Treatment method for relieving cold injury of harvested mangoes

Abstract

The invention belongs to the technical field of postharvest preservation of fruits and vegetables, and particularly relates to a treatment method for relieving cold injury of postharvest mangoes, which comprises the following steps: treating mango fruits with an n-butyl alcohol aqueous solution, air-drying, and storing in a low-temperature environment. The cold injury index of the mangoes can be remarkably reduced through n-butyl alcohol treatment, and symptoms such as black spots are relieved; related enzyme activity is inhibited, and cell membranes are protected; the activity of antioxidant enzyme is improved, and the oxidation resistance is enhanced; energy metabolism is regulated and controlled; and the expression level of membrane lipid degradation genes can be lowered, and the expression level of resistance genes can be increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of postharvest preservation of fruits and vegetables, and particularly relates to a treatment method for reducing postharvest chilling injury of mangoes. Background Art

[0002] Mango (Mangifera indica L.) is a typical tropical fruit tree, with its fruit having a rich flavor and abundant nutrition, and occupying an important position in the global fruit market. As a climacteric fruit, mango has active metabolism after harvest, is extremely prone to softening and rotting, has poor storage and transportation performance, and is likely to cause high postharvest losses. Therefore, in order to extend the shelf life and maintain the commercial quality, cold storage preservation, as a conventional and widely used technical means in postharvest fruit treatment, is generally used in the storage and transportation process of mangoes. In the existing cold storage system, the storage temperature is usually set at 13°C.

[0003] However, as a tropical fruit, mango is extremely sensitive to low temperature, and its suitable storage temperature should generally not be lower than 13°C. When the storage temperature is lower than this critical value, the fruit is extremely prone to chilling injury, manifested as a series of physiological disorders such as peel browning, fruit surface depression, flesh browning and black spots, resulting in significant deterioration of the fruit appearance, texture and flavor, and seriously affecting the commercial value and storage and transportation adaptability. Although appropriately increasing the storage temperature can effectively alleviate the occurrence of chilling injury, it will also accelerate the ripening and senescence of the fruit, significantly shorten the shelf life, and it is difficult to meet the basic requirements of long-distance transportation and commercial circulation for the shelf life. The above contradictions lead to a dilemma in the temperature control during the cold storage preservation of mangoes, and there is an urgent need to find an effective postharvest regulation means that takes into account both extending the shelf life and alleviating chilling injury.

[0004] To alleviate the chilling injury of mangoes, existing research and application attempts include treatment methods such as physical regulation, chemical preservation and biomembrane materials. For example, in the physical method, the intermittent warming technology restores the normal temperature for a short time during cold storage to activate the fruit defense mechanism, thereby slowing down the low temperature stress response; in the chemical means, exogenous hormone and antioxidant treatments, 1-methylcyclopropene (1-MCP) treatment are used to delay fruit senescence or regulate the stress response signaling pathway. Although the above methods can reduce the degree of chilling injury to a certain extent, they generally have limitations such as complex operation, high treatment cost, adverse effects on flavor or texture, and some chemical preservatives have problems such as difficult dose control, poor treatment uniformity and unclear action mechanism, and it is difficult to meet the green, safe and efficient industrialization requirements.

[0005] Studies have shown that the occurrence of chilling injury is closely related to the stability of the cell membrane system, and membrane lipid metabolism disorder is an important physiological basis for mango chilling injury. Among them, phospholipase D and phospholipase C are key enzymes involved in membrane lipid degradation, catalyzing the conversion of phospholipids to phosphatidic acid and diacylglycerol, respectively. When the above enzymes are overactivated, it will cause the loss of membrane phospholipid components, destroy the structural integrity and selective permeability of the cell membrane, and induce a series of chilling injury symptoms. Therefore, regulating the activity of key enzymes in membrane lipid metabolism and stabilizing the structure of the membrane system are important technical paths to alleviate mango chilling injury.

[0006] As a phospholipid metabolism regulator with a simple structure and a wide range of sources, n-butanol has been shown to inhibit phospholipase D activity in model plant systems, blocking its catalytic transphospholipidation reaction, thereby intervening in the process of membrane lipid degradation. However, existing studies have mostly focused on model plants such as Arabidopsis thaliana. There is still a lack of systematic verification and practical application data on the role of n-butanol in alleviating chilling damage in fruit and vegetable products, especially mango, a tropical fruit. The effective concentration range, action time window, and synergistic mechanism of n-butanol treatment with low-temperature storage conditions are still unclear, making it difficult to directly apply it to post-harvest preservation industry practices.

[0007] Therefore, there is an urgent need to develop an n-butanol regulation strategy that can be applied to mango postharvest processing, is easy to operate, and is compatible with the existing cold chain system. On the premise of ensuring fruit quality and food safety, it can achieve effective regulation of membrane lipid metabolism, thereby improving the fruit's ability to adapt to low temperature environments, reducing the risk of chilling damage, extending the storage and transportation shelf life, and improving the post-harvest preservation level. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a treatment method for reducing chilling injury of post-harvest mangoes, comprising treating mango fruits with an aqueous solution of n-butanol, air-drying the mango fruits, and storing them in a low temperature environment.

[0009] In some preferred embodiments, the mango fruit is washed with tap water to remove surface stains before being treated with n-butanol, and then dried naturally.

[0010] In some preferred embodiments, the temperature at which the mango fruit is treated with the n-butanol aqueous solution is 20-30°C.

[0011] In some preferred embodiments, the temperature at which the mango fruit is treated with the n-butanol aqueous solution is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0012] In some preferred embodiments, the mass concentration of the n-butanol aqueous solution is 0.5% to 2%.

[0013] In some preferred embodiments, the mass concentration of the n-butanol aqueous solution is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%.

[0014] In some preferred embodiments, the mango fruits are soaked in the n-butanol aqueous solution and gently stirred, and the treatment time is 2 to 10 minutes.

[0015] In some preferred embodiments, the mango fruits are soaked in the n-butanol aqueous solution and gently stirred, and the treatment time is 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes.

[0016] In some preferred embodiments, the air-drying step is carried out under ventilation conditions for 10 to 120 minutes.

[0017] In some preferred embodiments, the air-drying step is carried out under ventilation conditions for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes.

[0018] In some preferred embodiments, the weight of the mango fruits is 200 to 500 grams.

[0019] In some preferred embodiments, the weight of the mango fruits is 200 grams, 300 grams, 400 grams, 500 grams.

[0020] In some preferred embodiments, the storage step is carried out in an environment with a temperature of 2 to 8 °C.

[0021] In some preferred embodiments, the storage temperature is 2 °C, 2.5 °C, 3 °C, 3.5 °C, 4 °C, 4.5 °C, 5 °C, 5.5 °C, 6 °C, 6.5 °C, 7 °C, 7.5 °C, 8 °C.

[0022] The storage step is carried out in an environment with a relative humidity of 80% to 90%.

[0023] In some preferred embodiments, the relative humidity is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%.

[0024] The second aspect of the present invention provides the use of n-butanol with a mass concentration of 0.5 to 2% in the preparation of a treatment agent for inhibiting postharvest chilling injury of mangoes.

[0025] By treating mangoes after harvest with an aqueous solution of n-butanol and combining appropriate treatment time and storage conditions, the present invention achieves the technical effects of effectively reducing chilling injury, maintaining membrane stability, and enhancing antioxidant capacity. The specific beneficial effects are as follows: (1) The n-butanol treatment in the present invention can effectively alleviate the chilling injury symptoms caused by low-temperature stress, significantly reduce the browning of the mango epidermis and tissue damage, and thus improve the fruit's resistance to chilling injury.

[0026] (2) The n-butanol treatment in the present invention can significantly delay the process of fruit softening under low-temperature storage conditions, effectively maintain the texture characteristics of mangoes, and extend the storage period and marketable time of the fruits.

[0027] (3) The n-butanol treatment in the present invention helps to delay the deterioration of the appearance quality of mango fruits, improve the commercial performance during storage, and maintain the market value of the fruits.

[0028] (4) The n-butanol treatment in the present invention can stabilize the cell membrane structure, maintain the integrity of the membrane system, and enhance the antioxidant defense ability of mangoes, thereby synergistically slowing down the degradation of the membrane system and significantly improving the physiological stability and quality retention ability of the fruits during low-temperature storage.

[0029] (5) The n-butanol treatment in the present invention enhances the antioxidant capacity and physiological stability of mangoes through multi-pathway synergistic effects. Specifically, n-butanol significantly reduces the accumulation of reactive oxygen species, inhibits the generation of superoxide anions and hydrogen peroxide, and alleviates the damage of oxidative stress to fruit cells; at the same time, it increases the activity of antioxidant enzymes, delays the decline of catalase activity, enhances the activity of phenylalanine ammonia-lyase, and improves the stress resistance of the fruits.

[0030] (6) The n-butanol treatment in the present invention alleviates the chilling injury response caused by low-temperature stress by regulating membrane lipid metabolism, enhances the activity of phospholipase A, and inhibits the activities of phospholipase C, phospholipase D, and lipoxygenase, helping to maintain the stability of the fruit membrane system and improve the physiological quality during storage.

[0031] (7) The n-butanol treatment in the present invention can effectively regulate the energy metabolism process of mango fruits during low-temperature storage, manifested as a moderate decrease in the contents of ATP, ADP, and AMP and an earlier advancement of the process of increasing the energy charge value, thereby improving the energy supply efficiency and metabolic stability of cells and enhancing the fruit's adaptability to low-temperature stress.

[0032] (8) The n-butanol treatment in the present invention can significantly down-regulate the gene expression related to membrane lipid degradation (such as MiPLDα, MiPLC6, MiLOX3.1), inhibit the activities of phospholipase D, phospholipase C, and lipoxygenase; at the same time, up-regulate the expression of genes such as MiPLA2, MiATPγ2, MiDGK7, etc., enhance membrane stability and energy metabolism ability, and reduce the chilling injury of mangoes during low-temperature storage. Brief Description of the Drawings

[0033] Figure 1 Figure A is a comparison diagram of the appearance of freezing injury between the n-butanol treatment group and the control group during the 30-day cold storage of mangoes at 4°C. Figure B is a line graph showing the change in the chilling injury index of mangoes over time. Figure C is a line graph showing the change in the color difference of mangoes over time. Figure D is a line graph showing the change in the hardness of mangoes over time. Figure E is a line graph showing the change in the electrolyte leakage rate of mangoes over time. Figure F is a line graph showing the change in the MDA content of mangoes over time.

[0034] Figure 2 Figure A is a line graph showing the change in the O 2- content of mangoes over time. Figure B is a line graph showing the change in the H2O2 content of mangoes over time. Figure C is a line graph showing the change in the superoxide dismutase activity of mangoes over time. Figure D is a line graph showing the change in the ascorbate peroxidase activity of mangoes over time. Figure E is a line graph showing the change in the PAL activity of mangoes over time. Figure F is a line graph showing the change in the catalase activity of mangoes over time.

[0035] Figure 3 Figure A is a line graph showing the change in the PLA activity of mangoes over time. Figure B is a line graph showing the change in the phospholipase C activity of mangoes over time. Figure C is a line graph showing the change in the phospholipase D activity of mangoes over time. Figure D is a line graph showing the change in the lipoxygenase activity of mangoes over time.

[0036] Figure 4 is a bar graph showing the expression levels of the membrane lipid metabolism enzyme genes MiPLDβ, MiPLDα, MiPLC6, MiPLC2, MiLOX3.1, MiDGK7, MiDGK5, MiPLA2, and MiPLA1 in mangoes.

[0037] Figure 5 Figure A is a line graph showing the change in the ATP content of mangoes over time. Figure B is a line graph showing the change in the ADP content of mangoes over time. Figure C is a line graph showing the change in the AMP content of mangoes over time. Figure D is a line graph showing the change in the energy charge of mangoes over time.

[0038] Figure 6 is a bar graph showing the expression levels of the energy cycle genes MiATPγ1, MiATPγ2, and MiAMP in mangoes. Detailed Embodiments

[0039] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further elaborate on the present invention.

[0040] Term Explanation: (1) Chilling Injury Index: An indicator used to measure the degree of damage to plants under low-temperature stress. It is usually graded according to tissue browning, the area of necrotic spots, or the degree of physiological metabolic disorders. The higher the value, the more severe the chilling injury.

[0041] (2) n-Butanol: A four-carbon straight-chain alcohol, belonging to polar organic solvents, commonly used for lipid extraction in biochemical experiments or as a reaction medium, with slight toxicity.

[0042] (3) Phospholipase A (PLA): An enzyme that hydrolyzes the ester bonds at the sn-1 or sn-2 positions of phospholipids, divided into two categories: PLA1 and PLA2. Among them, PLA2 releases polyunsaturated fatty acids and participates in cell signal transduction and inflammatory responses.

[0043] (4) Phospholipase C (PLC): An enzyme that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol trisphosphate. Both of them participate in calcium ion mobilization and protein kinase C activation as second messengers.

[0044] (5) Phospholipase D (PLD): An enzyme that hydrolyzes phospholipids to generate phosphatidic acid and free polar head groups. PA plays a regulatory role in membrane dynamics, cytoskeleton reorganization, and stress responses.

[0045] (6) Lipoxygenase (LOX): An enzyme that catalyzes the oxygenation reaction of polyunsaturated fatty acids to generate hydroperoxides. The products participate in jasmonic acid synthesis and plant stress responses.

[0046] (7) Electrolyte Leakage Rate: An indicator to measure the degree of cell membrane damage, evaluating membrane integrity by detecting the proportion of leaked electrolytes in total electrolytes. The higher the value, the more severely the membrane is damaged.

[0047] (8) Malondialdehyde (MDA): One of the end products of membrane lipid peroxidation, and its content reflects the degree of cell oxidative damage, commonly measured by the thiobarbituric acid method.

[0048] (9) Superoxide Dismutase (SOD): A key antioxidant enzyme that catalyzes the dismutation of superoxide anions into hydrogen peroxide and oxygen, divided into isoenzymes such as Cu / Zn-SOD, Mn-SOD, and Fe-SOD, which is the first line of defense in the reactive oxygen species scavenging system.

[0049] (10) Catalase (CAT): An enzyme that decomposes hydrogen peroxide into water and oxygen. It is mainly located in peroxisomes, preventing oxidative damage caused by the accumulation of hydrogen peroxide, and working in concert with superoxide dismutase, POD, etc. to maintain the redox balance.

[0050] (11) Superoxide (O2-): A reactive oxygen species generated by the single-electron reduction of oxygen molecules. It has strong oxidizing properties and can damage biological macromolecules. Superoxide dismutase can convert it into hydrogen peroxide and oxygen.

[0051] (12) Hydrogen Peroxide (H2O2): A reactive oxygen species that participates in cell regulation as a signaling molecule at low concentrations and induces oxidative stress at high concentrations. It can be decomposed by catalase or ascorbate peroxidase.

[0052] (13) Ascorbate Peroxidase (APX): An ascorbate-dependent peroxidase that catalyzes the reduction of hydrogen peroxide to water while oxidizing ascorbic acid to monodehydroascorbic acid. It is a key enzyme in the plant antioxidant system.

[0053] (14) Reference Gene: A stably expressed gene used to correct differences between samples in gene expression analysis. Common ones include Actin, GAPDH, UBQ, etc., ensuring the reliability of the quantitative results of target genes.

[0054] (15) Chilling Injury Area: The percentage of the total area of plant tissues that shows necrosis or browning under low-temperature stress, used to visually evaluate the degree of chilling injury, often quantified in combination with image analysis software.

[0055] (16) Ferrous Sulfate Colorimetry: A method for determining the content of hydrogen peroxide by the reaction of ferrous ions with potassium thiocyanate to form a red complex. It is applicable to the quantitative analysis of hydrogen peroxide in plant oxidative stress research.

[0056] (17) Hydroxylamine Hydrochloride Colorimetry: A method for determining the content of superoxide by using hydroxylamine to react with superoxide to generate nitrite ions, which then react with sulfanilic acid and naphthylamine to develop color. It is commonly used in the analysis of superoxide dismutase activity.

[0057] (18) Linoleic Acid Colorimetry: A method for determining the activity of lipoxygenase or the degree of lipid peroxidation based on the oxidation of linoleic acid catalyzed by lipoxygenase to generate conjugated dienes.

[0058] (19) Hydrogen Peroxide Colorimetry: Commonly used titanium reagent method or ferrous sulfate method. By forming a yellow complex between hydrogen peroxide and a specific reagent, the absorbance is measured at 415 nm to quantify hydrogen peroxide.

[0059] (20) NBT Colorimetry: A classic method for quantifying the content of O2- or the activity of SOD (inhibition rate method) by using superoxide to reduce nitroblue tetrazolium chloride to form a blue formazan precipitate and measuring the absorbance at A560 nm.

[0060] (21) Phenylalanine Ammonia-Lyase (PAL): A key enzyme in plant secondary metabolism that catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid. It is the starting enzyme of the phenylpropanoid metabolic pathway, participates in the synthesis of secondary metabolites such as lignin and flavonoids, and plays an important role in plant disease resistance and stress response.

[0061] (22) L-Phenylalanine Colorimetric Method: An analytical method for quantifying the activity of phenylalanine ammonia-lyase by measuring the change in absorbance of trans-cinnamic acid at a wavelength of 290 nm. This method is based on the reaction of phenylalanine ammonia-lyase catalyzing the deamination of L-phenylalanine to form trans-cinnamic acid and is a commonly used technique for studying plant secondary metabolism and stress resistance.

[0062] (23) High Performance Liquid Chromatography (HPLC): An analytical technique based on the separation of the mobile phase driven by a high-pressure pump through a chromatographic column, featuring high resolution, high sensitivity, and high selectivity. It is widely used in the qualitative and quantitative analysis of biomolecules such as plant hormones and secondary metabolites.

[0063] (24) Enzyme-Linked Immunosorbent Assay (ELISA): An immunoassay technique based on the principle of specific antigen-antibody binding. By using an enzyme-labeled secondary antibody to catalyze a colorimetric reaction to quantitatively detect the content of the target protein or hormone, it features high specificity and high sensitivity.

[0064] (25)RNAprep Pure Plant Kit: A kit specifically designed for extracting high-quality total RNA from plant tissues. It uses unique lysis buffer and adsorption column technologies to effectively remove interfering substances such as polysaccharides and polyphenols. Combined with DNase treatment, it can ensure the integrity and purity of RNA, and is suitable for subsequent molecular biology experiments such as fluorescence quantitative PCR.

[0065] (26)Color Difference: A parameter that quantifies the difference between a sample and a standard color through the CIE-Lab color system, including three dimensions: lightness (L), red-green value (a), and yellow-blue value (b*). It is commonly used to evaluate fruit maturity, leaf senescence, or tissue browning caused by chilling injury.

[0066] (27)Quantitative Real-Time PCR (qRT-PCR): A technique for quantifying the expression level of specific genes by real-time monitoring of fluorescence signals. It uses SYBR Green dye or TaqMan probes to detect the accumulation of PCR products, and calculates the initial template amount through the cycle threshold. It features high sensitivity and high accuracy and is the gold standard for gene expression analysis.

[0067] (28)Energy Charge (EC): An important indicator reflecting the energy state of cells, indicating the distribution of high-energy phosphate bonds in the adenylate pool. Its value ranges from 0 to 1. The higher the energy charge value, the better the energy state of the cells and the more active the metabolic activities.

[0068] (29)Energy Metabolism: The process in living organisms that generates ATP through metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation, providing energy for various life activities of cells and being finely regulated by environmental stresses and internal regulatory networks.

[0069] (30)Reactive Oxygen Species (ROS): A class of highly reactive molecules derived from oxygen molecules, including superoxide anions, hydrogen peroxide, and hydroxyl radicals. At low concentrations, they participate in cell regulation as signaling molecules, while at high concentrations, they cause oxidative damage.

[0070] (31)Thiobarbituric Acid Method (TBA method): A commonly used colorimetric analysis method for quantitatively determining the degree of lipid peroxidation in biological samples through the specific condensation reaction of thiobarbituric acid with malondialdehyde.

[0071] (32) Ascorbic Acid (AsA): An important water-soluble antioxidant, also known as vitamin C, which participates in the ascorbate-glutathione cycle as an electron donor, can directly scavenge reactive oxygen species and regenerate other antioxidants, playing a core role in the plant antioxidant defense system.

[0072] (33) Nitroblue Tetrazolium Chloride (NBT): A pale yellow dye that can be reduced by superoxide anions to form a water-insoluble blue formazan precipitate, commonly used for the determination of superoxide dismutase activity or in situ detection of reactive oxygen species accumulation in tissues.

[0073] (34) L-Phenylalanine: An essential aromatic amino acid, which is a key precursor in the phenylpropanoid metabolic pathway of plants. It is deaminated to form trans-cinnamic acid under the catalysis of phenylalanine ammonia-lyase, and is also the main raw material for protein synthesis and various secondary metabolites.

[0074] (35) trans-Cinnamic Acid: The first intermediate in the phenylpropanoid metabolic pathway, which is formed by the deamination of L-phenylalanine catalyzed by phenylalanine ammonia-lyase. It is an important precursor for the synthesis of various plant secondary metabolites such as lignin and flavonoids.

[0075] (36) Phenylpropanoid Pathway: An important secondary metabolic pathway in plants, which uses L-phenylalanine as the starting substrate and generates various secondary metabolites such as lignin, flavonoids, and anthocyanins through a series of enzymatic reactions, playing a key role in plant structure support, disease resistance defense, and stress response.

[0076] (37) Adenylate Pool: The general term for three adenylates, ATP, ADP, and AMP, in cells. The composition ratio reflects the energy state of the cell, which is quantified by the energy charge value and is the core index for regulating cell energy metabolism and stress response.

[0077] (38) ATP (Adenosine Triphosphate): Adenosine triphosphate, the main energy currency in cells, contains two high-energy phosphate bonds and releases a large amount of energy when hydrolyzed, providing energy support for various life activities and being the core molecule of energy metabolism.

[0078] (39)ADP (Adenosine Diphosphate): Adenosine diphosphate, the product of ATP hydrolysis, contains a high-energy phosphate bond and can be regenerated into ATP through substrate-level phosphorylation or oxidative phosphorylation, playing a key role in the process of energy conversion and transfer.

[0079] (40)AMP (Adenosine Monophosphate): Adenosine monophosphate, the product of further hydrolysis of ADP, does not contain a high-energy phosphate bond and can react with ATP to generate two molecules of ADP through the catalysis of adenylate kinase. It is an important indicator molecule of the cell energy state.

[0080] (41)Hydroxyl Radical (·OH): The most reactive member of reactive oxygen species, with extremely strong oxidizing ability, can be generated from hydrogen peroxide through the Fenton reaction, and can attack biological macromolecules indiscriminately, resulting in serious oxidative damage.

[0081] Material source: (1)Mango fruits: Several Guiqi mangoes, with a single fruit weight of about 300 grams, were transported to the laboratory within 24 hours after harvesting.

[0082] (2)Reagents: 0.5 - 2% n-butanol, deionized water, hydroxylamine hydrochloride, sulfanilic acid solution, α-naphthylamine solution, ferrous sulfate, Ti(SO4)2, concentrated ammonia water, sulfuric acid, ascorbic acid, hydrogen peroxide, phosphate buffer, thiobarbituric acid, nitroblue tetrazolium, methionine, riboflavin, L-phenylalanine, ether, ELISA kit.

[0083] (3)Instruments: GY-1 type hardness tester, spectrophotometer, conductivity meter, centrifuge, constant temperature water bath, ELISA microplate reader, electronic balance, high performance liquid chromatograph, magnetic stirrer.

[0084] Example 1: Treatment method for alleviating postharvest chilling injury of mango (1)Experimental steps: Pretreat the mangoes under normal temperature conditions (i.e., about 20 - 30°C). First, rinse the surface of the mangoes with tap water to remove visible stains, and then air dry naturally. Select 168 mangoes and divide them into two groups on average: one group as the treatment group and the other group as the control group.

[0085] In the treatment group, 84 mangoes were completely immersed in an aqueous solution of 1% n-butanol by mass volume fraction. During the treatment process, maintain room temperature conditions and apply gentle stirring, and the treatment time is 3 minutes; in the control group, an equal number of mangoes were immersed in deionized water, and other treatment conditions were the same as those in the treatment group.

[0086] After the treatment, all mangoes were placed in a ventilated environment and naturally dried for 60 minutes to ensure that there was no obvious residual liquid on the surface. Subsequently, the mangoes were laid flat in a single layer in plastic baskets and transferred to a refrigerated environment for storage. The parameters of the cold storage were set as a temperature of 4 ± 0.5 °C and a relative humidity controlled at 85 - 90%. The entire storage period was 30 days.

[0087] During the storage period, it was set to sample once every 5 days, and the following indicators were measured and analyzed respectively: chilling injury index, fruit firmness, color change, cell membrane integrity, antioxidant enzyme activity, key enzyme activities related to membrane lipid metabolism, expression levels of related genes, and energy metabolism levels, etc. The systematic monitoring of the above parameters was used to evaluate the effects of different treatment methods on the low-temperature storage adaptability of mangoes.

[0088] (2) Determination of chilling injury index: Chilling injury symptoms were evaluated by regular observation and grading. The chilling injury symptoms on the mango epidermis were visually inspected every 5 days and scored according to the 0 - 4 grade standard: grade 0 indicates no chilling injury symptoms; grade 1 is that the chilling injury area is less than 10%, showing slight brown spots; grade 2 is that obvious brown spots appear in the area of 10 - 25%; grade 3 is that large areas of depressions appear in the area of 25 - 50%; grade 4 is that more than 50% of the area shows severe browning or water-soaked lesions. The chilling injury index was calculated by the following formula:

[0089] The experimental results are as Figure 1 shown in A and B: Under the condition of low-temperature storage at 4 °C, obvious chilling injury symptoms began to appear in the mango fruits of the control group from the 15th day, manifested as obvious aggravation of the brown spots on the peel and large-area tissue depression, and continued to deteriorate during the subsequent storage process. In contrast, only a small amount of brown spots were observed in the 1% n-butanol treatment group at the same time point, the tissue surface structure remained relatively intact, the overall browning degree was significantly reduced, and the chilling injury index measured at each time point was significantly lower than that of the control group.

[0090] Conclusion: n-Butanol treatment can effectively relieve the chilling injury symptoms induced by low-temperature stress, significantly reduce the browning of the mango epidermis and tissue damage, and improve the cold resistance of the fruits.

[0091] (3) Determination of fruit firmness: The fruit firmness was measured according to the previous method. A GY-1 type hardness tester with a probe diameter of 5 mm was used. Three different measuring points were selected at the equatorial part of each fruit for puncture measurement, and the obtained values were recorded and the average value was taken as the firmness value of the fruit. The results were expressed in units of Newton (N).

[0092] The experimental results are as Figure 1As shown in Figure D: With the extension of storage time, the fruit hardness of the control group of mangoes showed a significant downward trend, showing a texture change of rapid softening. In contrast, the rate of decrease in fruit hardness in the n-butanol treatment group was significantly slowed down. Especially in the interval from the 20th day to the 30th day, the hardness value remained basically constant, showing an overall linear downward trend without obvious softening phenomenon.

[0093] Conclusion: n-Butanol treatment can significantly delay the fruit softening process under low-temperature storage, effectively maintain the texture characteristics of mangoes, and thus extend the fruit storage period and its commercial sale time.

[0094] (4)Analysis of peel color change: The peel color change was quantitatively analyzed using a spectrophotometer. The measured parameters included L value (brightness), a value (red-green value), and b value (yellow-blue value). Samples were taken every five days, and the L, a, and b values were recorded and compared with the L, a, and b values on the 0th day of storage to calculate the change in brightness, the change in red-green value, and the change in yellow-blue value. The degree of color change (ΔE) was calculated by the following formula:

[0095] where ΔL, Δa, and Δb are the differences from the L, a, and b values on the 0th day, respectively, and ΔE represents the overall degree of color change.

[0096] The experimental results are as Figure 1 shown in Figure C: During the entire storage period, the color difference (ΔE) of the fruits in the control group and the n-butanol treatment group showed an upward trend, but the ΔE value at each storage time point in the treatment group was always significantly lower than that in the control group. This result indicates that n-butanol treatment can effectively inhibit the amplitude of fruit color change during low-temperature storage and maintain the stability of the peel color.

[0097] Conclusion: n-Butanol treatment helps to delay the deterioration of fruit appearance quality and improve the commercial performance during storage.

[0098] (5)Membrane integrity index: Membrane integrity was evaluated by the electrolyte leakage rate and the malondialdehyde content.

[0099] The method for measuring electrolyte leakage refers to the previous method. Use a slicer to cut the middle part of the mango peel into thin slices with a uniform thickness (about 0.07 mm). Then, use a puncher to take 10 small round slices with the same diameter from the pulp area of the thin slices and place them on a sieve. After rinsing the small round slices 3 times with deionized water, gently wipe the surface moisture with filter paper, place them in a 50 mL beaker, accurately add 20 mL of 0.25 mol / L mannitol solution, measure the electrolyte leakage rate of the solution, and record it as C0. Seal it with plastic wrap and place it in a constant temperature shaker for shaking for 1.5 h. After completion, measure the electrolyte leakage rate of the solution and record it as C1. After measurement, tightly seal the beaker mouth with plastic wrap and a rubber band, place it in boiling water at 100 °C for steaming for 20 min. After the solution in the beaker cools down, measure the electrolyte leakage rate again and record it as C2. Calculate the electrolyte leakage rate through the following formula:

[0100] The content of malondialdehyde was determined by the thiobarbituric acid colorimetric method. Weigh 0.2 g of the sample, dissolve it with 1.0 mL of 100 g / L TCA solution, vortex and mix well, then centrifuge at 4 °C and 1000 g for 20 min. Keep the supernatant at low temperature for later use. Take 250 μL of the supernatant (add 250 μL of 100 g / L trichloroacetic acid solution instead of the extract in the control blank tube) and add 250 μL of 0.67% thiobarbituric acid. After mixing, boil it in a boiling water bath for 20 min (add it to a 2.0 mL tube and seal it with a sealing film), take it out and cool it, then centrifuge again. Measure the absorbance values at 450, 532, and 600 nm respectively. The calculation formula for the MDA content is as follows:

[0101]

[0102] OD 450 ——Absorbance value at 450 nm wavelength; OD 532 ——Absorbance value at 532 nm wavelength; OD 600 ——Absorbance value at 600 nm wavelength; c——Concentration of malondialdehyde in the reaction solution (μmol / L); V——Total volume of the extract (mL); V S ——Volume used during measurement (mL); m——Sample mass (g).

[0103] The experimental results are as Figure 1As shown in Figure E: During the entire storage process, although the electrolyte leakage rate of both groups of fruits gradually increased with the prolongation of storage time, the leakage rate of the n-butanol treatment group was always lower than that of the control group and remained at a relatively low level. This phenomenon indicates that n-butanol treatment can effectively reduce the degree of increase in cell membrane permeability of fruits under chilling stress.

[0104] As Figure 1 shown in Figure F: During storage, the malondialdehyde content of the n-butanol treatment group was continuously lower than that of the control group at each time point, and the increase amplitude was relatively slow, indicating that this treatment can significantly inhibit membrane lipid peroxidation reaction and reduce the level of cell membrane oxidative damage.

[0105] Conclusion: n-butanol treatment can stabilize the cell membrane structure, maintain the integrity of the membrane system, and enhance the antioxidant defense ability of fruits, thereby synergistically slowing down the degradation of the membrane system and effectively improving the physiological stability and quality retention ability of mangoes during low-temperature storage.

[0106] (6)Detection of antioxidant system: The detection of the antioxidant system includes the determination of the content of reactive oxygen species and the activity of antioxidant enzymes.

[0107] The content of superoxide anion was determined by the hydroxylamine hydrochloride colorimetric method. Take 1 g of mango sample, add 1 mL of extraction buffer (containing 1 mmol / L ethylenediaminetetraacetic acid, 0.3% Triton X-100 and 2% polyvinylpyrrolidone), mix well, centrifuge at 4 °C and 12000 rpm for 20 min, collect the supernatant, and store it at 4 °C for later use. Take 0.5 mL of the supernatant, add 0.5 mL of 50 mmol / L phosphate buffer (pH 7.8) and 0.5 mL of 1 mmol / L hydroxylamine hydrochloride solution, mix well and incubate at 25 °C for 1 h. Then add 0.5 mL of 17 mmol / L sulfanilic acid and 0.5 mL of 7 mmol / L α-naphthylamine, mix well and react at 25 °C for 20 min. After taking out, measure the absorbance value at 530 nm. According to the difference between the measurement tube and the blank tube, the amount of substance of superoxide anion is obtained from the standard curve. The amount of substance of superoxide anion produced per minute per gram of sample is used as its production rate. For the reference blank, take 0.5 mL of the supernatant for the experiment without 1 h incubation, and the other experimental operations are the same. The unit of the superoxide anion production rate is expressed as nmol / (min·g), and the calculation formula is as follows:

[0108] n —— The amount of substance of O 2- in the solution obtained from the standard curve (μmol); V —— The total volume of the sample extract (mL); Vs —— The volume of the sample solution taken (mL); t —— The reaction time (min) of the sample and hydroxylamine; m —— The mass of the sample (g); The experimental results are as Figure 2 shown in A: During the entire low-temperature storage period, regardless of the time point, the superoxide anion content in the n-butanol treatment group was always lower than that in the control group. Although the superoxide anion levels in both groups increased over time, on the 30th day of storage, the superoxide anion content in the treatment group decreased by 22.88% compared to the control group.

[0109] Hydrogen peroxide was determined by the ferrous sulfate colorimetric method. Weigh 0.2 g of the sample, add 1.0 mL of pre-cooled acetone at 4 °C, vortex and mix well, then centrifuge at 4 °C and 8000 rpm for 10 min. Pipette 0.2 mL of the supernatant of the sample extract and place it on ice for later measurement. Add 0.2 mL of 100 μmol / mL H2O2 standard solution to the standard tube, add 0.2 mL of acetone to the control tube, and add 0.2 mL of the sample to the sample tube. Then, add 0.1 mL of 10% titanium tetrachloride and 0.2 mL of concentrated ammonia water to the control tube, standard tube, and sample tube respectively. After the precipitate forms, centrifuge at room temperature at 4000 rpm for 10 min, discard the supernatant and keep the precipitate. Add 1 mL of 2 mol / mL sulfuric acid to dissolve the precipitate, let it stand for more than 5 min, and measure the absorbance value at 415 nm. The calculation formula for the H2O2 content in the tissue is as follows:

[0110]

[0111]

[0112] W —— The fresh weight of the sample (g).

[0113] The experimental results are as Figure 2 shown in B: Within the first 20 days of storage, the hydrogen peroxide content in the n-butanol treatment group was lower than that in the control group, especially on the 15th day, and the difference between the groups was the most significant.

[0114] Determination of ascorbate peroxidase activity: Take 0.2 g of the sample and add 1 mL of ascorbate peroxidase extraction solution (containing 0.1 mol / L, pH 7.5 phosphate buffer, 0.1 mmol / L ethylenediaminetetraacetic acid, 1 mmol / L ascorbic acid, and 2% polyvinylpyrrolidone), mix well, centrifuge at 4 °C and 12,000 rpm for 30 min, collect the supernatant, and store it at 4 °C for later use. Take 100 μL of the supernatant and add it to 2.6 mL of APX reaction buffer (containing 50 mmol / L, pH 7.5 phosphate buffer, 0.1 mmol / L ethylenediaminetetraacetic acid, and 0.5 mmol / L ascorbic acid), with distilled water as the reference blank. Then quickly add 0.3 mL of 2 mmol / L H2O2 solution and start timing. Record the absorbance value of the reaction system at 290 nm at 15 s after the reaction starts, and then record it every 30 s for 6 consecutive points.

[0115] The experimental results are as Figure 2 shown in Figure C: The ascorbate peroxidase activity in the n-butanol treatment group was higher than that in the control group throughout the storage period, and gradually increased with the extension of time. Except on the 5th day, the differences between groups were significant at other time points, and the gap showed an expanding trend.

[0116] The superoxide dismutase level was determined using a kit provided by Solarbio Science & Technology Co., Ltd. Weigh 0.3 g of the sample, add 1 mL of the extraction solution and mix well, centrifuge at 4 °C and 8,000 rpm for 10 min, take the supernatant and store it at 4 °C for measurement, and record the absorbance value at 560 nm after the sample is measured. The experiment was repeated three times.

[0117] The experimental results are as Figure 2 shown in Figure D: The superoxide dismutase activity in the treatment group was always higher than that in the control group, and showed a gradually increasing trend during storage.

[0118] The activity of phenylalanine ammonia-lyase was determined by the L-phenylalanine colorimetric method. Weigh 0.2 g of the sample, add 1.0 mL of boric acid-borax extraction buffer (pH 8.8, 40 g / L polyvinylpyrrolidone, 2 mM ethylenediaminetetraacetic acid, and 5 mM β-mercaptoethanol), vortex to mix well, then centrifuge at 4°C and 12,000 rpm for 30 min. The supernatant was stored at low temperature for later use. Take 2 test tubes and add 1.5 mL of 50 mmol / L, pH 8.8 boric acid buffer and 0.25 mL of 20 mmol / L L-phenylalanine solution to each. Add 0.25 mL of the enzyme extract to one test tube, and add 0.25 mL of the inactivated enzyme solution that has been boiled for 5 min to the other test tube as a control. Then, place the 2 test tubes in a water bath at 37°C for 60 min. Immediately add 0.05 mL of 6 mol / L hydrochloric acid solution to each of the 2 reaction tubes to terminate the reaction at the end of the incubation. Using distilled water as the reference blank for zero adjustment, measure the absorbance values of the solutions in the sample reaction tube and the control reaction tube at a wavelength of 290 nm. According to the difference in absorbance values of the reaction solution (OD I -OD0), calculate the PAL activity. When the absorbance value of the reaction system of every gram (W) of fruit and vegetable tissue increases by 0.01 per hour, it is defined as 1 unit (U) of PAL activity. The calculation formula for PAL enzyme activity is as follows:

[0119] OD I —— Absorbance value of the reaction solution in the sample tube or the initial value of the reaction solution before incubation; OD0—— Absorbance value of the reaction solution in the control tube or the termination value of the reaction solution after incubation; V—— Total volume of the sample extract (mL); V S —— Volume of the sample extract taken during measurement (mL); t—— Enzyme-catalyzed reaction time (h); W—— Sample mass (g) The experimental results are as shown in Figure 2 E: The activity of phenylalanine ammonia-lyase showed a trend of "first increasing and then decreasing" with the storage time, reaching the peak on the 20th day. There were significant differences between the treatment group and the control group on the 10th, 15th, 20th, and 30th days. Among them, the PAL activity of the treatment group on the 20th day was 67.81 U / g, higher than 57.05 U / g of the control group.

[0120] Determination of catalase activity: Weigh 0.2 g of the sample, add 1.0 mL of sodium phosphate extraction buffer (pH 7.5 containing 5 mM dithiothreitol and 5% polyvinylpyrrolidone), vortex to mix well, then centrifuge at 4°C and 12,000 rpm for 30 min. The supernatant is stored at low temperature for later use. The reaction system includes 1.45 mL of 20 mM H2O2 and 50 μL of enzyme solution. Using distilled water as the reference blank, start recording the absorbance value of the reaction system at 240 nm at 15 s of the reaction, record one data point every 1 min, and continuously measure 6 points. An increase in absorbance value of 0.01 per gram of sample per minute is defined as 1 enzyme activity unit (U). The calculation formula for CAT enzyme activity is as follows:

[0121]

[0122] ΔOD 240 —— Change in absorbance value of the reaction per minute; OD 240F —— Final absorbance value of the reaction system; OD 240I —— Initial absorbance value of the reaction system; t F —— Reaction termination time (min); t I —— Reaction initial time (min); V - Total volume of the extraction solution (mL); V S —— Volume of the sample extraction solution taken during measurement (mL); m - Sample mass (g).

[0123] The experimental results are as Figure 2 shown in Figure F: During the entire low-temperature storage process, the catalase activity generally showed a downward trend. However, the catalase activity in the n-butanol treatment group was always higher than that in the control group, and the decline rate was slower. After 25 days, the difference between the groups was significant.

[0124] Conclusion: Treatment with n-butanol can enhance the antioxidant capacity and physiological stability of mangoes under low-temperature storage conditions through multi-path synergistic effects. On the one hand, this treatment can significantly reduce the accumulation level of reactive oxygen species, including effectively inhibiting the generation of superoxide anions and hydrogen peroxide, thereby reducing the damage caused by oxidative stress to fruit cells. On the other hand, n-butanol treatment significantly increases the activities of various antioxidant-related enzymes, including promoting the continuous increase in the activities of superoxide dismutase and ascorbate peroxidase, enhancing the reactive oxygen scavenging ability, delaying the decline in catalase activity, maintaining its degradation function for hydrogen peroxide, and enhancing phenylalanine ammonia-lyase activity, which is beneficial for regulating secondary metabolic responses and enhancing tissue stress resistance. Thus, n-butanol treatment effectively improves the oxidative response mechanism and quality retention ability of mangoes under low-temperature stress by simultaneously reducing the level of oxidizing substances and activating the endogenous antioxidant system.

[0125] (7) Activities of key enzymes in membrane lipid metabolism: The activities of key enzymes in membrane lipid metabolism were detected by ELISA.

[0126] The activity of plant phospholipase A was determined by ELISA (Hengyuan, HB153X-Pt, Shanghai, China); this kit uses the double antibody sandwich method to determine the level of plant phospholipase A in specimens. The microplate was coated with purified plant phospholipase A antibody to form a solid-phase antibody. Plant phospholipase A was added successively to the wells coated with monoclonal antibody, and then combined with HRP-labeled plant phospholipase A (PLA) antibody to form an antibody-antigen-enzyme-labeled antibody complex. After washing, substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid. The depth of color was positively correlated with the plant phospholipase A in the sample. The absorbance (OD value) was measured at a wavelength of 450 nm with an enzyme-labeled instrument, and the concentration of plant phospholipase A in the sample was calculated through the standard curve. The activity of plant phospholipase C was determined by ELISA (Hengyuan, HB151-Pt, Shanghai, China); the activity of plant phospholipase D was determined by ELISA (Hengyuan, HB150-Pt, Shanghai, China). The determination methods of plant phospholipase C activity and plant phospholipase D activity refer to the determination method of PLA.

[0127] The experimental results are as Figure 3 shown in A to C. The activity of plant phospholipase A in the n-butanol treatment group was always higher than that in the control group throughout the storage period and increased significantly after 20 days of storage, indicating that n-butanol can enhance the activity of plant phospholipase A in mango fruits under low-temperature stress. N-butanol treatment significantly inhibited the increasing trend of the activities of plant phospholipase C and plant phospholipase D, and their activity levels were lower than those in the control group throughout the storage period, suggesting that n-butanol may slow down the membrane lipid degradation during chilling injury by regulating the phospholipid signaling pathway.

[0128] As Figure 3As shown in Figure D, the lipoxygenase activity of mango fruits in the control group increased rapidly in the middle and late stages of storage, reaching 79.23 U / g on the 25th day, while the lipoxygenase activity of the n-butanol treatment group remained at a low level all the time, and the highest value was only 38.33 U / g, indicating that it played a positive role in delaying the process of membrane lipid peroxidation and reducing oxidative damage.

[0129] Conclusion: n-butanol treatment can relieve the chilling injury caused by low temperature stress at the level of membrane lipid metabolism regulation by enhancing the activity of phospholipase A and inhibiting the activities of phospholipase C, phospholipase D and lipoxygenase, which helps to maintain the stability of the fruit membrane system and improve the physiological quality during storage.

[0130] (8)Gene expression analysis: The real-time fluorescence quantitative PCR technique was used to analyze the expression levels of target genes. The specific experimental steps are as follows: RNA extraction and quality inspection: 0.1 g of mango peel was taken to extract total RNA. The extracted RNA samples were treated with DNase I enzyme to remove possible residual genomic DNA contamination. Subsequently, a micro nucleic acid and protein analyzer was used to measure the concentration and purity of RNA. A qualified sample had an A260 / A280 ratio between 1.8 and 2.0. At the same time, a 1.2% agarose gel electrophoresis method was used to detect the integrity of RNA samples to confirm that they met the requirements of subsequent experiments.

[0131] cDNA synthesis: 0.1 μg of total RNA qualified by quality inspection was taken as a template for reverse transcription reaction. The first-strand cDNA was synthesized according to the reaction system and procedures listed in the product manual for subsequent fluorescence quantitative PCR analysis.

[0132] qRT-PCR amplification and analysis: The SYBR Green fluorescence dye system was used for real-time fluorescence quantitative PCR amplification reaction, and the Actin gene was used as an internal reference for expression normalization analysis. Three technical replicates were set for each sample to ensure data reliability, and the 2 −ΔΔCt method was used to calculate the relative expression levels of target genes. The primer sequences and amplification parameters of each gene are shown in Table 1-3.

[0133]

[0134] Table 1 Results of fluorescence quantitative detection on the 5th day Conclusion: n-butanol treatment significantly down-regulated the expression of membrane lipid degradation-related genes such as MiPLDα, MiPLC6, and MiLOX3.1, inhibited the activities of phospholipase D, phospholipase C, and lipoxygenase; at the same time, up-regulated the expression of genes such as MiPLA2, MiATPγ2, and MiDGK7, enhanced membrane stability and energy metabolism ability, thus reducing the chilling injury during low-temperature storage of mangoes.

[0135] (9)Energy metabolism analysis: The indexes related to energy metabolism were determined by high performance liquid chromatography.

[0136] Weigh 20 g of the sample, add ultrapure water and grind it. After extraction in a boiling water bath, centrifuge at 8000 rpm, take the supernatant, and filter it with a 0.45 μm aqueous filter membrane for standby. Chromatographic conditions: Use a Sepax C18 chromatographic column with a column temperature of 30 °C; mobile phase A is a 50 mmol / L potassium phosphate buffer with a pH value of 6.5, and mobile phase B is methanol. The gradient elution program is set as follows: 0–10 min, 0% B; 10–15 min, 0%–20% B; 15–25 min, 20%–50% B. The flow rate is 0.8 mL / min, the detection wavelength is 254 nm, the injection volume is 10 μL, and the running time is set to 40 min. The contents of ATP, ADP, and AMP were qualitatively determined by retention time and quantitatively determined by the external standard method. The energy charge value was calculated according to the following formula:

[0137] The experimental results are as Figure 5 shown in A to C. The contents of the three in the n-butanol treatment group showed varying degrees of decrease at each time point: among them, the content of ATP decreased significantly at 30 days of storage, ADP decreased significantly in the early stage, and AMP was significantly lower than the control group at 5 days and 20 days of storage.

[0138] As Figure 5 shown in D, after treatment with n-butanol, the rising process of the energy charge value in mango fruits occurred earlier, and an increase in the energy metabolism level could be observed at 5 days of storage.

[0139] Conclusion: Treatment with n-butanol can effectively regulate the energy metabolism process of mango fruits during low-temperature storage, manifested as a moderate decrease in the contents of ATP, ADP, and AMP and an earlier increase in the energy charge value, thereby contributing to improving the energy supply efficiency and metabolic stability of cells at the initial stage of storage and enhancing the adaptability of fruits to low-temperature stress.

[0140] Example 2: Verification of n-butanol concentration gradient and soaking time Select 150 fresh Guixi mango fruits with intact appearance, uniform size, and no mechanical damage again, and randomly divide them into two groups, namely the concentration gradient treatment group and the time gradient treatment group, with 75 fruits in each group. After all fruits are thoroughly rinsed with tap water, leave them to dry at room temperature to remove the interference of water residue on the treatment effect.

[0141] The concentration gradient treatment group was further equally divided into five subgroups. The fruits were immersed in n-butanol aqueous solutions with mass-volume fractions of 0% (i.e., control, using deionized water), 0.5%, 1%, 1.5%, and 2% respectively, and the immersion time was uniformly set to 3 minutes to investigate the effects of different n-butanol concentrations on the low-temperature storage adaptability of mangoes.

[0142] The time gradient treatment group was equally divided into three subgroups. Under the condition of a fixed n-butanol concentration of 1%, the treatment times were set to 2 minutes, 5 minutes, and 10 minutes respectively to evaluate the influence law of treatment time on the fruit response.

[0143] After each treatment, the mangoes were placed in a ventilated environment to air dry for 60 minutes to allow the residual liquid on the surface to naturally volatilize. The dried fruits were placed in a single layer in plastic baskets and then transferred to a refrigerated environment with a set temperature of 4 ± 0.5°C and a relative humidity of 85% - 90% for storage for 30 days to simulate the low-temperature stress conditions during commercial storage and transportation.

[0144] During the storage process, sampling was carried out every 5 days. Each time, 6 fruits were randomly selected from each group as test samples to conduct relevant physiological and biochemical index determinations to systematically evaluate the low-temperature storage performance and quality retention effect of mango fruits under different n-butanol treatment conditions.

[0145] The experimental results are shown in Table 2 - 3: Table 2 Effects of n-butanol concentration gradient treatment on mango chilling injury (Fixed immersion for 3 minutes, air dried for 60 minutes, refrigerated at 4°C for 30 days) Conclusion: Among different n-butanol concentration treatments, a concentration of 1.0% is the best, significantly reducing the chilling injury index, electrolyte leakage rate, and MDA content of mangoes, while inhibiting the activity of phospholipase D and increasing the fruit hardness, effectively alleviating chilling injury.

[0146] Table 3 Effects of immersion time gradient on mango chilling injury (Fixed 1% n-butanol, air dried for 60 minutes, refrigerated at 4°C for 30 days) Conclusion: Under the condition of a fixed n-butanol concentration of 1%, the treatment effect is the best when immersed for 2 minutes, significantly reducing the chilling injury index and MDA content, and significantly increasing the fruit hardness, effectively alleviating chilling injury.

[0147] Comparative Example 1: Replace n-butanol with ethanol and isobutanol This comparative example was set up to verify the specificity of the technical effect of the n-butanol treatment described in the present invention in alleviating the chilling injury of mango fruits. A comparative experiment using alcohol compounds with similar structures was conducted, with 1% ethanol and 1% isobutanol replacing 1% n-butanol for treatment, and their effects were evaluated under the same conditions. Through this experiment, the alleviating effects of ethanol and isobutanol on mango chilling injury under the same treatment concentration and conditions can be systematically compared, thereby verifying that the n-butanol used in the present invention has an irreplaceable physiological regulation advantage among structural analogues, and reflecting the significance and difference of the technical solution of the present invention.

[0148] (1) Experimental steps: A total of 100 Guiqi mango fruits freshly harvested were selected in this comparative example. They had intact appearance, uniform size, and no mechanical damage. After all the fruits were rinsed with tap water, the surface moisture was naturally dried at room temperature. Subsequently, they were randomly and evenly divided into two groups, namely the ethanol treatment group and the isobutanol treatment group, with each group containing 50 fruits.

[0149] The mango fruits in the ethanol treatment group were soaked in an ethanol aqueous solution with a volume fraction of 1%, while the isobutanol treatment group was soaked in a 1% isobutanol aqueous solution. The soaking time was set to 3 minutes for both groups. After the treatment was completed, the fruits in each group were uniformly dried in a ventilated condition for 60 minutes to exclude the interference of surface residual liquid.

[0150] The dried fruits were all placed in a single layer in plastic baskets and transferred to a refrigerated environment with a temperature controlled at 4 ± 0.5 °C and a relative humidity of 85% - 90% for storage. The storage period was 30 days. During this period, 6 fruits were randomly selected from each group every 5 days as test samples to conduct tests on relevant indicators including chilling injury index, hardness, color change, membrane integrity, antioxidant enzyme activity, key enzyme activity of membrane lipid metabolism, gene expression level, and energy metabolism level.

[0151] (2) Experimental results: In terms of the evaluation of chilling injury symptoms, the chilling injury indices of the mango fruits in the ethanol group and the isobutanol group were both significantly lower than those of the control group, indicating that both replacement alcohols could, to a certain extent, reduce the tissue damage induced by low temperature. Among them, the degree of peel browning in the ethanol treatment group was the lightest, and that in the isobutanol group was slightly heavier, initially indicating that the spatial configuration differences in the carbon chain structures of alcohols have a significant impact on their physiological regulation abilities.

[0152] The hardness test results showed that both ethanol and isobutanol treatments effectively delayed the softening process of mango fruits during cold storage. Compared with the control group, the fruit hardness in the ethanol group remained excellent within the first 15 days of storage, reaching a maximum of 59 N, and the hardness value in the isobutanol group was slightly lower, at 58 N, but both were significantly better than 53 N in the control group, suggesting that both have a positive effect on the stability of the cell wall structure.

[0153] In terms of color retention, the fruit epidermal color uniformity and brightness maintenance time in the ethanol treatment group were long, the occurrence of browning was delayed, and the visual sensory score was significantly higher than that in the isobutanol group and the control group. Although isobutanol treatment also had a certain delaying effect, marginal dark brown spots appeared in the middle and late storage period (days 20 - 30), indicating that its inhibitory effect on pigment oxidation was relatively weak.

[0154] The results of membrane integrity detection further showed that both ethanol and isobutanol treatments could effectively alleviate the damage of fruit membrane lipid peroxidation. Compared with the control group, the malondialdehyde content in the ethanol group decreased significantly to 1.64 at 15 days, and that in the isobutanol group also decreased to 1.65. At the same time, the electrolyte leakage rates of both groups were controlled at 16%, which was significantly better than 17% of the control group, reflecting their obvious protective effect in maintaining the stability of the cell membrane system.

[0155] In terms of the activity of the antioxidant enzyme system, both ethanol and isobutanol treatments could induce the increase in the activities of enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase, enhancing the endogenous antioxidant defense ability of the fruit. Among them, the catalase activity in the ethanol group was the most stable in the early storage period. The catalase activity in the isobutanol group was slightly lower, but still significantly better than that in the control group, indicating its certain ability to relieve oxidative stress.

[0156] In terms of the regulation of membrane lipid metabolism, both ethanol and isobutanol could effectively inhibit the activities of enzymes related to membrane degradation. The decrease in the activity of phospholipase D in the ethanol group was the most significant, and the isobutanol group had a stronger inhibitory effect on the activity of phospholipase C. Both of them slowed down the process of membrane system structure disintegration to a certain extent.

[0157] The results of transcriptional level analysis further showed that both alternative alcohol treatments had a regulatory effect on the expression of genes related to membrane lipid metabolism. Ethanol and isobutanol treatments could both down - regulate the expression levels of membrane lipid degradation - related genes MiPLDβ and MiPLC6, and at the same time had an upward trend in the expression of membrane lipid resynthesis - related genes such as MiDGK5. The down - regulation amplitude of ethanol treatment was relatively larger, indicating that its effect intensity was more significant at the molecular regulation level; although the regulatory ability of isobutanol was slightly weaker, it also showed responsive changes at some targets, indicating its certain potential regulatory function in cold injury alleviation.

[0158] At the energy metabolism level, both ethanol and isobutanol treatments had a positive impact on the ATP metabolic state of the fruit. The energy charge value in the ethanol group increased significantly in the early storage period, and the contents of ADP and AMP decreased moderately, indicating that it was helpful to enhance the initial cell metabolic activity and the stability of energy supply; the EC value in the isobutanol group was also better than that in the control group, showing a certain potential for metabolic regulation. Both of them inhibited the energy depletion phenomenon caused by low - temperature stress to a certain extent and promoted the maintenance of cell physiological functions.

[0159] Conclusion: Although ethanol and isobutanol treatments can alleviate the chilling injury symptoms of mango fruits to a certain extent during low-temperature storage, such as reducing the chilling injury index, delaying the softening process, improving the stability of the membrane system, and enhancing the activities of antioxidant enzymes and the level of energy metabolism, the overall alleviation effect is significantly lower than that of the n-butanol treatment group. There are gaps especially in key physiological parameters such as the intensity of chilling injury inhibition, maintenance of cell membrane integrity, activation of the antioxidant system, and regulation of energy metabolism. This indicates that n-butanol has unique structural advantages and physiological regulation efficacy among alcohols, and can more efficiently enhance the adaptability of mango fruits to low-temperature stress and the ability to maintain quality through a multi-pathway synergistic mechanism, with irreplaceable technical optimality and significance.

[0160] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A postharvest treatment method for alleviating chilling injury of mangoes, characterized in that, It includes treating mango fruits with an aqueous solution of n-butanol, air-drying them, and storing them in a low-temperature environment.

2. The method according to claim 1, wherein Before the n-butanol treatment, the mango fruits are washed with tap water to remove surface stains and then naturally air-dried.

3. The method according to claim 1 or 2, characterized in that, The temperature for treating the mango fruits with the aqueous solution of n-butanol is 20 - 30°C, preferably 25°C.

4. The method according to claim 1, wherein The mass concentration of the aqueous solution of n-butanol is 0.5% - 2%, preferably 1%.

5. The method according to claim 1, characterized in that The mango fruits are soaked in the aqueous solution of n-butanol and gently stirred, and the treatment time is 2 - 10 minutes, preferably 3 minutes.

6. The method according to claim 1, wherein The air-drying step is carried out under ventilation conditions for 10 - 120 minutes, preferably 60 minutes.

7. The method according to claim 1, wherein The weight of the mango fruits is 200 - 500 grams, preferably 400 grams.

8. The method according to claim 1, wherein The storage step is carried out in an environment with a temperature of 2 - 8°C, preferably 4°C.

9. The method according to claim 1, characterized in that The storage step is carried out in an environment with a relative humidity of 80% - 95%, preferably 85% - 90%.

10. Use of the aqueous solution of n-butanol according to claim 1 or 4 in the preparation of a treatment agent for inhibiting postharvest chilling injury of mangoes.

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