Use of abscisic acid to reduce accumulation of methoxypyrazines in grapes or wine

Abstract

The present application relates to grape cultivation and wine brewing technical field, particularly to the application of abscisic acid in reducing the accumulation of methoxypyrazine substances in grape or wine, which effectively reduces the accumulation and content of methoxypyrazine substances in red wine grape and wine by selecting the period of leaf spraying, the concentration of leaf spraying agent and the number of leaf spraying, etc. The present application also provides a cultivation method for reducing the accumulation of methoxypyrazine substances in grape or wine, which is simple and easy to operate, reduces the labor intensity and improves the efficiency; and the dosage of abscisic acid is very small, which is environment-friendly and ecologically safe.

Description

Technical Field

[0001] This invention relates to the fields of grape cultivation and winemaking technology, and in particular to the application of abscisic acid in reducing the accumulation of methoxypyrazine compounds in grapes or wine. Background Technology

[0002] Aroma is a crucial indicator in the sensory evaluation of grapes and wines. A "green" or "unripe" aroma (indicating unripe fruit) significantly impacts consumer preferences and purchasing desire, and is a significant factor limiting the competitiveness and high-quality development of the wine market. The "green" aroma in red wines primarily originates from methoxypyrazine compounds in the grape berries, mainly including 3-isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP).

[0003] The "green" flavor in red wine is less affected by the winemaking and aging processes; therefore, addressing this flavor in grapes and wine through cultivation techniques is currently a crucial measure. Specifically, in production, this is mainly achieved through cultivation management methods such as leaf removal, canopy pruning, and reducing planting density. In China, however, leaf removal and canopy pruning still rely heavily on manual labor, resulting in low efficiency and high costs; while reducing planting density affects the overall yield of the vineyard.

[0004] Therefore, it is essential to consider an easy-to-operate and labor-saving method to reduce the accumulation of methoxypyrazine compounds in red wine grapes and wines, thereby reducing the "green" taste in red wines. Summary of the Invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an application of abscisic acid in reducing the accumulation of methoxypyrazine compounds in grapes or wine.

[0006] Another object of the present invention is to provide a cultivation method for reducing the accumulation of methoxypyrazine compounds in grapes or wine.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Application of abscisic acid in reducing the accumulation of methoxypyrazine compounds in grapes or wine;

[0009] The grapes mentioned are preferably red wine grape varieties such as Cabernet Sauvignon, Merlot, and Cabernet Franc, which have a strong "green" flavor.

[0010] The methoxypyrazines are preferably at least one of 3-isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP);

[0011] The preferred treatment concentration of abscisic acid is 100 mg / L;

[0012] The preferred treatment time for abscisic acid is when the grapes are in the mid-to-late stage of color change and the soluble solids content of the fruit is at 15-18°Brix.

[0013] The soluble solids content of the fruit is further preferably 16-17°Brix;

[0014] The preferred treatment method for abscisic acid is foliar spraying;

[0015] The abscisic acid treatment is preferably performed twice, with a two-week interval between the first and second treatments;

[0016] A cultivation method for reducing the accumulation of methoxypyrazine compounds in grapes or wine includes the following steps;

[0017] (1) During the grape growing process, when the grapes enter the middle and late stages of color change and the soluble solids of the fruit are at 15-18°Brix, abscisic acid solution is used as a foliar spray agent to carry out the first foliar spray on the grape plants.

[0018] (2) Two weeks after the first foliar spray, use abscisic acid solution as the foliar spraying agent to spray the grapevines a second time, and then manage them according to the usual methods.

[0019] The grapes mentioned in step (1) are preferably red wine grape varieties such as Cabernet Sauvignon, Merlot, and Cabernet Franc, which have a strong "green" flavor.

[0020] The mid-to-late stage of color change mentioned in step (1) refers to the coloring of 60%-80% of the grape fruits;

[0021] The abscisic acid solution described in steps (1) and (2) preferably comprises the following components: 100 mg / L abscisic acid and 0.1% (v / v) Tween-80;

[0022] The preferred dosage for foliar spraying in steps (1) and (2) is 80-120 mL per grapevine, which is determined by the size of the canopy.

[0023] The preferred time for foliar spraying in steps (1) and (2) is 5-7 pm on a windless, sunny day after sunset;

[0024] The foliar sprayer mentioned in steps (1) and (2) can be at least one of a manual backpack sprayer and a drone sprayer.

[0025] If severe weather such as rain occurs within 24 hours after foliar spraying in steps (1) and (2), a second spray should be applied after the weather clears up.

[0026] The principle of this invention:

[0027] In existing technologies, abscisic acid (ABA) is a key hormone regulating the ripening of non-climacteric fruits (such as grapes). It not only accelerates the accumulation of sugars and phenols in the fruit but also reduces acidity, thereby improving fruit quality. This invention, based on extensive research, has discovered that ABA, while promoting fruit ripening, can also reduce the accumulation of methoxypyrazines in grapes and their wines. The timing, concentration, and frequency of foliar spraying directly affect the accumulation of methoxypyrazines in grapes and their wines. This invention utilizes appropriate ABA application methods to effectively reduce the accumulation and content of methoxypyrazines in red wine grapes and wines.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] (1) In practical work, this invention found that when grapes enter the middle and late stages of color change and the soluble solids content of the fruit is 15-18°Brix, foliar spraying of abscisic acid can effectively reduce the accumulation and content of methoxypyrazines in grapes and their wines, providing new ideas and directions for improving the quality of grapes and wines.

[0030] (2) This invention provides a cultivation method to reduce the accumulation of methoxypyrazines in grapes or wine. This method can significantly reduce the content of methoxypyrazines in grapes and wine, effectively improving the problem of strong "green" taste in red wine grapes and wine. Moreover, this method does not require leaf removal, leaf pruning, or reduction of grape planting density. It is simple and easy to operate, reduces the intensity of manual labor, and improves efficiency. Furthermore, the amount of abscisic acid used is extremely small, making it environmentally friendly and ecologically safe.

[0031] (3) The cultivation method provided by the present invention effectively controls the accumulation and content of methoxypyrazines, which is convenient for promotion and application and can effectively improve the economic benefits of grape cultivation. Attached Figure Description

[0032] Figure 1 This is a graph showing the effect of different abscisic acid spray concentrations on methoxypyrazine compounds in wine grapes. In the graph, (A): IBMP content, (B): IPMP content, and (C): SBMP content.

[0033] Figure 2 This is a graph showing the effect of different abscisic acid spraying times on methoxypyrazine compounds in wine grapes. In the graph, (A): IBMP content, (B): IPMP content, and (C): SBMP content.

[0034] Figure 3 This is a graph showing the results of the analysis of the effects of different abscisic acid spraying times on methoxypyrazine compounds in wine grapes. In the graph, (A): IBMP content, (B): IPMP content, and (C): SBMP content.

[0035] Figure 4 This is a result analysis diagram of the effect of appropriate abscisic acid application on methoxypyrazine compounds in Cabernet Sauvignon wine, where: (A): IBMP content; (B): IPMP content; (C): SBMP content.

[0036] Figure 5 This is a radar chart for odor sensory evaluation, where: control is the control group, and AT: screening measures for abscisic acid application.

[0037] Figure 6 This is a diagram illustrating the experimental process of the present invention. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] Cabernet Sauvignon is the most widely planted red wine grape variety in China and the world; therefore, this example uses Cabernet Sauvignon grapes. Meanwhile, the three main substances contributing most to the "green" flavor in grapes and wine are 3-isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP). Therefore, the content of methoxypyrazine compounds is often expressed as the total amount of IBMP, IPMP, and SBMP.

[0040] Example 1: Screening of abscisic acid concentration

[0041] (1) Prepare abscisic acid solutions with concentrations of 25, 100 and 400 mg / L as foliar sprays. The abscisic acid solutions also contain 0.1% (v / v) Tween-80. A deionized water solution containing only 0.1% (v / v) Tween-80 was prepared as a control.

[0042] (2) The experiment was conducted at the vineyard of Tianming Minquan Winery in Minquan County, Henan Province, in late August 2021. The specific method was as follows: when the grapes entered the mid-to-late stage of color change (i.e., 60%-80% of the grape berries were colored) and the average soluble solids content of the berries was at 17°Brix, the experiment was carried out on a windless, sunny day from 5-7 pm after sunset. Grapevines with basically uniform growth were selected as test materials and divided into four treatments (each treatment was replicated three times, with 20 vines per replicate); among them, Control group: Each grapevine was uniformly sprayed with a deionized water solution containing only 0.1% (v / v) Tween-80; AT1 group: Each grapevine was uniformly sprayed with a 25 mg / L abscisic acid solution; AT2 group: Each grapevine was uniformly sprayed with a 100 mg / L abscisic acid solution; AT3 group: Each grapevine was uniformly sprayed with a 400 mg / L abscisic acid solution. Spraying was performed using a backpack sprayer. Figure 6 The spraying dosage is 100 mL per vine. After spraying, the grapevines should be managed according to conventional methods.

[0043] (3) Two weeks later, perform a second foliar spraying according to the method described in step (2). After the spraying is completed, manage the plant according to the usual method.

[0044] (4) Determination of IBMP, IPMP, and SBMP in grape berries, specifically including:

[0045] ① Sample pretreatment: During the harvest period, 20 bunches of grapes were randomly collected from each replicate. Immediately after collection, they were flash-frozen in liquid nitrogen and then transported to the laboratory under dry ice refrigeration and stored in a freezer at -40℃. For the determination of methoxypyrazines, 50 grapes were removed from the freezer from each replicate, the stems were removed, and the fruit weight was recorded. The samples were then flash-frozen in liquid nitrogen until completely frozen. After being completely frozen, the samples were ground into powder using a crusher and placed into 50mL centrifuge tubes. After being stored at 4℃ for about 2 hours, the samples were fully thawed and thoroughly mixed using a vortex mixer. The samples were then centrifuged at 4℃ and 8000r / min for 10 minutes. 5mL of the supernatant was then taken into a 20mL sample vial, 1g of NaCl was added, and the cap was tightened for GC-MS analysis.

[0046] ②GC-MS Detection: An Agilent 7890B-5977B GC-MS system was used with an HP-INNOWAX column (60m×0.25mm×0.25μm). Headspace solid-phase microextraction (SPE) was performed using an automated injection system. The vials were equilibrated by shaking at 50℃ for 30 min, followed by extraction with a headspace microextraction needle (50 / 30μm DVB / CAR / PDMS2CM) for 30 min in splitless mode. The temperature program was as follows: initial temperature 50℃, held for 5 min, increased to 130℃ at a rate of 3℃ / min, then increased to 220℃ at a rate of 25℃ / min, held for 5 min, for a total run time of 42.20 min. The carrier gas was high-purity helium (purity >99.999%) at a flow rate of 1.0 mL / min.

[0047] ③ Establishment of Standard Curve: The content of each component of methoxypyrazine was determined using the external standard method. Ten serial dilutions were performed using simulated grape juice solution (7 g / L tartaric acid, 200 g / L glucose). The standard curve (simulated grape juice) is as follows: IBMP: y = 47531x + 23525, R 2 =0.998; IPMP: y=29568x+425485, R 2 =0.995; SBMP: y=38585x+86452, R 2 =0.998;

[0048] See results Figure 1 Compared with the control group, AT1 group, and AT3 group, the AT2 group (i.e., 100 mg / L abscisic acid solution) had the lowest total amounts of IBMP, IPMP, and SBMP in grapes, with total amounts of IBMP, IPMP, and SBMP of 9.7 ng / L, 2.6 ng / L, and 4.5 ng / L, respectively. Therefore, the optimal dose of abscisic acid is 100 mg / L.

[0049] Example 2: Screening of the number of abscisic acid sprays

[0050] (1) Prepare an abscisic acid solution with a concentration of 100 mg / L (containing 0.1% (v / v) Tween-80) as a foliar spray, and prepare a deionized water solution containing only 0.1% (v / v) Tween-80 as a control.

[0051] (2) The experiment was conducted at the vineyard of Tianming Minquan Winery in Minquan County, Henan Province, in late August 2021. The specific method was as follows: when the grapes entered the mid-to-late stage of color change (i.e., 60%-80% of the grape fruits were colored) and the average soluble solids content of the fruit was at 17°Brix, the experiment was carried out at 5-7 pm on a windless sunny day after sunset. Grape plants with basically the same growth were selected as test materials and divided into 3 treatments (each treatment was replicated 3 times, and each replicate had 20 plants). Among them, the control group was sprayed with a deionized water solution containing only 0.1% (v / v) Tween-80 on the leaves of each grape plant; the AT1 group and the AT2 group were sprayed with a 100 mg / L abscisic acid solution on the leaves of each grape plant. The spraying was carried out by a manual backpack sprayer, and the spraying dose was 100 mL / plant. After the spraying, the grape plants were managed according to the conventional method.

[0052] (3) Two weeks later, the control group and AT1 group: each grape vine was evenly sprayed with a deionized water solution containing only 0.1% (v / v) Tween-80; AT2 group: each grape vine was evenly sprayed with an abscisic acid solution with a concentration of 100 mg / L; the specific spraying method, dosage and subsequent management method were the same as in step (2).

[0053] (4) The determination of IBMP, IPMP and SBMP in grape berries was the same as in Example 1.

[0054] See results Figure 2 Compared with the control group and the AT1 group, the total amounts of IBMP, IPMP and SBMP in grapes were lowest under the AT2 treatment (i.e., sprayed twice), with total amounts of IBMP, IPMP and SBMP of 9.7 ng / L, 2.6 ng / L and 4.5 ng / L, respectively. Therefore, the optimal number of sprays for abscisic acid application is 2.

[0055] Furthermore, this embodiment did not include a three-application test because if a third application were included, the time between the third application and harvest would be only one to two weeks, making it impossible to guarantee the effects of abscisic acid on the human body.

[0056] Example 3: Screening of abscisic acid spraying time

[0057] (1) Prepare a foliar spray of abscisic acid solution with a concentration of 100 mg / L (containing 0.1% (v / v) Tween-80), and prepare a deionized water solution containing only 0.1% (v / v) Tween-80 as a control.

[0058] (2) The experiment was conducted at the vineyard of Tianming Minquan Winery in Minquan County, Henan Province. The experiment began in early August 2022, on a windless, sunny day between 5 and 7 pm after sunset. The specific method was as follows: grapevines with similar growth were selected and divided into four treatments (each treatment was replicated three times, with 20 vines per replicate). Control 1 group: at the beginning of grape veraison (early August), each grapevine was uniformly sprayed with a deionized water solution containing only 0.1% (v / v) Tween-80. AT 1 group: at the beginning of grape veraison (early August), each grapevine was uniformly sprayed with a solution containing 100 mg / L of Tween-80. L of abscisic acid solution; Control 2 group: when the grapes entered the mid-to-late stage of veraison and the average soluble solids content of the fruit was 16°Brix (late August), the leaves of each grapevine were evenly sprayed with a deionized water solution containing only 0.1% (v / v) Tween-80; AT 2 group: when the grapes entered the mid-to-late stage of veraison and the average soluble solids content of the fruit was 16°Brix (late August), the leaves of each grapevine were evenly sprayed with a 100 mg / L abscisic acid solution; spraying was carried out using a backpack sprayer, with a spraying dose of 100 mL / vine, and the grapevines were managed according to conventional methods after spraying.

[0059] (3) Control1 group, Control2 group, AT1 group and AT2 group were sprayed for the second time two weeks after the first spraying, and the spraying method was the same as step (2);

[0060] (4) Determination of IBMP, IPMP, and SBMP in grape berries, specifically including:

[0061] ① Sample pretreatment: Same as in Example 1;

[0062] ②GC-MS detection: Same as in Example 1;

[0063] ③ Establishment of Standard Curve: The content of each component of methoxypyrazine was determined using the external standard method. Ten serial dilutions were performed using simulated grape juice solution (7 g / L tartaric acid, 200 g / L glucose). The standard curve (simulated grape juice) is as follows: IBMP: y = 31451x + 244382, R 2 =0.996; IPMP: y=23270x+43748, R 2 =0.997; SBMP: y=23270x+9017, R 2 =0.997;

[0064] See results Figure 3Compared with Control1, Control2 and AT1, the total amounts of IBMP, IPMP and SBMP were lowest under the AT2 treatment, with total amounts of IBMP, IPMP and SBMP of 10.1 ng / L, 5.6 ng / L and 4.6 ng / L, respectively. Therefore, the optimal time for abscisic acid application should be when the grapes are in the mid-to-late stage of color change and the average soluble solids content of the fruit is at 16°Brix.

[0065] Example 4: Application of abscisic acid in sensory analysis of methoxypyrazine and aroma in Cabernet Sauvignon wines

[0066] (1) Prepare an abscisic acid solution with a concentration of 100 mg / L (containing 0.1% (v / v) Tween-80) as a foliar spray, and prepare a deionized water solution containing only 0.1% (v / v) Tween-80 as a control.

[0067] (2) The experiment was conducted at the vineyard of Tianming Minquan Winery in Minquan County, Henan Province, in late August 2022. The specific method was as follows: when the grapes entered the mid-to-late stage of color change (i.e., 60%-80% of the grape fruits were colored) and the average soluble solids content of the fruit was at 16°Brix, the experiment was carried out at 5-7 pm on a windless sunny day after sunset. Grape plants with basically the same growth were selected as test materials and divided into two treatments (each treatment was replicated 3 times, and each replicate had 20 plants). Among them, the control group was sprayed with a deionized water solution containing only 0.1% (v / v) Tween-80 on the leaves of each grape plant; the AT group was sprayed with a solution of abscisic acid with a concentration of 100 mg / L on the leaves of each grape plant. The spraying was carried out by a manual backpack sprayer, and the spraying dosage was 100 mL / plant. After the spraying, the grape plants were managed according to the conventional method.

[0068] (3) Two weeks later, perform a second foliar spraying as described in step (2), and then manage it according to the usual method.

[0069] (4) Winemaking process: The wine is made according to the winery's conventional winemaking process. The specific operation is as follows: 20 kg of grapes are taken for each replicate, the stems are removed, the grapes are crushed and put into the fermentation tank, sodium metabisulfite and LallzymeEx pectinase (LallzymeEx, Lallemand, France) are added and stirred evenly. About 24 hours after entering the tank, activated Lalvin RC212 series commercial wine yeast (Lallemand, France) is added. Then, during the alcoholic fermentation process, the temperature is controlled between 25-27℃. When the specific gravity drops to 0.993-0.996 and the residual sugar is less than 2.5 g / L, the alcoholic fermentation ends. Then, the skins and pomace are separated, and the free-run juice is mixed with the pressed juice. 100 mL of wine sample is taken and stored in a -40℃ refrigerator.

[0070] (5) Determination of IBMP, IPMP and SBMP in wine:

[0071] ① Sample pretreatment: Take 5 mL of wine sample and place it directly into a sample vial, add 1 g NaCl, and tighten the cap for GC-MS analysis as described below;

[0072] ②GC-MS detection: Same as in Example 1;

[0073] ③ Establishment of standard curve: The content of each component of methoxypyrazine was determined using the external standard method. Standard solutions were prepared using simulated wine (10 degrees) and diluted 10 times. The standard curve (simulated wine) is as follows: IPMP: y = 90x + 2592, R 2 =0.995; SBMP: y=162x+10166, R 2 =0.993; IBMP: y=115x+3460, R 2 =0.995;

[0074] (6) Sensory Evaluation: Fifteen tasters (8 women and 7 men) aged 23-42 were invited to conduct aroma sensory evaluations of the two treated wines. Seven commonly used aroma descriptors were selected, including floral, fruity, green, spicy, toasty, mineral, and animalic aromas. Evaluators were asked to rate the intensity of the aromas: 0, 1, 2, 3, and 4 represent no aroma, weak aroma intensity, moderate aroma intensity, strong aroma intensity, and very strong aroma intensity, respectively. A score of 0.5 was allowed.

[0075] Experiments revealed that compared to conventional cultivation methods, treatment with 100 mg / L abscisic acid solution significantly reduced the total amounts of IBMP, IPMP, and SBMP in wine, especially IPMP. The total amounts of IBMP, IPMP, and SBMP were 4.10 ng / L, 0.13 ng / L, and 2.63 ng / L, respectively. Figure 4 ).

[0076] The sensory evaluation results are as follows: Figure 5 Among the results, the "green" flavor was significantly reduced, the "fruity" flavor was improved, and the floral, spicy, toasty, mineral, and animal aromas did not change significantly. These results indicate that the application of 100 mg / L abscisic acid solution has a significant effect, effectively controlling the accumulation and content of methoxypyrazines and improving the quality of wine to a certain extent.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of abscisic acid in reducing the accumulation of methoxypyrazine compounds in grapes or wine, characterized in that... The application includes the following steps: (1) During grape cultivation, when the grapes enter the mid-to-late stage of color change and the soluble solids content of the fruit is between 15-18... o During Brix, abscisic acid solution was used as the foliar spray agent for the first foliar spraying of the grapevines; (2) Two weeks after the first foliar spraying, use abscisic acid solution as the foliar spraying agent to spray the grapevines a second time, and then manage them according to the conventional method. The grapes mentioned are Cabernet Sauvignon, Merlot, or Cabernet Franc; The methoxypyrazines mentioned are at least one of 3-isobutyl-2-methoxypyrazine, 3-isopropyl-2-methoxypyrazine, and 3-sec-butyl-2-methoxypyrazine; The treatment concentration of abscisic acid is 100 mg / L; The dosage for foliar spraying in steps (1) and (2) is 80-120 mL for each grapevine. The foliar spraying time mentioned in steps (1) and (2) is 5-7 pm after sunset on a windless, sunny day.

2. The application according to claim 1, characterized in that: In step (1), when the grapes enter the mid-to-late stage of color change and the soluble solids content of the fruit is between 16-17... o During Brix, abscisic acid solution was used as the foliar spray for the first foliar application to the grapevines.

3. A cultivation method for reducing the accumulation of methoxypyrazine compounds in grapes or wine, characterized in that... It includes the following steps; (1) During grape cultivation, when the grapes enter the mid-to-late stage of color change and the soluble solids content of the fruit is between 15-18... o During Brix, abscisic acid solution was used as the foliar spray agent for the first foliar spraying of the grapevines; (2) Two weeks after the first foliar spraying, use abscisic acid solution as the foliar spraying agent to spray the grapevines a second time, and then manage them according to the conventional method. The grapes mentioned are Cabernet Sauvignon, Merlot, or Cabernet Franc; The methoxypyrazines mentioned are at least one of 3-isobutyl-2-methoxypyrazine, 3-isopropyl-2-methoxypyrazine, and 3-sec-butyl-2-methoxypyrazine; The abscisic acid solution described in steps (1) and (2) comprises the following components: 100 mg / L abscisic acid and 0.1% (v / v) Tween-80; The dosage for foliar spraying in steps (1) and (2) is 80-120 mL for each grapevine. The foliar spraying time mentioned in steps (1) and (2) is 5-7 pm after sunset on a windless, sunny day.

4. The cultivation method for reducing the accumulation of methoxypyrazine compounds in grapes or wine according to claim 3, characterized in that: The foliar sprayer described in steps (1) and (2) can be at least one of a manual backpack sprayer and a drone sprayer.

5. The cultivation method for reducing the accumulation of methoxypyrazine compounds in grapes or wine according to claim 3, characterized in that: If it rains within 24 hours after foliar spraying in steps (1) and (2), it is necessary to spray again after the weather clears up.

Images