A method for predicting the potential of wine fatty lactone generation based on fruit precursors

Abstract

The present application relates to the technical field of wine brewing, and particularly relates to a method for predicting the generation potential of wine aliphatic lactone based on fruit precursors. The concentration of aliphatic precursors in grape fruits is detected, and the generation potential of wine aliphatic lactone is obtained according to the concentration of the aliphatic precursors through a pre-trained potential prediction model. The prediction method can efficiently and accurately predict and evaluate the aroma potential of wine after fermentation according to the concentration of precursors in grape fruits before fermentation, so as to achieve the purpose of guiding harvesting and brewing.

Description

Technical Field

[0001] This invention relates to the field of winemaking technology, and more specifically, to a method for predicting the aliphatic lactone formation potential of wine based on fruit precursors. Background Technology

[0002] Aliphatic lactones in wine can impart key aromas such as sweetness and stone fruit aromas. Especially in products such as ice wine, late-harvest wine, and dried wine, lactones are the core flavor components that constitute their characteristic sweet aroma. Therefore, predicting the potential of fruit raw materials in terms of lactone formation is of great significance for assessing the ripeness of raw materials, determining the appropriate harvest time, and guiding production.

[0003] Since the direct precursors of aliphatic lactones in wine are unknown, the existing technology for assessing the potential of grape raw materials to generate aliphatic lactones generally adopts the method of detecting the lactone concentration after fermentation. The core of the technical operation is to process and ferment the grape raw materials to be tested into wine, and then perform metabolic component analysis on the finished wine. By measuring the content of aliphatic lactones in the final wine sample, the accumulation status and conversion potential of related precursors in the raw materials can be indirectly inferred and evaluated.

[0004] While existing technologies can provide the final results of lactone formation, they have significant drawbacks. First, the method is too time-consuming, typically taking more than a week from the start of fermentation to obtaining the test results, which is completely unsuitable for the timeliness required for grape harvesting decisions and can easily lead to missing the optimal harvesting window. Second, the complete fermentation experiment process is complex, consuming a lot of manpower, materials, and time, making it difficult to achieve rapid and low-cost evaluation of a large number of samples from different periods and plots of raw materials. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for predicting the aliphatic lactone formation potential of wine based on fruit precursors.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for detecting aliphatic precursors in grape fruit. The method involves preparing a solution containing grape juice from grape fruit, obtaining an eluent by solid-phase extraction of the solution containing grape juice, and detecting the eluent using high-performance liquid chromatography-triple tandem quadrupole mass spectrometry to obtain the concentration of the aliphatic precursor. In the solid-phase extraction, the volume ratio of dichloromethane used for elution to grape juice is 1:1. The aliphatic precursors include 4-oxohexanoic acid, 4-oxooctanoic acid, 4-oxonanoic acid, 4-oxodecanoic acid, 5-oxooctanoic acid, and 5-oxodecanoic acid.

[0007] Furthermore, the method for preparing the solution containing grape juice using grape fruit is as follows: crushing and soaking the grape fruit to obtain grape juice, diluting it with water and adding an internal standard substance to obtain the solution containing grape juice; the internal standard substance is 6,6,7,7-d4-nonanoic acid; The solution containing grape juice has a volume percentage of 75% grape juice and a concentration of 550 μg / L for the internal standard.

[0008] Furthermore, after adding the solution containing grape juice to the solid phase extraction column, water is first added for rinsing, and then dichloromethane is added for elution.

[0009] Furthermore, the solid-phase extraction column is a Cleanert PEP solid-phase extraction column; before performing the solid-phase extraction, the solid-phase extraction column is activated sequentially with 10 mL of dichloromethane, 10 mL of methanol and 10 mL of water.

[0010] Furthermore, in the high performance liquid chromatography-triple tandem quadrupole mass spectrometry method, the chromatographic column used is a Poroshell 120 SB-C18 column with specifications of 150 mm × 2.1 mm and 2.7 μm. During detection, the column temperature of the chromatographic column was 50℃, the injection volume was 10 μL, and the flow rate was 0.4 mL / min; the mobile phase A used was a 0.1% (w / w) aqueous solution of formic acid, and the mobile phase B was a 0.1% (w / w) solution of formic acid and acetonitrile. The elution procedure for high performance liquid chromatography is as follows: .

[0011] Furthermore, in the high performance liquid chromatography-triple tandem quadrupole mass spectrometry method, the mass spectrometry conditions are as follows: AJSESI ion source, negative ion mode, nebulizer gas pressure of 30 psi, drying gas flow rate of 7 L / min, and drying gas temperature of 250℃.

[0012] The present invention also provides an application of the detection method for aliphatic precursors as described above, wherein the concentration of aliphatic precursors in grape berries is detected by the detection method, and the content of aliphatic lactones in wine made from the grape berries is predicted.

[0013] The present invention also provides a method for predicting the aliphatic lactone formation potential of wine based on fruit precursors, wherein the fruit precursors are aliphatic precursors in grapes, and the prediction method is to detect the concentration of the aliphatic precursors in grapes using the detection method described above, and obtain the aliphatic lactone formation potential of the wine made from the grapes based on the concentration of the aliphatic precursors through a pre-trained potential prediction model.

[0014] Furthermore, the low prediction threshold and high prediction threshold of aliphatic lactones in the wine made from the grapes are obtained through the pre-trained potential prediction model, and the aliphatic lactone generation potential of the wine is obtained based on the low prediction threshold and the high prediction threshold.

[0015] Furthermore, when the low prediction threshold is greater than or equal to 1, the wine made from the grapes is determined to have a high potential for lactone formation. When the high prediction threshold is greater than or equal to 1, the wine made from the grapes is determined to have a medium lactone formation potential. When the high prediction threshold is less than 1, the wine made from the grapes is determined to have low lactone formation potential.

[0016] The beneficial effects of this invention are as follows: This invention constructs a predictive model for predicting the lactone content in post-fermentation wine based on aliphatic precursors in grape berries. By simply detecting the concentration of aliphatic precursors in grape raw material samples, the model can directly calculate the threshold of fatty acid lactones and assess and predict the lactone formation potential.

[0017] The prediction method of this invention can efficiently and accurately predict and evaluate the aroma potential of wine after fermentation based on the concentration of precursors in grapes before fermentation, thereby guiding harvesting and winemaking.

[0018] Compared to existing technologies that rely on fermentation experiments to evaluate aliphatic lactones, this invention is more timely, enabling immediate detection and efficient prediction during the raw material harvesting stage. This matches the immediacy required for grape harvesting decisions, assists in determining the optimal harvest time and winemaking process decisions, and avoids the drawbacks of complex fermentation experiments that consume large amounts of manpower, materials, and time. It achieves rapid and low-cost evaluation of a large number of samples from different periods and plots of raw materials. Compared to the extensive evaluation methods in existing technologies that rely on single physicochemical indicators or empirical judgments, this application establishes a quantitative conversion relationship between aliphatic lactone precursors and corresponding lactones, thereby achieving precise quantitative prediction of the γ- and δ-lactone synthesis potential in grape berries. Attached Figure Description

[0019] Figure 1 This is a graph showing the concentration changes of various fatty acid lactones during fermentation in Example 1 of the present invention. Figure 1 The af values ​​are, in order, γ-caprolactone, γ-nonalactone, δ-octolactone, γ-octolactone, δ-decanolide, and γ-decanolide; Figure 2 The dMRM scan chromatograms of each aliphatic lactone precursor in Example 2 of the present invention are shown. Detailed Implementation

[0020] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] The present invention discloses a method for detecting aliphatic precursors in grape fruit, comprising preparing a solution containing grape juice from grape fruit, obtaining an eluent by solid-phase extraction of the solution containing grape juice, and detecting the eluent by high performance liquid chromatography-triple tandem quadrupole mass spectrometry to obtain the concentration of aliphatic precursors; in the solid-phase extraction, the volume ratio of dichloromethane used for elution to grape juice is 1:1; the aliphatic precursors include 4-oxohexanoic acid, 4-oxooctanoic acid, 4-oxonanoic acid, 4-oxodecanoic acid, 5-oxooctanoic acid, and 5-oxodecanoic acid.

[0022] The method for detecting aliphatic precursors in grapes of the present invention can rapidly and simultaneously detect 4-oxohexanoic acid, 4-oxooctanoic acid, 4-oxonanoic acid, 4-oxodecanoic acid, 5-oxooctanoic acid, and 5-oxodecanoic acid, providing a reliable means for predicting the content of aliphatic lactones in wine using these aliphatic precursors.

[0023] The aliphatic precursor of the present invention can predict the aliphatic lactone content in wine, thereby effectively assessing the ripeness of grapes in the raw material, determining the appropriate harvest time, and guiding the winemaking process.

[0024] The following reaction formula (1) is the pathway for the asymmetric reduction cyclization of 4-oxoalkyl acid to generate aliphatic γ-lactone (R)-enantiomer, and reaction formula (2) is the pathway for the generation of aliphatic δ-lactone (R)-enantiomer from 5-oxoalkyl acid, where R represents an alkyl group with 1-8 carbon atoms.

[0025]

[0026] Experiments with the addition of grape juice during fermentation confirmed that 4-oxonanoic acid can be rapidly converted into γ-nonanolactone with a high bioconversion rate. Based on this clear conversion pathway and the theoretical mechanism of Mosandl and Günther, other aliphatic lactones in wine also have structurally similar oxo-fatty acid precursors, which are converted into lactones through the same reductive-lactoneization pathway.

[0027] As shown in Table 1, specifically, 4-oxohexanoic acid, 4-oxooctanoic acid, 4-oxonanoic acid, and 4-oxodecanoic acid are converted into potential precursors of γ-caprolactone, γ-octanoic acid, γ-nonanoic acid, and γ-decanoic acid, respectively; meanwhile, based on similar reduction pathways, 5-oxooctanoic acid and 5-oxodecanoic acid are converted into precursors of δ-octanoic acid and δ-decanoic acid, respectively.

[0028] Table 1. Structures of potential aliphatic lactone precursors in wine

[0029] Therefore, the aliphatic precursors 4-oxohexanoic acid, 4-oxooctanoic acid, 4-oxonanoic acid, 4-oxodecanoic acid, 5-oxooctanoic acid and 5-oxodecanoic acid from grapes can be used to effectively predict the aliphatic lactone content in wine.

[0030] In the detection method of the present invention, the method of preparing a solution containing grape juice by grape fruit is as follows: grape fruit is crushed and soaked to obtain grape juice, diluted with water and an internal standard is added to obtain a solution containing grape juice; the internal standard is 6,6,7,7-d4-nonanoic acid; in the solution containing grape juice, the volume percentage of grape juice is 75% and the concentration of the internal standard is 550 μg / L.

[0031] Preferably, the solid phase extraction column is a Cleanert PEP solid phase extraction column; before solid phase extraction, the solid phase extraction column is activated sequentially with 10 mL of dichloromethane, 10 mL of methanol and 10 mL of water.

[0032] Preferably, when using a Cleanert PEP solid-phase extraction column, the amount of water added for rinsing is 5 mL, and the amount of dichloromethane added is 15 mL.

[0033] In the high performance liquid chromatography-triple tandem quadrupole mass spectrometry method of the present invention, the chromatographic column used is a Poroshell 120 SB-C18 column with specifications of 150 mm × 2.1 mm and 2.7 μm.

[0034] During the detection, the column temperature was 50℃, the injection volume was 10 μL, and the flow rate was 0.4 mL / min. The mobile phase A used was a 0.1% formic acid aqueous solution, and the mobile phase B was a 0.1% formic acid acetonitrile solution. The elution procedure for high performance liquid chromatography is as follows: .

[0035] Preferably, in the high performance liquid chromatography-triple tandem quadrupole mass spectrometry method, the mass spectrometry conditions are as follows: using an AJS ESI ion source, negative ion mode, nebulizer gas pressure of 30 psi, drying gas flow rate of 7 L / min, and drying gas temperature of 250℃.

[0036] For the qualitative and quantitative processes of each precursor, conventional calculation methods can be used.

[0037] Specifically, the standards for each precursor were tested using the same detection method described above, and the detection results of the test sample were compared with the chromatographic and mass spectrometric peaks of the standards to qualitatively detect each precursor.

[0038] Furthermore, for each precursor, multiple concentration gradients were used for detection to establish a standard curve. The detection results of the test sample were then substituted into the standard curve for calculation to obtain the quantitative detection results of the concentration of each precursor.

[0039] The prediction method of this invention obtains the aliphatic lactone formation potential of wine based on the concentration of aliphatic precursors through a pre-trained potential prediction model.

[0040] Preferably, the low prediction threshold and high prediction threshold of aliphatic lactones are obtained by a pre-trained potential prediction model, and then the aliphatic lactone generation potential of wine is obtained based on the low prediction threshold and high prediction threshold.

[0041] Specifically, the calculation formula for this model is as follows: (1) (2) In equations (1) and (2), AR L Indicates a low prediction threshold, AR H Indicates a high prediction threshold, C i K represents the concentration of an aliphatic precursor. Li Indicates low conversion parameter, K Hi T represents the high conversion parameter. i This indicates the olfactory threshold of the compound corresponding to number i, which can be found in publicly available literature.

[0042] The specific prediction method is as follows: when the low prediction threshold is greater than or equal to 1, the wine is judged to have high lactone formation potential; when the high prediction threshold is greater than or equal to 1, the wine is judged to have medium lactone formation potential; when the high prediction threshold is less than 1, the wine is judged to have low lactone formation potential.

[0043] The effects of the present invention will be illustrated by specific embodiments below.

[0044] Example 1: Validation of precursor fermentation and transformation In this embodiment, a fermentation experiment was conducted by adding a predicted precursor to simulated juice to clarify the conversion relationship between the precursor and the corresponding aliphatic lactone.

[0045] The specific experimental method involved adding 300 mL of simulated grape juice to a 500 mL Erlenmeyer flask equipped with a fermentation plug. Two potential addition experiments with different concentration gradients were designed, as shown in Table 2. Yeast (D254) was added at a concentration of 200 ppm, along with 60 mg / L SO2 (1 mL / L H2SO3), and alcoholic fermentation was carried out at 18–22 °C. The fermentation process was assessed by determining reducing sugar using the DNS method and CO2 loss by weight method. Fermentation was considered complete when the sugar concentration was below 2 g / L. At days 0, 4, 8, and 12, and at the fermentation endpoint, 20 mL samples were taken from the fermentation broth after shaking, centrifuged, and the supernatant was analyzed. Simulated grape juice without added oxoalkyl acids served as a control, and each treatment was repeated three times.

[0046] Table 2. Amounts of various oxoalkyl acids added in simulated grape juice

[0047] Note: "-" indicates that no yeast or oxoalkyl acid was added, and "+" indicates that oxoalkyl acid was added.

[0048] During yeast alcoholic fermentation, 5-oxooctanoic acid is converted to δ-octanolactone, 4-oxooctanoic acid is converted to γ-octanolactone, 5-oxodecanoic acid is converted to δ-decanolactone, and 4-oxodecanoic acid is converted to γ-decanolactone. Figure 1 The final conversion rates in the Pre-H group were 89.45%, 80.07%, 82.47%, and 76.93%, respectively, while the final conversion rates in the Pre-L group were 86.41%, 92.27%, 83.79%, and 91.13%, respectively.

[0049] Example 2: Construction of a targeted quantitative method for aliphatic lactone precursors (1) Extraction of precursors: Weigh 50 g of grapes stored at -80℃, remove the stems and seeds under liquid nitrogen protection, add 0.5 g of D-gluconolactone and 1 g of PVPP, and crush into powder using a fruit grinder; soak the powder in a sealed environment at 4℃ for 4 h, and then centrifuge at 4℃ and 8000 r / min for 10 min, and collect the clear grape juice from the top layer.

[0050] After filtering the grape juice through a sieve, 15 mL was measured and mixed with 5 mL of water. 10 μL of internal standard (1100 mg / L 6,6,7,7-d4-nonanoic acid) was added. The mixture was then passed through a 500 mg / 6 mL Cleanert PEP solid-phase extraction column activated with 10 mL dichloromethane, 10 mL methanol, and 10 mL water. 5 mL of deionized water was added to remove sugars, acids, and large polar molecules. Finally, 15 mL of chromatographically pure dichloromethane was added for elution. The collected eluent was dried over 1 g of anhydrous sodium sulfate, then dried under nitrogen. The eluent was reconstituted with 500 μL of methanol, filtered through a 0.22 μm organic filter membrane, and transferred to a 2 mL sample vial for analysis.

[0051] (2) Precursor scanning method: High-performance liquid chromatography-quadrupole mass spectrometry (HPLC-QqQ-MS / MS) analysis was performed using an Agilent 1290-6470 triple quadrupole HPLC-MS / MS system with a Poroshell 120 SB-C18 column (150 mm × 2.1 mm, 2.7 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The optimized elution program was as follows: 0–8 min, 15%–55% B; 8–9 min, 55%–100% B; 9–10 min, hold at 100% B. The column temperature was 50 °C, the injection volume was 10 μL, and the flow rate was 0.4 mL / min.

[0052] The mass spectrometry conditions were as follows: an AJS ESI ion source, negative ion mode, nebulizer gas pressure of 30 psi, drying gas flow rate of 7 L / min, and drying gas temperature of 250℃. For each substance, a full scan was first used to determine the retention time and precursor ions. Then, a product ion scan was used to determine the optimal product ions for each substance, establishing a dynamic multiple reaction monitoring (dMRM) method. Figure 2 Then, the MassHunter Optimizer software (Agilent, Santa Clara, CA, USA) was used to optimize the multiple reaction monitoring (MRM) parameters of the compound, and the Fragmentor parameter and fragmentation voltage corresponding to the maximum abundance of the target ion were selected (Table 3).

[0053] (3) Methods for quantifying precursors: Standard solutions of different concentration gradients were prepared in simulated dry wine, simulated ice wine, and simulated grape juice using oxidized fatty acid standards. The standard solutions were extracted and detected using the same method. The retention time and quantitative / qualitative ions of the target substances are shown in Table 2. A yx standard curve was established based on the ratio of the peak area of ​​the target substance to the peak area of ​​the internal standard and the concentration of the target substance (Table 4).

[0054] Table 3 Scanning information for oxidized fatty acids and internal standards

[0055] Table 4. Standard curves and method evaluation of oxidized fatty acids

[0056] Integrate the peaks of the target substance and the internal standard in the sample separately, and substitute the ratio of the peak area of ​​the target substance to the peak area of ​​the internal standard in the sample into the corresponding standard curve to calculate the concentration of the target substance.

[0057] Example 3: Establishment of a predictive model for the fatty acid lactone formation potential of grape fruit The concentration of oxidized fatty acids (C) in grape berries was obtained by detecting oxidized fatty acids using the method described in Example 2. i Based on the conversion rates of the Pre-H and Pre-L groups obtained from the precursor fermentation and transformation verification in Example 1, K was obtained. Hi and K Li .

[0058] The following formula for the prediction model is established, and the concentration of oxo-fatty acids is substituted into the formula to calculate AR. L and AR H .

[0059]

[0060]

[0061] Calculations were performed based on the parameters in Table 5 according to the fatty acid ester type, and raw material evaluation was conducted according to the lactone formation potential evaluation table (Table 6).

[0062] Table 5 Calculation Parameters

[0063] Table 6 Grape Raw Material Aliphatic Lactone Formation Potential Ranking

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting aliphatic precursors in grape berries, characterized in that, A solution containing grape juice was prepared using grape fruit. The solution containing grape juice was then subjected to solid-phase extraction to obtain an eluent. The eluent was detected using high-performance liquid chromatography-triple tandem quadrupole mass spectrometry to obtain the concentration of the aliphatic precursor. In the solid-phase extraction, the volume ratio of dichloromethane used for elution to grape juice is 1:

1. The aliphatic precursors include 4-oxohexanoic acid, 4-oxooctanoic acid, 4-oxonanoic acid, 4-oxodecanoic acid, 5-oxooctanoic acid, and 5-oxodecanoic acid.

2. The method for detecting aliphatic precursors according to claim 1, characterized in that, The method for preparing a solution containing grape juice using grape fruit involves crushing and soaking the grape fruit to obtain grape juice, diluting it with water, and adding an internal standard to obtain the solution containing grape juice; the internal standard is 6,6,7,7-d4-nonanoic acid. The solution containing grape juice has a volume percentage of 75% grape juice and a concentration of 550 μg / L for the internal standard.

3. The method for detecting aliphatic precursors according to claim 2, characterized in that, After adding the solution containing grape juice to the solid phase extraction column, water is first added for rinsing, and then dichloromethane is added for elution.

4. The method for detecting an aliphatic precursor according to claim 3, characterized in that, The solid-phase extraction column is a Cleanert PEP solid-phase extraction column; before performing the solid-phase extraction, the solid-phase extraction column is activated sequentially with 10 mL of dichloromethane, 10 mL of methanol and 10 mL of water.

5. A method for detecting an aliphatic precursor according to any one of claims 1-4, characterized in that, In the high performance liquid chromatography-triple tandem quadrupole mass spectrometry method, the chromatographic column used is a Poroshell 120 SB-C18 column with specifications of 150 mm × 2.1 mm and 2.7 μm. During detection, the column temperature of the chromatographic column was 50℃, the injection volume was 10 μL, and the flow rate was 0.4 mL / min; the mobile phase A used was a 0.1% (w / w) aqueous solution of formic acid, and the mobile phase B was a 0.1% (w / w) solution of formic acid and acetonitrile. The elution procedure for high performance liquid chromatography is as follows: 。 6. A method for detecting an aliphatic precursor according to any one of claims 1-4, characterized in that, In the high performance liquid chromatography-triple tandem quadrupole mass spectrometry method, the mass spectrometry conditions are as follows: AJS ESI ion source, negative ion mode, nebulizer gas pressure of 30 psi, drying gas flow rate of 7 L / min, and drying gas temperature of 250℃.

7. The application of a method for detecting aliphatic precursors as described in any one of claims 1-6, characterized in that, The concentration of aliphatic precursors in grapes was detected using the aforementioned detection method, and the content of aliphatic lactones in wines made from the grapes was predicted.

8. A method for predicting the aliphatic lactone formation potential of wine based on fruit precursors, characterized in that, The fruit precursor is an aliphatic precursor in grape fruit. The prediction method is to detect the concentration of the aliphatic precursor in grape fruit using the detection method described in any one of claims 1-6, and obtain the aliphatic lactone formation potential of the wine made from the grape fruit by means of a pre-trained potential prediction model based on the concentration of the aliphatic precursor.

9. The method for predicting the aliphatic lactone formation potential of wine based on fruit precursors according to claim 8, characterized in that, The low prediction threshold and high prediction threshold of aliphatic lactones in the wine made from the grapes are obtained by the pre-trained potential prediction model, and the aliphatic lactone generation potential of the wine is obtained based on the low prediction threshold and the high prediction threshold.

10. The method for predicting the aliphatic lactone formation potential of wine based on fruit precursors according to claim 9, characterized in that, When the low prediction threshold is greater than or equal to 1, the wine made from the grapes is determined to have a high potential for lactone formation. When the high prediction threshold is greater than or equal to 1, the wine made from the grapes is determined to have a medium lactone formation potential. When the high prediction threshold is less than 1, the wine made from the grapes is determined to have low lactone formation potential.

Images