Antioxidant substance-based detection method for kiwifruit infected with botryosphaeria dothidea

Abstract

The invention discloses a method for detecting kiwi fruits infected with botryosphaeria dothidea based on antioxidant substances. The ratio of the superoxide dismutase activity to the flavonoid content in the kiwi fruits is used for distinguishing the fruits infected by botryosphaeria dothidea and the fruits not infected by the botryosphaeria dothidea in the storage process, and the fruits with the ratio of the superoxide dismutase activity to the flavonoid content in the to-be-detected sample being 310 or above are diseased fruits infected by the botryosphaeria dothidea; the fruits with the ratio of the superoxide dismutase activity to the flavonoid content being lower than 250 are healthy fruits. The method is simple to operate and reliable in detection result, and can provide reference for judging whether the kiwi fruits are infected by pathogenic bacteria or not in the storage process.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and specifically relates to a method for detecting kiwifruit fruits infected with Botrytis cinerea based on antioxidant substances Technical Background

[0002] kiwi( Chinese gooseberry ), also known as sheep peach vine, kiwi fruit, etc., belongs to the Ericales ( Ericales ) Actinidiaceae Actinidiaceae ) Actinidia Actinidia ), is a perennial dioecious vine. Kiwifruit is a berry with a high juice content and delicious taste. Studies have shown that kiwifruit is rich in nutrients, rich in vitamins, dietary fiber, sugar, organic acids, amino acids and a variety of trace elements necessary for the human body. It has antioxidant, anti-inflammatory, antibacterial, anti-hypertensive, anti-hypercholesterolemia, neuroprotective and other effects, and also helps promote intestinal health. However, most kiwifruit varieties are prone to softening, rotting and other problems after harvest, which leads to the loss of flavor and even edible value. In particular, soft rot caused by infection with Botrytis cinerea has caused serious economic losses to the storage, transportation and sales of kiwifruit, which is one of the main reasons restricting the development of my country's kiwifruit industry.

[0003] Botrytis cinerea Botryosphaeria dothidea ) belongs to the phylum Ascomycota and is a pathogenic fungus widely distributed throughout the world. It has a wide range of hosts and can cause rot in many fruits such as kiwifruit, apples, blueberries, and pears, as well as cankers in fruit trees. Botrytis cinerea can infect kiwifruit through tiny wounds, pores, or natural openings on the surface of the fruit, causing the disease. The lesions on the fruit infected by Botrytis cinerea appear as round or oval water-soaked lesions. Because Botrytis cinerea is latent and the kiwifruit peel has a certain thickness and toughness, forming a physical barrier, it is difficult to determine whether the fruit is infected by pathogens based on appearance alone.

[0004] Superoxide dismutase and flavonoids are important antioxidant components in kiwifruit. SOD can catalyze the dismutation reaction of superoxide anion free radicals and effectively remove free radicals. Flavonoids can also remove various free radicals such as hydroxyl free radicals and hydrogen peroxide free radicals, reducing the damage of oxidative stress to the fruit. They act together on the fruit, have complementary effects in anti-oxidation and enhancing the overall resistance of the fruit, and can enhance the resistance of the fruit to pathogenic fungi by reducing oxidative stress and inflammatory reactions. Summary of the invention

[0005] The purpose of the present invention is to provide a detection method based on antioxidant substances for kiwifruit infected with Botrytis cinerea, and to distinguish between fruits infected with Botrytis cinerea and uninfected fruits during storage by using the ratio of superoxide dismutase activity and flavonoid content in kiwifruit.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows: The invention discloses a method for detecting kiwifruit fruits infected with Botrytis cinerea based on antioxidant substances. The method comprises the following steps: detecting antioxidant substances in kiwifruit fruits, determining superoxide dismutase activity and flavonoid content in the fruits, and calculating their ratios. The tested samples having a ratio of superoxide dismutase activity to flavonoid content of more than 310 are diseased fruits infected with Botrytis cinerea, and the tested samples having a ratio of superoxide dismutase activity to flavonoid content of less than 250 are healthy fruits.

[0007] The strain number of the Botrytis cinerea is ACCC 38020.

[0008] The superoxide dismutase activity was measured by nitroblue tetrazolium photoreduction method.

[0009] The specific determination steps are as follows: 1 g of kiwifruit sample was homogenized in 5 mL of PBS buffer on ice and centrifuged at 10,000 rpm for 25 min at 4 °C using a high-speed centrifuge. The supernatant was the crude enzyme extract. 0.1 mL of the crude enzyme extract was mixed with 2.9 mL of the reaction solution and reacted at 25 °C and 3000 lx for 25 min. After the reaction, the A 560 The absorbance value at .

[0010] The calculation method of superoxide dismutase activity is: SOD activity (U·g -1 )=(A CK -A 560 )×V / (0.5×A CK ×W×V t ), where V is the total extraction volume, W is the fresh weight of the sample, and V t To measure volume.

[0011] The reaction solution specifically consists of 1.5 mL PBS, 0.3 mL methionine solution, 0.3 mL nitroblue tetrazolium solution, 0.3 mL EDTA-Na2, 0.2 mL sterile water, and 0.3 mL riboflavin.

[0012] The flavonoid content was determined by 1% hydrochloric acid methanol extraction method.

[0013] The specific determination steps are as follows: Take 2 g of kiwifruit sample, add a small amount of 1% hydrochloric acid methanol solution, grind in ice bath and dilute to 10 mL, place at 4 °C for 20 min and filter, and measure the filtrate by ELISA. 325 The absorbance value at 1 / 2 was taken as the relative content of flavonoids.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes the ratio of superoxide dismutase activity and flavonoid content in kiwifruit to distinguish fruits infected with Botrytis cinerea and uninfected fruits during storage. The discrimination method of the present invention has the following characteristics: simple and rapid operation, accurate and reliable results, and can provide a basis for evaluating the quality changes of kiwifruit fruits during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A6, A8, B6, B8 represent diseased fruit stored for 6 days, diseased fruit stored for 8 days, healthy fruit stored for 6 days, and healthy fruit stored for 8 days, respectively. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below in conjunction with specific examples, but the implementation methods of the present invention include but are not limited to the scope represented by the following examples. Example

[0017] A method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances. The ratio of superoxide dismutase activity and flavonoid content in kiwifruit is used to distinguish between fruits infected with Botrytis cinerea and fruits not infected during storage. Fruits infected with Botrytis cinerea in samples with a ratio of superoxide dismutase activity to flavonoid content above 310 are diseased fruits infected with Botrytis cinerea, and fruits infected with Botrytis cinerea in samples with a ratio of superoxide dismutase activity to flavonoid content below 250 are healthy fruits.

[0018] The specific method is: Pick the hyphae of Botrytis cinerea in the center of potato dextrose agar (PDA) medium, put it in a 25℃ incubator and culture it for 5-7 days. Use a sterilized cork puncher to cut the Botrytis cinerea cake with a diameter of 6 mm at the edge of the hyphae for later use.

[0019] On the day of harvest, kiwifruit was soaked in 2% ethanol solution for 5 minutes, naturally air-dried and sealed for one week to accelerate the softening of kiwifruit. Then the kiwifruit was divided into two groups. The diseased fruits infected by Botrytis cinerea were the fruits inoculated with 6 mm Botrytis cinerea cake at the equator, and the healthy fruits were the fruits not inoculated with cake. After 6-8 days of storage, the peeled pulp samples at the equator of the fruit were taken for testing.

[0020] The superoxide dismutase activity and flavonoid content were determined and their ratio was calculated. The samples with a ratio of superoxide dismutase activity to flavonoid content above 310 were diseased fruit samples infected by Botrytis cinerea, and the samples with a ratio of superoxide dismutase activity to flavonoid content below 250 were healthy fruit samples.

[0021] Superoxide dismutase activity was determined by nitroblue tetrazolium photoreduction method. 1 g of kiwifruit sample was homogenized in 5 mL of PBS buffer (pH = 7.8) in an ice bath, and then centrifuged at 10,000 rpm for 25 min at 4 °C using a high-speed centrifuge. The supernatant was the crude enzyme extract. 0.1 mL of crude enzyme extract was mixed with 2.9 mL of reaction solution (1.5 mL of PBS, 0.3 mL of methionine solution, 0.3 mL of nitroblue tetrazolium solution, 0.3 mL of EDTA-Na2, 0.2 mL of sterile water, and 0.3 mL of riboflavin). After mixing, react at 25 °C and 3000 lx for 25 min (blank is dark conditions at the same temperature). After the end, A was measured. 560 The absorbance value at the position of the superoxide dismutase activity is calculated as follows: SOD activity (U·g -1 )=(A CK -A 560 )×V / (0.5×A CK ×W×V t ), where V is the total extraction volume, W is the fresh weight of the sample, and V t To measure volume.

[0022] The flavonoid content was determined by 1% hydrochloric acid methanol extraction. Take 2 g of kiwifruit sample, add a small amount of 1% hydrochloric acid methanol solution, grind it in an ice bath and dilute it to 10 mL, place it at 4°C for 20 min, filter it, and measure the filtrate in A by an enzyme marker. 325 The absorbance value at 1 / 2 was taken as the relative content of flavonoids.

[0023] The ratio of superoxide dismutase activity to flavonoid content in the examples is shown in Figure 1 (A6, A8, B6, B8 represent diseased fruits stored for 6 days, diseased fruits stored for 8 days, healthy fruits stored for 6 days, and healthy fruits stored for 8 days, respectively).

[0024] The present invention uses the ratio of superoxide dismutase activity to flavonoid content as a standard for distinguishing diseased fruits infected by Botrytis cinerea and healthy fruits, that is, samples to be tested with a ratio of superoxide dismutase activity to flavonoid content of more than 310 are diseased fruit samples infected by Botrytis cinerea, and samples to be tested with a ratio of superoxide dismutase activity to flavonoid content of less than 250 are healthy fruit samples.

Claims

1. A method for detecting antioxidant substances in kiwifruit infected with Botrytis cinerea, characterized in that: By detecting the antioxidant substances in kiwi fruit, the superoxide dismutase activity and flavonoid content in the fruit were determined, and their ratio was calculated. If the ratio of superoxide dismutase activity to flavonoid content in the test sample was above 310, it was diseased fruit infected by Botrytis cinerea, and if the ratio of superoxide dismutase activity to flavonoid content was below 250, it was healthy fruit.

2. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 1, characterized in that: The strain number of the Botrytis cinerea is ACCC 38020.

3. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 2, characterized in that: The superoxide dismutase activity was measured by nitroblue tetrazolium photoreduction method.

4. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 3, characterized in that: The specific determination method steps are as follows: 1 g of kiwifruit sample was homogenized in 5 mL of PBS buffer on ice and centrifuged at 10,000 rpm for 25 min at 4 °C using a high-speed centrifuge. The supernatant was the crude enzyme extract. 0.1 mL of the crude enzyme extract was mixed with 2.9 mL of the reaction solution and reacted at 25 °C and 3000 lx for 25 min. After the reaction, the A 560 The absorbance value at .

5. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 4, characterized in that: The calculation method of superoxide dismutase activity is: SOD activity (U·g -1 )=(A CK -A 560 )×V / (0.5×A CK ×W×V t ), where V is the total extraction volume, W is the fresh weight of the sample, and V t To measure volume.

6. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 4, characterized in that: The reaction solution specifically consists of 1.5 mL PBS, 0.3 mL methionine solution, 0.3 mL nitroblue tetrazolium solution, 0.3 mL ETA-Na2, 0.2 mL sterile water, and 0.3 mL riboflavin.

7. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 2, characterized in that: The flavonoid content was determined by 1% hydrochloric acid methanol extraction method.

8. The method for detecting kiwifruit infected with Botrytis cinerea based on antioxidant substances according to claim 7, characterized in that: The specific determination method steps are as follows: Take 2 g of kiwifruit sample, add a small amount of 1% hydrochloric acid methanol solution, grind in ice bath and dilute to 10 mL, place at 4 °C for 20 min and filter, and measure the filtrate by ELISA. 325 The absorbance value at 1 / 2 was taken as the relative content of flavonoids.

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