Application of chlorogenic acid in improvement of tomato fruit quality

Abstract

The invention relates to the technical field of tomato growth promoters, in particular to application of chlorogenic acid to improvement of tomato fruit quality, and the dosage of the chlorogenic acid is 25-100 mol / L by mass concentration; the application method comprises the following steps: preparing the chlorogenic acid into a chlorogenic acid aqueous solution, and spraying the chlorogenic acid aqueous solution on tomato fruits once every 7 days for 4 times. Chlorogenic acid is externally applied, so that the content of lycopene and the content of carrots are increased, the content of chlorophyll is reduced, and color turning of tomato fruits is promoted; external application of chlorogenic acid can increase the total yield of tomato fruits and reduce the hardness of the tomato fruits; the soluble sugar content and the sugar-acid ratio of the tomato fruits as well as the soluble solid content and the vitamin C content of the fruits are improved, the growth and maturity of the tomato fruits are promoted, and the flavor and the nutrient content of the tomato fruits are improved. The application and development of the chlorogenic acid in the maturation and quality of the pre-harvest tomato fruits are promoted, and the problems that the fruit ripening agent in the current market contains hormone, is high in price, has potential toxicity and the like are solved.

Description

Technical Field

[0001] The invention relates to the technical field of tomato growth promoters, and in particular to the application of chlorogenic acid in improving tomato quality. Background Art

[0002] With the continuous improvement of living standards, people have higher and higher requirements for the quality of fruits. They not only need to have good nutritional value, but also be delicious and beautiful. Tomato is an annual or perennial herbaceous plant of the genus Solanum in the Solanaceae family. It is an economic crop widely planted worldwide. It contains a variety of bioactive ingredients and has high nutritional value. However, in the process of tomato cultivation, problems such as poor coloring and sour taste are often encountered, which not only affects the appearance and flavor of tomatoes, but also affects its market economic value. It can be seen that we need a safe and effective substance to promote the color change and ripening of tomato fruits and improve the quality of tomato fruits. At present, most of the ripening agents on the market are hormones, including abscisic acid, gibberellin, ethephon and methyl jasmonate. It is well known that some plant hormones such as abscisic acid are still mainly chemically synthesized, which is expensive, restricting their application in agricultural and horticultural production. Long-term excessive exposure to ethephon will be toxic to the eyes and skin. In addition, if used too early or inappropriately, it will also inhibit the growth and development of fruits.

[0003] Chlorogenic acid (CGA) is a polyphenolic compound composed of caffeic acid and quinic acid. It is the main phenolic substance in the flesh of many fruits such as tomatoes, peaches, pears, strawberries and grapes. It is reported that exogenous chlorogenic acid can prolong the post-harvest storage time of fruits such as nectarines, apples and pears; it also plays an important role in post-harvest preservation and disease prevention and control of grapes. However, there are few studies on the growth and development of fruits. If chlorogenic acid can be studied and promoted in the ripening and quality of pre-harvest tomato fruits, it can greatly promote the research on chlorogenic acid and the development of fruit ripening agents.

[0004] Therefore, the prior art needs to be further improved. Summary of the invention

[0005] The present invention aims to solve the above problems and provides the application of chlorogenic acid in improving the quality of tomato fruit. Chlorogenic acid, as an important secondary metabolite, is used to prematurely change the color and ripen tomato fruits before harvest and improve their fruit quality. It solves the problems of fruit ripening agents currently on the market containing hormones, being expensive and potentially toxic.

[0006] Specifically, the technical solution provided by this application is as follows: The application of chlorogenic acid in improving the quality of tomato fruit, the amount of chlorogenic acid used is 25-100 µmol / L; the method of use is to prepare the chlorogenic acid into a chlorogenic acid aqueous solution, and spray the tomato fruit once every 7 days for 4 times.

[0007] Further preferably, the dosage of chlorogenic acid is a mass concentration of 50 µmol / L. Verified by laboratory potted and greenhouse planting data, the dosage of 50 µmol / L significantly improves tomato color change, tomato fruit quality and other indicators. In addition, after measuring the relative expression of tomato fruit ripening-related transcription factor genes, the relative expression of tomato fruit ethylene synthase genes, and the relative expression of lycopene synthase genes, the data showed that the corresponding gene expression levels were significantly improved.

[0008] Furthermore, the method of use is to prepare the chlorogenic acid into a chlorogenic acid aqueous solution, which can be sprayed during the greening stage, swelling stage and color change stage of tomato fruits.

[0009] The preparation method of the chlorogenic acid aqueous solution is as follows: weigh 0.177 g of chlorogenic acid, dilute to 50 mL with distilled water, and prepare a mother solution with a concentration of 10 mmol / L. Measure 1 mL of the 10 mmol / L mother solution and dilute to 100 mL with distilled water to obtain a 25 µmol / L chlorogenic acid aqueous solution. Dilute the mother solution in the same way to obtain 50 µmol / L and 100 µmol / L chlorogenic acid aqueous solutions.

[0010] Furthermore, the chlorogenic acid can be used in plant growth promoters.

[0011] The present invention has the following advantages / benefits: 1. The chlorogenic acid (CGA) used in the present invention is a polyphenol compound composed of caffeic acid and quinic acid. It is the main phenolic substance in the pulp of many fruits such as tomatoes, peaches, pears, strawberries and grapes. It is an important secondary metabolite and is safer than chemically synthesized hormones. The present invention promotes the application and development of chlorogenic acid in the ripening and quality of pre-harvest tomato fruits, and solves the problems of fruit ripening agents currently on the market containing hormones, being expensive and potentially toxic.

[0012] 2. The experimental data of the present invention proves that the exogenous application of chlorogenic acid increases the lycopene content and carotenoid content, reduces the chlorophyll content, and thus promotes the color change of tomato fruit; the exogenous application of chlorogenic acid can increase the total yield of tomato fruit and reduce the hardness of tomato fruit; increase the soluble sugar content and sugar-acid ratio of tomato fruit, as well as the soluble solids content and vitamin C content of the fruit, promote the growth and ripening of tomato fruit, and enhance the flavor and nutrient content of tomato fruit.

[0013] 3. In addition, the results of the detection of the expression of ripening-related genes showed that exogenous chlorogenic acid can induce high expression of ripening-related transcription factors and ethylene synthase genes in tomatoes. It is speculated that exogenous chlorogenic acid can promote the ripening of tomato fruits and improve their quality by regulating the ethylene synthesis pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0015] Figure 1 : Coloration of tomato fruit after treatment with chlorogenic acid in Example 1 of the present invention; Figure 2 : Coloration of tomato fruit after treatment with chlorogenic acid in Example 2 of the present invention; Figure 3 : Effects of exogenous chlorogenic acid treatment of the present invention on chlorophyll content (A), carotenoid content (B), and lycopene content (C) of tomato fruit; Figure 4 : Effects of exogenous chlorogenic acid treatment of the present invention on tomato fruit firmness (A), total yield (B) and fruit shape index (C); Figure 5 : Effects of exogenous chlorogenic acid treatment of the present invention on soluble sugar content (A), titratable acid content (B), and sugar-acid ratio (C) of tomato fruit; Figure 6 : Effects of exogenous chlorogenic acid treatment of the present invention on soluble solid content (A) and vitamin C content (B) of tomato fruit; Figure 7 Effect of exogenous chlorogenic acid treatment on ripening-related genes in tomato fruit SlNAC1 , SlNAC4 , SlFUL1 , SlRIN , SlNOR The influence of relative expression level; Figure 8 :The effect of exogenous chlorogenic acid treatment on ethylene synthesis related genes SlACS2 , SlACS4 , SlACSIA , SlACO4 The influence of relative expression level; Fig. 9 : The exogenous chlorogenic acid treatment of the present invention has an effect on the expression of lycopene synthase gene in tomato fruit SlFPPS1 , SlGGPS , SlPSY1 , SlPd , SZD , SlCRTISO The effect of relative expression. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0017] Example 1: Chlorogenic acid concentration screening experiment 1. Experimental Materials tomato( Solanum lycopersicum )'Micro Tom' variety, which is preserved by the Laboratory of Plant Stress Physiology and Molecular Biology of Qingdao Agricultural University.

[0018] Chlorogenic acid (RQ524R320) was purchased from Shanghai Ruiyong Biotechnology Co., Ltd. (RYON, Shanghai).

[0019] 2. Experimental methods 1) Material cultivation: Tomato seeds were sown in seedling trays covered with nutrient soil and cultured in a greenhouse at a temperature of 25°C, a humidity of 55%, and a photoperiod of 16 / 8 h. After two weeks, single tomato seedlings with uniform growth were selected and transplanted into round pots with a diameter of about 10 cm for subsequent treatment.

[0020] 2) Experimental treatment: Control group: Tomato fruits were sprayed with distilled water Treatment group 1: Tomato fruits were sprayed with 25 µmol / L chlorogenic acid aqueous solution Treatment group 2: Tomato fruits were sprayed with 50 µmol / L chlorogenic acid aqueous solution Treatment group 3: Tomato fruits were sprayed with 100 µmol / L chlorogenic acid aqueous solution Treatment method: Select 'Little Tom' tomato plants with basically the same growth and no disease or damage, and spray different test groups on their flower spikes at the peak flowering period. Spray once every 7 days, for a total of 7 times, with at least 3 replicates for each treatment, and take photos before each spraying.

[0021] 3. Experimental data 1) Effect of exogenous chlorogenic acid treatment on tomato fruit coloring Chlorogenic acid was applied to tomato fruits at the peak flowering stage, and the coloration was observed every 7 days. Figure 1 As shown in the figure, compared with the control, after treatment with 50 µmol / L chlorogenic acid in treatment group 2, tomato fruits began to color at 35 days after flowering (35 DAF), while the control group and treatment groups 1 and 3 did not start to color until 42 days after flowering. It can be inferred that exogenous chlorogenic acid can promote early coloring of tomato fruits, and 50 µmol / L chlorogenic acid has the best effect.

[0022] Example 2: Greenhouse verification experiment of chlorogenic acid 1. Experimental Materials The test material was greenhouse tomato 'Jingcai No. 8', which was from Caocun, No. 137, Tieqishan Road, Chengyang District, Qingdao City, Shandong Province (36°28′N, 120°46′E).

[0023] Chlorogenic acid CGA (RQ524R320) was purchased from Shanghai Ruiyong Biotechnology Co., Ltd. (RYON, Shanghai).

[0024] 2. Experimental methods 1) Material cultivation: Select tomato seedlings with good growth and basically consistent 5-leaf 1-heart stage and plant them in the greenhouse in mid-September 2023. The planting row spacing is 25 cm and the spacing is 85 cm. The greenhouse temperature is 10-20 o C, humidity is 65~75%, and managed according to normal field management mode throughout the growth cycle.

[0025] 2) Experimental treatment: Control group: Tomato fruits were sprayed with distilled water Treatment group: Tomato fruits were sprayed with 50 µmol / L chlorogenic acid aqueous solution Treatment method: After preliminary concentration screening in the laboratory, the optimal chlorogenic acid treatment concentration was determined to be 50 µmol / L; with clear water treatment as the control, three replicates were set. Tomato fruits at the green fruit stage (about 35 days after flowering), swelling stage (about 45 days after flowering) and color change stage (about 55 days after flowering) were selected for spraying. Spraying was performed once every 7 days, for a total of 4 times, with at least 3 replicates for each treatment. The fruits were picked 28 days after treatment to measure the fruit quality and other related indicators.

[0026] 3) Determination of fruit quality indicators and other related indicators: Measurement indicators: chlorophyll content, carotenoid content, lycopene content, firmness, yield, fruit shape index, soluble sugar content, titratable acid content, sugar-acid ratio, soluble solids content, vitamin C content, relative expression of tomato fruit ripening-related transcription factor genes, relative expression of tomato fruit ethylene synthase genes, and relative expression of lycopene synthase genes.

[0027] The chlorophyll and carotenoid contents in the fruits were determined by 95% ethanol extraction method (Zhang et al, 2024; Wang et al, 2023).

[0028] The lycopene content was determined by extraction colorimetry (Zhang Lianfu et al., 2001).

[0029] The horizontal and vertical diameters of tomato fruits were measured with a vernier caliper (ABS digital vernier caliper). The ratio of the vertical diameter to the horizontal diameter of the fruit was calculated based on the fruit height and the fruit transverse diameter, i.e., the fruit shape index, according to the following formula: fruit shape index = vertical diameter / horizontal diameter.

[0030] The weight of a single fruit was measured using an electronic balance (Mettler AE 200), and the total yield was calculated based on the number of fruits produced by the tomato plants.

[0031] The fruit hardness was measured using a fruit hardness meter (GY-1).

[0032] The soluble solids content of the fruit was determined using a PAL-1 saccharimeter.

[0033] The vitamin C content of the fruit was determined using the sodium 2-6-dichloroindophenol method (Arya et al, 2000).

[0034] The pH value or acidity of the fruit was measured using a Mettler benchtop pH meter.

[0035] The soluble sugar content of the fruit was determined by anthrone colorimetry (Sun et al, 2018).

[0036] The titratable acid content of the fruit was determined by acid-base titration (Yang Ximeng et al., 2024). The sugar-acid ratio of the fruit was calculated by combining the ratio of soluble solids to titratable acid. The calculation was performed according to the following formula: sugar-acid ratio = soluble solids / titratable acid.

[0037] The sample RNA was extracted by the modified cetyltrimethylammonium bromide (CTAB) method according to the method of Li Xidong et al. (Li Xidong et al., 2011). The first strand of cDNA was synthesized by reverse transcription using the EasyScript One-StepgDNA Removal and cDNA Synthesis SuperMix reverse transcription kit of Beijing Quanshi Biotechnology Co., Ltd. according to the instructions. Quantitative primers were designed online based on the tomato gene sequence in the NCBI database and the Primer-BLAST tool (Table 1). The quantitative primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The real-time fluorescence quantitative PCR reagent was selected from ArtiCan ATM SYBR qPCR Mix, system: 10 μL iQ TM SYBR Green Supermix, 2 μL cDNA, 0.4 μL upstream and downstream primers (10 μml / L), 7.2 μL ddH2O, reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 45 cycles, each sample was repeated 3 times. SlActin The internal reference gene was used for standardization and calibration, and the data were analyzed using 2 -ΔΔCT Method for analysis.

[0038] Table 1 Primer sequences Table 1 Primer Sequences

[0039] 3. Experimental data 1) Effect of exogenous chlorogenic acid treatment on tomato fruit coloring Chlorogenic acid was applied to tomato fruits at the green stage, expansion stage, and color change stage, and the coloration was observed 28 days after application of chlorogenic acid. Figure 2 As shown in the figure, after treatment with 50 umol / L chlorogenic acid, there was no significant difference in the coloring of tomato fruits in the green fruit stage compared with the control, while the coloring degree of tomato fruits in the expansion stage was better than that of the control group. The color change period was the same as the expansion period and the coloring phenomenon was more obvious. It can be inferred that the exogenous application of chlorogenic acid can promote the early coloring of tomato fruits in the expansion and color change periods.

[0040] We further tested the chlorophyll content, carotenoid content, and lycopene content, which are indicators related to tomato fruit coloration. The results are as follows: Figure 3 As shown in AC; Among them, Figure 3 -A, compared with the control, the chlorophyll content in tomato fruits during the expansion and color change stages decreased significantly after treatment with chlorogenic acid. Figure 3-B and 3-C, the carotenoid and lycopene contents increased significantly, especially the change of lycopene, which increased nearly 50 times during the expansion period ( Figure 3 -C).

[0041] It can be concluded that exogenous chlorogenic acid may promote the coloring of tomato fruit by reducing the chlorophyll content of the fruit and increasing the content of carotenoids and lycopene during the expansion and color change periods.

[0042] 2) Effects of exogenous chlorogenic acid treatment on tomato fruit firmness, total yield and fruit shape index from Figure 4 It can be seen that after chlorogenic acid treatment, the firmness of tomato fruit during the expansion and color change stages decreased significantly compared with the control, decreasing by 38.25% and 44.36% respectively ( Figure 4 A), while the total yield increased significantly during the green fruit stage, expansion stage and color change stage, increasing by 27.25%, 51.29% and 22.29% respectively ( Figure 4 B). However, exogenous chlorogenic acid treatment had no significant effect on the fruit shape index of tomato fruit ( Figure 4 C). The above results indicate that exogenous chlorogenic acid can increase the total yield of tomato fruit and reduce the firmness of tomato fruit.

[0043] 3) Effects of exogenous chlorogenic acid treatment on sugar and acid content of tomato fruit Depend on Figure 5 The results showed that the soluble sugar content and sugar-acid ratio of tomato fruit increased significantly after the application of chlorogenic acid during the color change period, increasing by 9.32% and 21.87% respectively, while there was no significant difference in the titratable acid content of tomato fruit. The above results indicate that exogenous chlorogenic acid may improve the flavor of tomato fruit by increasing the soluble sugar content and sugar-acid ratio of tomato fruit during the color change period.

[0044] 4) Effects of exogenous chlorogenic acid treatment on nutritional indicators of tomato fruit like Figure 6 As shown in the figure, compared with the control, the soluble solids and vitamin contents of tomato fruits treated with chlorogenic acid increased during the expansion and color change periods. The soluble solids content of tomato fruits treated with chlorogenic acid increased by 12.94% and 20.88% during the expansion and color change periods, respectively, both reaching significant differences; the vitamin C content of tomato fruits increased by 66.46% and 22.70% during the expansion and color change periods, respectively, both reaching significant differences. It is speculated that exogenous chlorogenic acid improves the nutritional value of tomato fruits by increasing the soluble solids and vitamin C content in tomato fruits.

[0045] 5) Effects of exogenous chlorogenic acid treatment on the expression of transcription factors and ethylene synthase genes related to tomato fruit ripening The relative expression of tomato fruit ripening-related transcription factor genes during the color change period after exogenous chlorogenic acid treatment was detected by fluorescence quantitative PCR. Figure 7 As shown, compared with the control, chlorogenic acid treatment significantly promoted SlNAC1 , SlNAC4 , SlFUL1 , SlRIN , SlNOR expression.

[0046] Tomato is a climacteric fruit, and ethylene is a key factor affecting the ripening of climacteric fruits. To this end, we used fluorescent quantitative PCR to detect the relative expression of ethylene synthase genes in tomato fruits at the color change stage after exogenous chlorogenic acid treatment. The results are shown in Figure 2. Figure 8 As shown in Figure 2, chlorogenic acid treatment significantly promoted the expression of key enzyme genes for ethylene synthesis. SlACS2 , SlACS4 , SlACS1 , SlACO4 expression.

[0047] It can be speculated that exogenous chlorogenic acid may promote the ripening and coloring of tomato fruit by affecting the expression of transcription factor genes related to tomato fruit ripening during the color change period, and then regulating the expression of key genes of ethylene synthase.

[0048] 6) Effect of exogenous chlorogenic acid treatment on the expression of lycopene synthase gene Depend on Figure 3 -C showed that the changes of lycopene in tomato fruits during the expansion and color change stages after chlorogenic acid treatment were the most obvious, and its increase during the expansion stage was nearly 50 times. Therefore, the relative expression of lycopene synthase gene during the expansion stage was further detected. Fig. 9 , and found that their expression levels were increased, especially the phytoene synthase gene ( SlPSY1 ) increased most significantly.

[0049] Therefore, exogenous chlorogenic acid may promote the coloring of tomato fruit by reducing the chlorophyll content of the fruit and increasing the content of carotenoids and lycopene during the expansion and color change stages.

[0050] In summary, exogenous chlorogenic acid increased the lycopene content and carotenoid content, reduced the chlorophyll content, and promoted the coloring of tomato fruits; it can increase the total yield of tomato fruits and reduce the hardness of tomato fruits; it can increase the soluble sugar content and sugar-acid ratio of tomato fruits, as well as the soluble solids content and vitamin C content of fruits, promote the growth and maturation of tomato fruits, and improve the flavor and nutrient content of tomato fruits. The results of the detection of the expression of ripening-related genes showed that exogenous chlorogenic acid can induce high expression of ripening-related transcription factors and ethylene synthase genes in tomatoes. It is speculated that exogenous chlorogenic acid can promote the ripening of tomato fruits and improve their quality by regulating the ethylene synthesis pathway. The above experiments show that a 50 μM chlorogenic acid aqueous solution can promote the color change and ripening of tomato fruits and improve the quality of tomato fruits.

[0051] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. The application of chlorogenic acid in improving the quality of tomato fruit, characterized in that: The dosage of chlorogenic acid is a mass concentration of 25-100 µmol / L; the method of use is to prepare the chlorogenic acid into a chlorogenic acid aqueous solution, and spray the tomato fruit once every 7 days for 4 times.

2. The use of chlorogenic acid in improving tomato fruit quality according to claim 1, characterized in that: The chlorogenic acid is used in an amount with a mass concentration of 50 µmol / L.

3. The use of chlorogenic acid in improving the quality of tomato fruit according to claim 1, characterized in that: The method of use is to prepare the chlorogenic acid into a chlorogenic acid aqueous solution, which can be sprayed during the greening stage, swelling stage and color change stage of tomato fruits.

4. Use of the chlorogenic acid according to any one of claims 1 to 3 in a plant growth promoter.

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