, CAT, POD activity: SOD activity determination: 0.3 g sample was added to 1.8 mL 0.1 mol L -1 , pH 7.8 PBS buffer (containing 5 mmolL -1 The slurry was ground in DTT and 5% PVP and centrifuged at 12000 × g for 30 min at 4 °C. The reaction system included 1.7 mL 0.05 mol L -1 , pH 7.8 PBS buffer, 0.3 mL 0.13 mol L -1 Methionine (MET) solution, 0.3 mL 0.75 mmol L -1 Nitroblue tetrazolium (NBT) solution, 0.3 mL 0.1 mmol L -1 EDTA-Na2 solution, 0.3 mL 0.02 mmol L -1 The reaction mixture was reacted under 4000 lx fluorescent light for 15 min, and then the absorbance was measured at 560 nm. The inhibition rate of NBT photoreduction was 50% as one SOD activity unit (U), and the unit was expressed as U g -1 FW.

[0043] CAT activity assay: 0.3 g sample was added to 1.8 mL 0.05 mol L -1 , pH 7.5 PBS buffer (containing 5 mmol L -1 DTT and 5% PVPP), centrifuged at 12000 × g for 30 min at 4 °C, and the reaction system included 0.05 mol L -1 , pH 7.5 PBS buffer, 0.02 mol L -1 H2O2 and 100 μL crude enzyme extract were added, and the absorbance was measured at 240 nm. The amount of enzyme that decomposed 1 μmol H2O2 per minute was defined as one CAT activity unit (U), and the unit was expressed as U g -1 FW.

[0044] POD activity assay: 0.3 g sample in 1.8 mL 1 mmol L -1The slurry was ground in acetic acid-sodium acetate buffer (containing 1 mmol polyethylene glycol, 4% PVPP and 1% Triton X-100) at pH 5.5 and centrifuged at 12000 × g for 30 min at 4 °C. The reaction system included 3 mL of 25 mmol L -1 Guaiacol solution, 0.5 mL enzyme extract and 0.5 mol L -1 H2O2 solution: After adding H2O2 solution, the absorbance value was measured at 470 nm immediately. The absorbance change value per gram of sample per minute increased by 0.1 as one POD activity unit (U), and the unit is expressed as U g -1 FW.

[0045] like Fig. 9 The figure shows the changing trend of CAT activity during storage of kiwifruit. Regardless of the control group or theanine treatment, the CAT activity showed a trend of increasing first and then decreasing as the storage time continued. The activity began to show significant differences between the control group and the treatment group on the 6th day (P<0.05). Regardless of the control group or the treatment group, the CAT enzyme activity reached its peak on the 6th day, and then the enzyme activity showed a downward trend. At this time, the CAT activity of the treatment group was 36.00% higher than that of the control group. Since the downward trend of the treatment group was significantly lower than that of the control group in the later storage period, there was still a significant difference between the control group and the treatment group on the 12th day (P<0.05), and the enzyme activity under theanine treatment was 35.71% higher than that of the control group.

[0046] like Fig.10 The figure shows the changing trend of SOD activity during storage of kiwifruit. The changing trend of SOD activity under theanine treatment during storage was the same as that of the control group, showing a trend of first decreasing and then increasing. At 0-3d, the increase in both the treatment group and the control group was relatively gentle. At 3-6d, the increase in enzyme activity under theanine treatment was greater than that in the control group, and there was a significant difference between the two (P<0.05). At the 6th day, the SOD activity of the control group was 11.67% lower than that of the treatment group. At the 9th day of storage, the enzyme activity of both the treatment group and the control group reached a peak, and then the enzyme activity decreased. At 9-12d, although the downward trend of enzyme activity under theanine treatment was higher than that of the control group, it still showed that the enzyme activity of the treatment group was higher, and the effect was better. At the 12th day of storage in the late stage, the enzyme activity of the treatment group was 28.76% higher than that of the control group, and the significant difference still existed (P<0.05).

[0047] like Fig.11The figure shows the changing trend of POD activity during storage of kiwifruit. During the entire storage period, the POD activity showed the same trend under the treatment of theanine and the control group, showing a trend of first increasing and then decreasing. On the 6th day, there was a significant difference in the enzyme activity between the two groups (P<0.05), and the enzyme activity of the treatment group was higher than 66.70%. On the 9th day, the enzyme activity of the treatment group and the control group reached a peak, and the peak value under theanine treatment was significantly higher than that of the control group, and then began to decline. In the later stage of storage, there was still a significant difference between the treatment group and the control group (P<0.05).

[0048] The above results indicate that under the stress of Botrytis cinerea, ROS burst in kiwifruit in the early stage of storage directly inhibits the growth of Botrytis cinerea, and in the later stage of storage, the activity of antioxidant enzymes in the fruit is enhanced, which is one of the mechanisms by which theanine treatment induces disease resistance in kiwifruit.

Claims

1. The application of natural inducers in fruit preservation is characterized in that: A natural inducer is used as an active ingredient to inhibit the growth of gray mold on fruits and induce the immune system of the fruits to enhance active oxygen metabolism; the natural inducer is theanine; and the fruit is kiwi fruit.

2. The use of the natural inducer according to claim 1 in fruit preservation, characterized in that: The active oxygen metabolism includes hydrogen peroxide content and superoxide anion free radical generation rate. Under the treatment of theanine, the hydrogen peroxide content is reduced and the superoxide anion free radical generation rate is increased.

3. The use of the natural inducer according to claim 1 in fruit preservation, characterized in that: The active oxygen metabolism also includes antioxidant enzymes, which include catalase, superoxide dismutase, and peroxidase. Under the treatment of theanine, the activity of catalase, superoxide dismutase, and peroxidase are all improved.

4. The use of the natural inducer according to any one of claims 1 to 3 in fruit preservation, characterized in that: Theanine concentration is 1-20 mmol L -1 In the range of 10 mmol L -1 Theanine has the best effect in inhibiting the growth of Botrytis cinerea hyphae.

5. The use of the natural inducer according to any one of claims 1 to 3 in fruit preservation, characterized in that: Theanine is used to regulate the active oxygen metabolism of kiwifruit at a concentration of 1-10 mmol L -1 In the range of 10 mmol L -1 Theanine has the best effect in regulating the metabolism of active oxygen in kiwi fruit.

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