Apple storage agent and application thereof in improving quality of apple fruits in storage period

Abstract

The invention provides an apple storage agent and application thereof in improving the quality of apple fruits during storage, and belongs to the technical field of fruit storage and preservation. The apple storage agent disclosed by the invention is prepared from a tea residue extract or epigallocatechin gallate, the content of epigallocatechin gallate in the tea residue extract is 62.94 mg / g to 131.43 mg / g; the concentration of the epigallocatechin gallate is 2 to 10 [mu] mol / L. By adopting the tea residue extract, not only can the greasy degree of the apples be reduced, but also the storage period of the apples is prolonged by influencing the generation of ethylene, reducing the accumulation of ester substances and increasing the accumulation of alkane substances. The EGCG is adopted, so that the greasy phenomenon of the apples in the storage period can be inhibited, the ethylene release rate is inhibited, the glossiness of the apples is improved, the fruit respiration is inhibited, the ethylene pathway in plant hormones and the glycolysis pathway in glycometabolism are inhibited, and the storage period of the apples is prolonged.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit storage and fresh-keeping, and in particular relates to an apple storage agent and application thereof in improving the quality of apple fruits during storage. Background Art

[0002] my country ranks first in the world in both apple production and consumption. The quality determines the flavor and taste of apples, which in turn directly affects the economic income of farmers. Therefore, how to improve the quality of fruits and save costs is an eternal theme in the fruit industry. At present, research on promoting plant growth and improving fruit quality by applying exogenous substances is relatively common. Epigallocatechin gallate (EGCG) is the main pharmacologically active ingredient in tea and is very friendly to the human body. EGCG can also help plants resist abiotic stress. The tea residue left after multiple brewing of tea is usually discarded as kitchen waste. Many beverage factories also produce a large amount of tea residue waste every year, resulting in serious waste of resources. Studies have shown that the EGCG content in the tea residue after tea soaking is still very high. The usual way to deal with the tea residue is to directly discard it, but this will cause a waste of beneficial components in the tea residue. Therefore, tea residue and the main active ingredient EGCG in the tea residue are explored, in order to provide a reference for the recycling of tea waste on fruit trees. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an apple storage agent and its use in improving the quality of apple fruit during storage, by reducing the degree of greasiness of apples, weakening the respiration of apples, and reducing the release of ethylene through tea residue extract or EGCG, thereby slowing down the decay of apples during storage and extending the storage period of apples.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] An apple preservative comprising a tea residue extract or epigallocatechin gallate;

[0006] The content of epigallocatechin gallate in the tea residue extract is 62.94-131.43 mg / g;

[0007] The concentration of the epigallocatechin gallate is 2-10 μmol / L.

[0008] Preferably, the method for preparing the tea residue extract comprises the following steps:

[0009] The tea leaves are mixed with boiling water and brewed for 2 to 5 times, and then filtered, dried, ground and sieved to obtain a tea residue extract.

[0010] Preferably, the mass volume ratio of the tea leaves to boiling water is 0.5-6 g:1 L;

[0011] The drying temperature is 60-70°C and the drying time is 18-30h;

[0012] The mesh number of the sieving is 300 to 500 meshes.

[0013] The invention also provides application of the apple storage agent in improving the quality of apple fruits during storage.

[0014] Preferably, the apples are placed in the apple preservative and treated with a vacuum pump before being stored in a controlled atmosphere.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention adopts tea residue extract to not only reduce the greasiness of apples, but also slow down the decay of apples during storage and prolong the storage period of apples by affecting the production of ethylene on the one hand and reducing the accumulation of ester substances and increasing the accumulation of alkane substances on the other hand.

[0017] The present invention adopts EGCG to inhibit the greasy phenomenon of apples during storage, inhibit the ethylene release rate of apples during storage, slow down physiological maturity, and thus prolong the storage time of apples.

[0018] The present invention adopts EGCG to significantly improve the glossiness of apples during storage, inhibit the respiration of fruits during storage, inhibit the consumption of soluble solids, and slow down the corruption of fruits.

[0019] The present invention uses EGCG treatment to affect the expression of some genes in the wax synthesis pathway and inhibit the ethylene pathway in plant hormones and the glycolysis pathway in sugar metabolism. It further shows that EGCG inhibits the oiliness of the fruit, weakens the respiration of apples, reduces the release of ethylene, reduces the sugar consumption in apples, and prolongs the storage period of apples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the EGCG content in three green tea residue extracts;

[0021] Figure 2 The test results of 'Fuji' apples treated with tea residue extract in vacuum infiltration (A is the effect of T3 and CK3 after 32 days of controlled atmosphere storage, B is the wax structure of the fruit peel after 10 days of storage for T3 and CK3, C is the ethylene release rate after 10 days of storage for T3 and CK3, and D is the glossiness of the fruit surface after 10 days of storage for T3 and CK3);

[0022] Figure 3 Effects of tea residue extract on storage wax components of 'Fuji' apples;

[0023] Figure 4 is the effect of EGCG on the oiliness of 'Fuji' apples during storage;

[0024] Figure 5 is the effect of EGCG on the ethylene release rate of 'Fuji' apples during storage;

[0025] Figure 6 is the effect of EGCG on fruit water loss, glossiness, and fruit surface temperature during storage of 'Fuji' apples (where A is fruit water loss, B is glossiness, and C is fruit surface temperature);

[0026] Figure 7 is the effect of EGCG on oxygen content during storage of ‘Fuji’ apples;

[0027] Figure 8 is the transcriptome analysis result of EGCG-treated 'Fuji' apple (among which, A is the KEGG enrichment analysis of differentially expressed genes between the EGCG-treated and control groups, B is the annotation of differentially expressed genes and metabolites in the cutin, lignin and wax biosynthesis pathways treated with EGCG (Note: red marks represent up-regulated genes, green marks represent down-regulated genes), C is the differentially expressed gene expression pattern in the starch and sucrose metabolism and glycolysis / saccharification pathways treated with EGCG in apples, and D is the annotation of differentially expressed genes in the MAPK signaling pathway treated with EGCG in plants). DETAILED DESCRIPTION

[0028] The invention provides an apple preservative, comprising a tea residue extract or epigallocatechin gallate; the content of epigallocatechin gallate in the tea residue extract is 62.94-131.43 mg / g; the concentration of the epigallocatechin gallate is preferably 2-10 μmol / L, more preferably 4-8 μmol / L, and even more preferably 5 μmol / L.

[0029] In the present invention, the preparation method of the tea residue extract comprises the following steps:

[0030] The tea leaves are mixed with boiling water and brewed for 2 to 5 times, and then filtered, dried, ground and sieved to obtain a tea residue extract.

[0031] In the present invention, the mass volume ratio of the tea leaves and boiling water is preferably 0.5-6 g:1 L, and more preferably 1-5 g:1 L; the number of brewing is preferably 3-4 times; the drying temperature is preferably 60-70° C., and more preferably 62-68° C.; the drying time is preferably 18-30 h, and more preferably 20-28 h; the sieving mesh size is preferably 300-500 mesh, and more preferably 350-450 mesh.

[0032] The present invention also provides the use of the apple storage agent in improving the quality of apples during storage. In the present invention, apples are placed in the apple storage agent and treated with a vacuum pump before being stored in a controlled atmosphere.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Materials used in the present invention:

[0035] Zhuyeqing, Anji White Tea and West Lake Longjing tea were purchased online at prices of 0.136 yuan / g, 0.396 yuan / g and 0.432 yuan / g respectively.

[0036] 'Fuji' apples were purchased from an orchard in Yantai, Shandong. After the fruits were picked, those with uniform size, no damage or disease, uniform color, and uniform growth period were selected and bagged, and then stored in a controlled atmosphere.

[0037] Example 1

[0038] Determination of EGCG content in tea residue extract

[0039] (1) Preparation of tea residue extract: 5 g of each of Zhuyeqing, Anji Baicha and West Lake Longjing tea leaves were weighed and brewed with 1 L of 100°C hot water. After cooling to room temperature, the mixture was brewed again with 100°C hot water. This process was repeated three times. The mixture was then dried (65°C, 24 h), ground and sieved through a 400-mesh sieve and stored in a refrigerator at 4°C.

[0040] (2) EGCG content determination: The EGCG content in the extracts of three kinds of tea residues, namely, Zhuyeqing, Anji White Tea and West Lake Longjing, was determined by Shandong Guocangjian Biotechnology Co., Ltd. (each type of tea residue was repeated three times).

[0041] Experimental results: Figure 1 As shown. Figure 1 It can be seen that the EGCG content in the extract of Zhuyeqing tea residue is the highest, reaching 131.43 mg / g, followed by the extract of Anji white tea residue at 85.05 mg / g, and the extract of West Lake Longjing tea residue has the least EGCG content, which is 62.94 mg / g. Therefore, the extract of Zhuyeqing tea residue was selected for subsequent experiments.

[0042] Example 2

[0043] Effect of Tea Residue Extract on Storage of Fuji Apple

[0044] By comparing the prices of three types of tea, Zhuyeqing, West Lake Longjing and Anji White Tea, and measuring the EGCG content in the tea residues produced after brewing, Zhuyeqing was finally selected as the test material, and a 1g / L tea residue extract suspension was prepared to treat apples.

[0045] Tea residue extract suspension treatment (T3) 'Fuji' apples were treated by vacuum infiltration method, vacuum pump distilled water was used as control (CK3), and T3 and CK3 were treated and stored in a controlled atmosphere incubator at 16℃. (30 apples were treated with tea T3 and CK3 respectively).

[0046] 1. 'Fuji' apples were treated with tea residue extract suspension and then stored in controlled atmosphere for 32 days.

[0047] Experimental results: Figure 2 As shown in A, the fruit surface of CK3 showed obvious wilting and brown necrotic parts, while the tea residue extract suspension had a lighter degree of wilting and almost no brown necrotic tissue.

[0048] 2. Determine the ethylene release rate after 10 days of controlled atmosphere storage: Place the apples in a 1.2L sealed container for 6-8 hours, and then use a gas chromatograph to detect the ethylene release rate of the apples. Set the parameters to DINJ: 80℃, column box: 70℃, DFID1: 200℃. Activate for 1h, then set the parameters to DINJ: 80℃, column box: 70℃, DFID1: 140℃. After cooling, extract 1mL of gas from the sealed container for detection.

[0049] The ethylene release rate was calculated according to the formula.

[0050]

[0051] C: ethylene content determined by gas chromatography (μmL / mL);

[0052] V: volume of sealed container (mL);

[0053] V1: apple sample volume (mL);

[0054] t: sealing time (h);

[0055] m: mass of apple (g).

[0056] Experimental results: Figure 2 As shown in C in the figure. After 10 days of storage, the ethylene release rate of the tea residue extract suspension was significantly lower than that of CK3, and CK3 was 2.22 times that of the tea residue extract suspension. The amount of ethylene release directly affects the storage property of the fruit. Generally speaking, the fruit with high ethylene release has poor storage property, while the fruit with low ethylene release has good storage property. This shows that the tea residue extract can slow down the decay of apples during storage by inhibiting the release of ethylene.

[0057] 3. Observation of waxy structure on the surface of apples by scanning electron microscopy after 10 days of controlled atmosphere storage: Select 3 peels from the same position of apple fruits with different treatments, place them in a clean glass petri dish, pour a small amount of liquid nitrogen into them and cool them quickly. After the liquid nitrogen evaporates completely, put them in a vacuum freeze dryer, wait for the machine to cool down to -40℃, and freeze dry them for more than 8 hours. The dried peels are placed in a desiccator, and then the samples are prepared. The samples are glued to the small stage of the scanning electron microscope, and ion spraying is performed using a JFC-1600 ion sputtering instrument (Japan Electronics Co., Ltd.). Scanning electron microscopy observations are performed at the Zeiss Launch Center (Germany). A representative 500x field of view is used for observation and data preservation.

[0058] Experimental results: Figure 2 As shown in B. Scanning electron microscopy observation of the surface wax structure revealed that the degree of greasiness of apples was reduced after treatment with tea residue extract compared with the control.

[0059] 4. Fruit gloss: The gloss meter (3nh, Shenzhen, China) should be perpendicular to the fruit surface. Three different positions of each fruit were selected for measurement. 20 apples were tested for each treatment and control.

[0060] Experimental results: Figure 2 As shown in D. The glossiness test results showed that the tea residue extract had no significant effect on the glossiness of the stored apple surface after treatment.

[0061] 5. Perform GC-MS analysis on the wax component content on the fruit surface. The specific steps are as follows:

[0062] Before wax extraction, the diameter and longitudinal diameter of the fruit should be measured to calculate the surface area of ​​the fruit, and the fruit should be extracted in 600 mL of chloroform three times, each time for 1 min.

[0063] ① Apple wax extraction was performed according to the method of Yang et al. (2017) (Yang, Y., Zhou, B., Zhang, J., Wang, C., Liu, C., Liu, Y., Zhu, X., and Ren, X. Relationships between cuticular waxes and skin greasiness of apples during storage. Postharvest Biology and Technology, 2017, 131: 55-67.) with some modifications. The whole apple peel was placed in a beaker and soaked in 30 mL of chloroform for 30 seconds at room temperature. Each sample was extracted three times. After extraction, the three extracts were mixed, filtered, and transferred to a rotary evaporator. Chloroform was evaporated into a waste liquid bottle through a vacuum condenser. 4 mL of chloroform and 1 mL of methanol were added to the evaporator to dissolve the white wax crystals. The dissolved concentrate was transferred to a 25 mL serum bottle. The serum bottle was blown dry with stable nitrogen gas on a nitrogen blower and then stored in a -20°C refrigerator. The treatment and control were repeated 3 times each.

[0064] ② Wax derivatization: Dissolve the wax in the bottle in a ratio of chloroform: methanol = 10:1, aspirate 1 mL and blow dry, add 200-400 μL of derivatization (BSTFA) reagent in a 70°C oven, immediately seal the mouth with film, and shake to mix. Derivatize in a 70°C metal bath for 40 minutes, shake once every 10 minutes, and blow dry with nitrogen under heating conditions at 50°C. Add 500 μL of chromatographic grade chloroform and 25 μL of n-tetracosane as internal standard. Use a 0.45 mm organic filter membrane to filter the sample into a brown loading bottle.

[0065] ③GC-MS loading analysis: GC-MS analysis was performed using a triple quadrupole gas chromatograph-mass spectrometer (Shimadzu, Japan). The GC parameters were set to an injection port temperature of 280°C and a column box temperature of 70°C. Other settings were based on the method of Wang Yongxu (2019). The MS parameters were set to an ion source temperature of 200°C, an interface temperature of 250°C, and a solvent delay time of 7 min.

[0066] ④ Wax component analysis: The retrieval database used was NIST17. The chemical structural formulas of the substances compared by computer retrieval were manually interpreted, and classification analysis was performed according to the different functional groups of the chemical structural formulas of the substances. The peak area normalization method and internal standard quantification method were used to calculate the relative substance content, and the wax component content per unit area was calculated based on the surface area of ​​the apple.

[0067] Statistical analysis Experimental data were recorded using Microsoft Excel 2019, graphing software was Graphpadprism 9, and gene differential expression heatmaps were produced using Origin Pro 202 Learning Edition software. Data processing and analysis were performed using IBM SPSS Statisticsdan one-way analysis of variance and Graphpad prism 9 one-way analysis of variance and T-test to test the significant differences of variables. *(P<0.05), **(P<0.01), ***(P<0.001) and different lowercase letters (P<0.05) were used to indicate significant differences between groups.

[0068] Experimental results: Figure 3 Compared with the control, the ester substances in the apple wax treated with tea residue extract decreased by 56.57%, and the alkanes increased by 87.34%. There was no significant difference in the contents of other components such as fatty acids, aldehydes, primary alcohols, ketones and total wax. It is speculated that during fruit storage, tea residues affect the production of ethylene on the one hand, and increase the accumulation of alkanes on the other hand by reducing the accumulation of ester substances, thereby extending the storage period of apples.

[0069] Example 3

[0070] Effects of EGCG on the storage of Fuji apples

[0071] The experiment was divided into 2 treatments:

[0072] Control group: sprayed with distilled water.

[0073] EGCG treatment group: (EGCG was purchased from Shanghai Solebow Biotechnology Co., Ltd., with a purity of 98%)

[0074] 'Fuji' apples were treated with vacuum pump in 5μmol / L EGCG aqueous solution, and vacuum pump distilled water was used as control. After treatment, they were also stored in a controlled atmosphere in an incubator at 16℃ (30 apples were treated with EGCG and distilled water, respectively).

[0075] 1. Observe the wax structure on the surface of apples using a scanning electron microscope. For specific methods, see Example 2.

[0076] Experimental results: Figure 4As shown. EGCG aqueous solution treatment of apples not only inhibits surface greasy, but also improves some storage indicators of the fruit. The wax morphology on the surface of apples was regularly observed by scanning electron microscopy. The results showed that no obvious greasy phenomenon was observed in EGCG treatment after 5 days of storage, while obvious oil droplets and aggregated liquid wax appeared in the control water treatment, with a high degree of greasy. After 10 days of storage, EGCG-treated apples showed oil droplets of liquid wax and began to show greasy phenomenon. The greasy degree of water-treated apples was further aggravated, and the wax continued to accumulate and thicken. After 40 days, both the treatment and the control entered the late stage of greasy and wax accumulation appeared. The results of scanning electron microscopy showed that EGCG can inhibit the greasy phenomenon of apples during storage.

[0077] 2. Determination of ethylene release rate

[0078] As a respiratory climacteric fruit, apple undergoes respiratory climacteric during the ripening process, and an ethylene release peak occurs at the same time. The appearance of the ethylene release peak is an important sign that the apple fruit enters the ripening process, and the fruit begins to rot after the respiratory peak. The level of ethylene release directly affects the storage property of the fruit. Generally speaking, the fruit with high ethylene release has poor storage property, while the fruit with low ethylene release has good storage property. The determination method is shown in Example 2.

[0079] Experimental results: Figure 5 Compared with the control, EGCG treatment reduced the ethylene release rate of apples by 30.75%, which indicates that EGCG can inhibit the ethylene release rate of apples during storage, slow down physiological ripening, and thus prolong the storage time of apples.

[0080] 3. Determination of fruit gloss, water loss rate and fruit surface temperature during storage.

[0081] (1) Fruit gloss: The gloss meter (3nh, Shenzhen, China) was used to measure the glossiness of the fruit. Three different positions of each fruit were selected for measurement. Twenty apples were tested for each treatment and control.

[0082] (2) Fruit water loss rate: The water loss rate of the fruit was detected using the weight loss method. The fruit weight was measured every 1 hour at room temperature using a 1 / 10,000 electronic balance.

[0083] (3) Fruit surface temperature: The apples were taken out from a storage environment at 16°C and placed at room temperature for 2 h. The surface temperature was measured using an ultraviolet thermal imager.

[0084] Experimental results: Figure 6 As shown. Figure 6It can be seen that EGCG has no significant effect on fruit water loss during storage. EGCG treatment significantly improves the glossiness of apples during storage. Oiliness increases the temperature of the fruit by increasing the respiration intensity of the fruit, and the surface temperature of the fruit increases by about 2 degrees compared with the fruit without obvious oiliness. These results all indicate that EGCG treatment inhibits the formation of fruit oiliness, and oiliness promotes ethylene production, enhances the respiration of the fruit, and then increases the consumption of soluble solids and accelerates fruit corruption.

[0085] 4. Fruit respiration intensity: The respiration intensity is tested by cumulative measurement method. The apple fruit is sealed in a 1.2L sealed container for 4-6 hours. Then a gas analyzer is used to detect the oxygen content in the container. The respiration intensity is analyzed by the oxygen consumption.

[0086] Experimental results: Figure 7 The apples were sealed in a container and the oxygen content was tested to reflect the strength of the fruit respiration. Compared with the control, the oxygen content of the apples treated with EGCG was 2 The content increased by 5.83%, which indicated that EGCG treatment inhibited the respiration of fruits during storage.

[0087] 5. Transcriptome data analysis of the effects of EGCG on apple oiliness and storage quality

[0088] To clarify whether EGCG treatment affects fruit respiration, reduces sugar consumption and prolongs storage period by inhibiting oiliness, transcriptome analysis was performed on EGCG-treated 'Fuji' apples.

[0089] RNA sequencing of fruit EGCG-treated and control samples was completed by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. KOBAS software was used for KEGG signaling pathway enrichment analysis.

[0090] Experimental results: Figure 8 As shown in the results, EGCG treatment during apple storage affects the expression of some genes in the wax synthesis pathway and inhibits the ethylene pathway in plant hormones and the glycolysis pathway in sugar metabolism. This further indicates that EGCG inhibits the oiliness of the fruit, weakens the respiration of apples, reduces the release of ethylene, and reduces the sugar consumption in apples, so EGCG treatment prolongs the storage period of apples.

[0091] The invention adopts tea residue extract or EGCG which can not only reduce the greasiness of apples and weaken the respiration of apples, but also reduce the amount of ethylene release, slow down the decay of apples during storage, and prolong the storage period of apples.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An apple preservative, characterized in that: including tea residue extract or epigallocatechin gallate; The content of epigallocatechin gallate in the tea residue extract is 62.94-131.43 mg / g; The concentration of the epigallocatechin gallate is 2-10 μmol / L.

2. The apple preservative according to claim 1, characterized in that: The preparation method of the tea residue extract comprises the following steps: The tea leaves are mixed with boiling water and brewed for 2 to 5 times, and then filtered, dried, ground and sieved to obtain a tea residue extract.

3. The apple preservative according to claim 2, characterized in that: The mass volume ratio of the tea leaves to the boiling water is 0.5-6 g:1 L; The drying temperature is 60-70°C and the drying time is 18-30h; The mesh number of the sieving is 300 to 500 meshes.

4. Use of the apple preservative according to any one of claims 1 to 3 for improving the quality of apple fruits during storage.

5. The use according to claim 4, characterized in that: The apples are placed in the apple storage agent and treated with a vacuum pump and then stored in a controlled atmosphere.

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