Apple fruit key aroma substance identification method based on sensory omics

Abstract

The invention discloses an apple fruit key aroma substance identification method based on sensory omics, and relates to the technical field of aroma substance detection, and the method specifically comprises the following steps: S1, detecting volatile compounds in apple fruits through comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC * GC-QTOFMS); s2, carrying out gas chromatography-olfactory analyzer (GC-O) and odor activity value (OAV) analysis to characterize characteristic aroma compounds in the apple fruits; and S3, determining key characteristic aroma compounds in the apple fruits through aroma recombination and omission experiments. According to the present invention, the aroma substance extraction and enrichment are performed on the apple by using the solid phase micro-extraction, the organic solvent is not required, the operation is simple and easy, the comprehensive two-dimensional gas chromatography-tandem time-of-flight mass spectrometry can comprehensively analyze the volatile compounds in the apple fruit within the short time compared with the common chromatography-tandem mass spectrometry, the peak capacity is significantly improved, and the determination result is significant. The separation and analysis efficiency is obviously superior to that of a traditional gas chromatograph-mass spectrometer.

Description

Technical Field

[0001] The present invention relates to the technical field of aroma substance detection, and specifically provides a method for identifying key aroma substances in apple fruits based on sensory omics. Background Technique

[0002] Apples have become one of the most popular fruits due to their rich nutrient content and good storage and transportation properties. Currently, apples are one of the fruits with the highest yields globally. In 2022, the cultivated area of apples in China reached 2.1 million hectares, with a yield of 47 million tons, ranking first in the world in terms of cultivated area and yield of apples. The volatile compounds in apple fruits jointly constitute the aroma characteristics of apples, which significantly affect consumers' preference for apples. Currently, more than 300 volatile compounds have been identified from different apple varieties, including esters, alcohols, aldehydes, terpenes, and ketones. The main reasons affecting the types and contents of apple volatile compounds are apple varieties, followed by the differences in volatile compounds caused by fruit maturity and the quality differences of fruits due to different cultivation and storage measures. Sensory omics, also known as molecular sensory science, can qualitatively and quantitatively analyze the aroma components in foods, judge characteristic aroma-active substances through GC-O technology, and supplement aroma recombination and omission tests to identify the overall aroma characteristics of foods. Currently, it has been widely applied to the analysis of characteristic aromas of various foods, such as spices, tea, fruits, dairy products, and alcoholic products.

[0003] Comprehensive two-dimensional gas chromatography (GC×GC) is a technical means widely used for the separation and identification of volatile compounds in complex sample mixtures. It uses two chromatographic columns with different polarities connected by a modulator to enhance the separation degree and identification accuracy of volatile compounds. At the same time, comprehensive two-dimensional gas chromatography (GC×GC) combined with high-resolution mass spectrometry technology can identify the components of volatile compounds in complex foods, conduct accurate qualitative and quantitative analysis on them, and evaluate the overall aroma component composition of samples. Currently, GC×GC-TOFMS has been widely applied in the construction of food flavor fingerprints, variety and origin identification, quality monitoring, and target compound analysis.

[0004] Currently, the evaluation of the quality of new apple varieties mostly relies on basic physical and chemical index levels, and there is a lack of evaluation from the level of key aroma substances. Therefore, this study aims to propose a new method for analyzing the key characteristic aromas of apple fruits by using sensory omics technology, providing a theoretical reference for the breeding of future excellent apple varieties and the regulation of fruit quality. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a method for identifying key aroma substances in apple fruits based on sensory omics, solving the problems raised in the above background technique.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions: A method for identifying key aroma compounds in apple fruits based on sensory omics, comprising the following steps:

[0007] S1: Detect volatile compounds in apple fruits by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-QTOFMS);

[0008] S2: Then perform gas chromatography-olfactometry (GC-O) and odor activity value (OAV) analysis to characterize the characteristic aroma compounds in apple fruits;

[0009] S3: Determine the key characteristic aroma compounds in apple fruits through aroma recombination and omission experiments.

[0010] Preferably, the specific method for detecting volatile compounds in apple fruits by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-QTOFMS) in S1 comprises the following steps:

[0011] S1.1: Sample preparation: After removing the cores of apple fruits, cut them into small pieces and grind them under liquid nitrogen. Add polyvinylpyrrolidone (PVPP) and D-glucono-δ-lactone in a mass ratio of 100:1:1. The mixture is extracted in the dark at 4°C for 24 h, and then centrifuged at 4°C and 8000×g for 15 min. Collect the supernatant and store it at -40°C for further testing;

[0012] S1.2: Extraction of volatile compounds: Place the sample extract, sodium chloride, and internal standard solution in a headspace vial and extract volatile compounds by headspace solid-phase microextraction (HS-SPME);

[0013] S1.3: Analysis of volatile compounds: Analyze volatile compounds in apples using comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-QTOFMS);

[0014] S1.4: Qualitative analysis of volatile compounds: Screen and verify the data based on retention indices, matching degrees, aroma descriptions, and standard product information, etc., and qualitatively analyze volatile compounds;

[0015] S1.5: Quantitative analysis of volatile compounds: Quantify volatile compounds by constructing a standard curve of the compounds.

[0016] Preferably, in S1.2, the sample extract is 5 mL, sodium chloride is 1 g, and the internal standard is 10 μL of 4-methyl-2-pentanol (324.4 μg / L);

[0017] The headspace vial containing the sample was equilibrated in a multi-functional autosampler at 250 r / min and 45 °C for 10 min, and then a solid-phase microextraction needle (50 / 30 μm, DVB / CAR / PDMS) that had been aged was inserted into the headspace vial for extraction for 30 min. Subsequently, the solid-phase microextraction needle was inserted into the gas chromatography injection port and desorbed for 3 min in the splitless injection mode.

[0018] Preferably, the instrument conditions for GC×GC-QTOFMS analysis in S1.3 are as follows:

[0019] GC×GC system conditions: The first-dimensional chromatographic column is DB-WAX (30 m × 0.25 mm × 0.25 μm), and the second-dimensional chromatographic column is DB-17MS (1.85 m × 0.180 mm × 0.18 μm); the injection port temperature is 250 °C, and the splitless injection mode; the column oven temperature program is an initial temperature of 50 °C, held for 1 min, heated at 3 °C / min to 230 °C and held for 2 min; helium (99.999%) is used as the carrier gas, and the constant flow rate is 1 mL / min; the transfer line temperature is 260 °C; the two chromatographic columns are connected by an SV series modulation column (1.3 m × 0.25 mm, C6-C40), and a solid-state thermal regulator is used for heating and cooling, and the modulation period is 4 s;

[0020] QTOFMS system conditions: The full scan mode is used during the acquisition process, the mass scan range is 45 - 500 m / z, and the acquisition rate is 50 Hz; the temperatures of the ion source and the quadrupole are set to 230 °C and 150 °C respectively, the EI ionization energy is 70 eV, and the solvent delay time is 2.0 min.

[0021] Preferably, in S1.4, the Canvas software workstation is used to process the mass spectrometry information, and the minimum peak detection signal-to-noise ratio is set to 10; the mass spectrometry data is compared with the NIST20 standard library; the actual retention index (RI) value is calculated using a n-alkane standard solution; the odors and mass spectrometry diagrams of standard compounds are compared to accurately identify volatile compounds.

[0022] Preferably, in S1.5, each standard solution is dissolved in methanol to prepare a mixed standard solution, and the mixed standard solution is diluted in a 2n gradient and continuously diluted into 9 solutions with different concentrations, and 4-methyl-2-pentanol (10 μL, 324.4 μg / L) is added as an internal standard. Then, the standard compounds are extracted under the same conditions as the sample. By plotting the ratio of the peak areas of the standard compounds and the internal standard and their concentration ratios, the corresponding standard curve is obtained. All analyses are performed in triplicate. For compounds without a standard curve, the standard curve of compounds with a similar substance type and a similar number of carbon atoms is used for quantification.

[0023] Preferably, the GC-O in S2 is measured by an aroma intensity method, which is composed of 6 experts with GC-O experience. The odor attributes, aroma intensity, and start and end times are recorded when the material flows out of the olfactory detection port. The aroma intensity is expressed from 0 (none) to 4 (very strong), and the final aroma intensity value is expressed as an average value.

[0024] Preferably, the odor activity value (OAV) in S2 is calculated using the following formula: OAV=(Ci / OTi), Ci is the concentration of compound i in apple fruit, and OTi is the odor threshold of the compound in water.

[0025] Preferably, the aroma reconstitution experiment in S3 is to dissolve the characteristic aroma compound standards detected in apples by GC-O and OAV in methanol, dissolve them in simulated solutions according to corresponding measured concentrations, perform sensory evaluation on the reconstituted samples, and compare the differences between them and the original samples.

[0026] Preferably, the aroma omission experiment in S3 is based on aroma recombination, by omitting an aroma-active compound from the complete recombination model, preparing different omission models, conducting aroma omission experiments, and using a three-point test method to compare the differences between each omission model and the corresponding original recombination model.

[0027] The present invention provides a method for identifying key aroma substances in apple fruit based on sensory omics, which has the following beneficial effects:

[0028] 1. This sensory omics-based method for identifying key aroma substances in apple fruit uses solid phase microextraction to extract and enrich the aroma substances in apples. It does not require organic solvents and is simple and easy to operate. Compared with ordinary chromatography-tandem mass spectrometry analysis, comprehensive two-dimensional gas chromatography-tandem time-of-flight mass spectrometry can comprehensively analyze the volatile compounds in apple fruit in a shorter time, with significantly improved peak capacity, and significantly better separation and analysis efficiency than traditional gas chromatography-mass spectrometry.

[0029] 2. The sensory omics-based method for identifying key aroma substances in apple fruit uses high-resolution mass spectrometry to qualitatively characterize the volatile components in thin-skinned melons, adopts standard library matching, precise molecular weight, and retention index to ensure the reliability of the qualitative results, and accurately quantifies the characteristic aroma substances in apples through corresponding standard products.

[0030] 3. The method for identifying key aroma substances in apple fruit based on sensory omics, the present invention identifies characteristic aroma substances in apples through an olfactometer combined with gas chromatography, and determines key characteristic aroma substances through OAV value analysis, aroma recombination and omission tests.

[0031] 4. The method for identifying key aroma substances in apple fruits based on sensory omics. The present invention adopts the method of sensory omics, which can accurately analyze the key characteristic aroma substances in apple fruits of different varieties, clarify the aroma compounds that mainly contribute to the excellent aroma flavor of apples, and provide a theoretical reference for the breeding of future excellent apple varieties and the regulation of fruit quality. Description of the Drawings

[0032] Figure 1 3D chromatogram of aroma substances in Ruoyang apples in the specific embodiment of the present invention;

[0033] Figure 2 3D chromatogram of aroma substances in Ruixue apples in the specific embodiment of the present invention;

[0034] Figure 3 3D chromatogram of aroma substances in Ruixianghong apples in the specific embodiment of the present invention;

[0035] Figure 4 Radar chart of the apple aroma recombination model in the specific embodiment of the present invention. Detailed Description of the Invention

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0037] Please refer to Figures 1 to 4 , the present invention provides a technical solution: a method for identifying key aroma substances in apple fruits based on sensory omics, including the following steps:

[0038] S1: Detect volatile compounds in apple fruits by comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GC×GC-QTOFMS);

[0039] The specific method includes the following steps:

[0040] S1.1: Sample preparation: After removing the cores of apple fruits, cut them into small pieces and grind them under liquid nitrogen. Add polyvinylpyrrolidone (PVPP) and D-glucono-δ-lactone according to a mass ratio of 100:1:1. The mixture is extracted in the dark at 4°C for 24 hours, and then centrifuged at 4°C and 8000×g for 15 minutes. Collect the supernatant and store it at -40°C for later measurement;

[0041] S1.2: Extraction of volatile compounds: Place the sample extract, sodium chloride, and internal standard solution in a headspace vial and extract volatile compounds by headspace solid-phase microextraction (HS-SPME);

[0042] S1.3, Volatile Compound Analysis: Analyze the volatile compounds in apples using comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GC×GC-QTOFMS);

[0043] S1.4, Qualitative Analysis of Volatile Compounds: Screen and verify the data based on retention indices, matching degrees, aroma descriptions, and standard product information, etc., and qualitatively analyze the volatile compounds;

[0044] S1.5, Quantitative Analysis of Volatile Compounds: Quantify the volatile compounds by constructing a standard curve of the compounds.

[0045] In S1.2, the sample extract is 5 mL, sodium chloride is 1 g, and the internal standard is 10 μL of 4-methyl-2-pentanol (324.4 μg / L);

[0046] The headspace vial containing the sample is equilibrated in a multi-functional autosampler at 250 r / min and 45 °C for 10 min, and then the solid-phase microextraction needle (50 / 30 μm, DVB / CAR / PDMS) that has been aged is inserted into the headspace vial for extraction for 30 min. Subsequently, the solid-phase microextraction needle is inserted into the gas chromatography injection port and desorbed for 3 min in the splitless injection mode.

[0047] The instrument conditions for the GC×GC-QTOFMS analysis in S1.3 are as follows:

[0048] GC×GC System Conditions: The first-dimensional chromatographic column is DB-WAX (30 m × 0.25 mm × 0.25 μm), and the second-dimensional chromatographic column is DB-17MS (1.85 m × 0.180 mm × 0.18 μm); the injection port temperature is 250 °C, and the splitless injection mode; the column oven temperature program is as follows: the initial temperature is 50 °C, held for 1 min, heated at 3 °C / min to 230 °C and held for 2 min; helium (99.999%) is used as the carrier gas, and the constant flow rate is 1 mL / min; the transfer line temperature is 260 °C; the two chromatographic columns are connected by an SV series modulation column (1.3 m × 0.25 mm, C6-C40), and a solid-state thermal regulator is used for heating and cooling, and the modulation period is 4 s;

[0049] QTOFMS System Conditions: The full scan mode is adopted during the acquisition process, the mass scan range is 45 - 500 m / z, and the acquisition rate is 50 Hz; the temperatures of the ion source and the quadrupole are set at 230 °C and 150 °C respectively, the EI ionization energy is 70 eV, and the solvent delay time is 2.0 min.

[0050] In S1.4, a Canvas software workstation was used to process mass spectrometry information, and the minimum peak detection signal-to-noise ratio was set to 10; the mass spectrometry data was compared with the NIST20 standard library; the actual retention index (RI) value was calculated using a n-alkane standard solution; the odors and mass spectrometry diagrams of standard compounds were compared to accurately identify volatile compounds.

[0051] In S1.5, each standard solution was dissolved in methanol to prepare a mixed standard solution. The mixed standard solution was serially diluted in a 2n gradient to form 9 different concentrations of solutions, and 4-methyl-2-pentanol (10 μL, 324.4 μg / L) was added as an internal standard. Then, the standard compounds were extracted under the same conditions as the samples. By plotting the ratio of the peak areas of the standard compounds to the internal standard and their concentration ratios, the corresponding standard curves were obtained. All analyses were performed in triplicate. For compounds without a standard curve, the standard curves of compounds with similar substance types and similar carbon atom numbers were used for quantification.

[0052] S2: Then, gas chromatography-olfactometry (GC-O) and odor activity value (OAV) analysis were performed to characterize the characteristic aroma compounds in apple fruits;

[0053] In S2, GC-O was determined using the aroma intensity method and consisted of 6 experts with GC-O experience. When the substances flowed out of the olfactory detection port, the odor attributes, aroma intensity, and start and end times were recorded. The aroma intensity was represented from 0 (none) to 4 (very strong), and the final aroma intensity value was represented by the average.

[0054] In S2, the odor activity value (OAV) was calculated using the following formula: OAV = (Ci / OTi), where Ci is the concentration of compound i in apple fruits and OTi is the odor threshold of the compound in water.

[0055] S3: Through aroma recombination and omission experiments, the key characteristic aroma compounds in apple fruits were determined;

[0056] In the aroma recombination experiment in S3, the standard products of the characteristic aroma compounds detected by GC-O and OAV in apples were dissolved in methanol and dissolved in the simulation solution according to the corresponding measured concentrations. The recombinant samples were subjected to sensory evaluation to compare their differences with the original samples.

[0057] In the aroma omission experiment in S3, based on aroma recombination, different omission models were prepared by omitting one aroma-active compound from the complete recombinant model, and the aroma omission experiment was carried out. The three-point test method was used to compare the differences between each omission model and the corresponding original recombinant model.

[0058] Example 2: Based on Example 1; Please refer to Figures 1 to 4, the following test methods were used to identify and analyze the key characteristic aroma substances in the apple fruits of three varieties, namely Ruiyang (CNA20151468.2), Ruixue (CNA20151469.1), and Ruixianghong (CNA20191003428).

[0059] (1) Extraction of volatile compounds

[0060] After removing the cores of apple fruits, they were cut into small pieces and ground under liquid nitrogen. Polyvinylpyrrolidone (PVPP) and D - glucono - δ - lactone were added in a mass ratio of 100:1:1. The mixture was extracted in the dark at 4°C for 24 h, and then centrifuged at 4°C and 8000×g for 10 min. The supernatant was collected and stored at - 40°C for further analysis.

[0061] Volatile compounds were analyzed by headspace solid - phase microextraction combined with gas chromatography - mass spectrometry (HS - SPME - GC - MS). 5 mL of the apple fruit sample extract, 1 g of sodium chloride, and 10 μL of the internal standard 4 - methyl - 2 - pentanol (324.4 μg / L) were placed into a 20 mL headspace vial. The sample was placed in a multi - functional autosampler and equilibrated at 250 r / min and 45°C for 10 min. Then, the pretreated solid - phase microextraction needle (50 / 30 μm, DVB / CAR / PDMS) was inserted into the headspace vial for extraction for 30 min. Subsequently, the solid - phase microextraction needle was inserted into the gas chromatography injection port and desorbed in the splitless injection mode for 3 min.

[0062] (2) Analysis of volatile compounds

[0063] A GC×GC - QTOFMS instrument was used to analyze volatile compounds. The gas chromatography system consisted of two chromatographic columns. The first chromatographic column was a polar DB - WAX (30 m×0.25 mm×0.25 μm), and the second chromatographic column was a medium - polar DB - 17MS (1.85 m×0.180 mm×0.18 μm). The two chromatographic columns were connected by an SV series modulation column (1.3 m×0.25 mm, C6 - C 40) are combined for the aggregation and release of volatile compounds, and a solid-state thermal regulator is configured for heating and cooling phases. The volatile compounds are desorbed at 250 °C for 3.0 min in the gas chromatography injection port, and the injection mode is splitless mode. The initial temperature of the column oven is set at 50 °C, held for 1 min, and then heated to 230 °C at a rate of 3 °C / min and held for 2 min. The transfer line temperature is set at 260 °C, and helium (99.999%) is used as the carrier gas with a constant flow rate of 1 mL / min. The modulation period of the solid-state thermal regulator is 4 s, and the cold zone temperature is set at -50 °C. The time-of-flight mass spectrometry settings are as follows: The acquisition process uses the full-scan mode, the mass scan range is 45 - 500 m / z, and the acquisition rate is 50 Hz. The temperatures of the ion source and the quadrupole are set at 230 °C and 150 °C respectively, the EI ionization energy is 70 eV, and the solvent delay time is 2.0 min.

[0064] (3) Qualitative analysis of volatile compounds

[0065] The obtained mass spectrometry information is analyzed using the Canvas software workstation. After setting the minimum peak detection signal-to-noise ratio to 10, the mass spectrometry data of each aroma compound is compared with the NIST20 standard library. Then, C7 - C 40 A series of alkane standard solutions were analyzed, and the actual retention index (RI) values were calculated. The actual RI values were compared with the standard RI values of the target compounds to verify their consistency. In addition, sensory evaluators recorded the odor descriptions and compared them with the aroma descriptions of the standard compounds. Finally, the qualitative analysis was verified with standard compounds.

[0066] (4) Quantitative analysis of volatile compounds

[0067] The quantitative analysis of volatile compounds is carried out by constructing a standard curve. Each standard solution is dissolved in methanol to prepare a mixed standard solution. The mixed standard solution is serially diluted by a factor of 2 n to continuously dilute into 9 solutions with different concentrations, and 4-methyl-2-pentanol (10 μL, 324.4 μg / L) is added as an internal standard. Then, the standard compounds are extracted under the same conditions as the samples. By plotting the ratio of the peak areas of the standard compounds and the internal standard and their concentration ratios, all analyses are performed in triplicate. For compounds without a standard curve, the standard curve of compounds with similar substance types and similar carbon atom numbers is used for quantification. For all the characteristic aroma substances determined in this method, quantification is carried out through the standard curves of the corresponding compounds.

[0068] (5) GC-O analysis

[0069] In the comprehensive two-dimensional gas chromatography system, it is split into two parts after the one-dimensional DB-WAX gas chromatography column to connect the olfactometer and the two-dimensional chromatography column. For olfactory detection, the aroma intensity method is used. Each detection member needs to record the odor description, aroma intensity, and start and end times when the effluent flows out of the olfactory detection port. The aroma intensity judgment is quantitatively measured using a five-point scale, ranging from 0 (none) to 4 (very strong). The final aroma intensity index is represented by the average value.

[0070] (6) Odor activity value analysis

[0071] Characteristic aroma substances do not solely depend on their concentrations. The odor threshold of the compound should also be considered. Therefore, odor activity values are used to determine its contribution to the overall odor of the sample. The OAV is calculated using the following formula: OAV = (Ci / OTi), where Ci is the concentration of compound i in apple fruits, and OTi is the odor threshold of the compound in water.

[0072] (7) Aroma recombination and omission

[0073] An apple juice simulation solution is prepared using distilled water containing 70 g / L fructose, 70 g / L glucose, 5 g / L malic acid, and 50 mg / L ascorbic acid. The standard products of the main aroma-active compounds identified by GC×GC-QTOFMS, GC-O, and OAV in the apple fruits of the three varieties of Ruoyang, Ruixue, and Ruixianghong are dissolved in methanol and dissolved in the simulation solution according to the corresponding measured concentrations. Sensory evaluation is carried out on the recombinant samples to compare their differences with the original samples.

[0074] Based on the aroma recombination model, different omission models are prepared by omitting one characteristic aroma substance from the complete recombination model for aroma omission experiments. The three-point test method is used to compare the differences between each omission model and the corresponding original recombination model. A total of 18 panel members participated in the omission test. If 10 panel members answer correctly, the result is marked as significant (p≤0.05); if 12 panel members answer correctly, the result is marked as highly significant (P≤0.01); if ≥13 panel members answer correctly, the result is marked as very significant (p≤0.001).

[0075] In this example, the volatile compound components and contents in apples of 3 varieties were obtained under the above analysis conditions. The 3D chromatograms are as Figure 1 , Figure 2 and Figure 3As shown, a total of 138 volatile compounds were detected. Then, 23 characteristic aroma substances were identified by GC-O method, and their contents in different apple varieties are shown in Table 1. The contents of all characteristic aroma substances were quantified by the standard curves of the aroma substances shown in Table 2. As shown in Table 3 are the odor intensity values and OAV values of the 23 identified characteristic aroma substances, and the characteristic aroma substances in different apple varieties were determined by these. As Figure 4 Shown is the sensory evaluation radar chart of the aroma recombination experiment, indicating that the identified characteristic aroma substances can successfully simulate the aroma of apple fruits. Finally, the key characteristic aroma substances were identified through the aroma omission experiment. As shown in Table 4, a total of 11 key characteristic aroma substances were identified, namely ethyl butyrate, isopropyl butyrate, 2-methylbutyl acetate, hexyl acetate, linalool, n-hexanal, trans-2-hexenal, (E,E)-2,4-nonadienal, 1-octen-3-one, damascenone, and geranyl acetone.

[0076] Table 1 Contents of Characteristic Aroma Substances in Apples

[0077]

[0078]

[0079]

[0080] Table 2 Quantitative Curves of Key Characteristic Aroma Compounds in Apples

[0081]

[0082]

[0083] Table 3 GC-O and OAV Values of Characteristic Aroma Substances in Apples

[0084]

[0085]

[0086]

[0087] Table 4 Results of Aroma Omission Experiment

[0088]

[0089]

[0090]

[0091] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention should cover within the protection scope of the present invention by making equivalent substitutions or changes according to the technical solution and inventive concept of the present invention.

Claims

1. A sensory omics-based method for identifying key aroma substances in apple fruit, characterized in that: The following steps are involved: S1: Detection of volatile compounds in apple fruit by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-QTOFMS); S2: Gas chromatography-olfactometer (GC-O) and odor activity value (OAV) analysis were performed to characterize the characteristic aroma compounds in apple fruit; S3: Identify key characteristic aroma compounds in apple fruit through aroma recombination and omission experiments.

2. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 1, characterized in that: The specific method of detecting volatile compounds in apple fruit by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-QTOFMS) in S1 comprises the following steps: S1.

1. Sample preparation: After core removal, apple fruit was cut into small pieces and ground under liquid nitrogen. Polyvinylpyrrolidone (PVPP) and D-gluconolactone were added in a mass ratio of 100:1:

1. The mixture was extracted at 4°C in the dark for 24 h, then centrifuged at 4°C, 8000×g for 15 min, and the supernatant was collected and stored at -40°C for testing. S1.2, extraction of volatile compounds: the sample extract, sodium chloride and internal standard solution were placed in a headspace bottle to extract volatile compounds by headspace solid phase microextraction (HS-SPME); S1.

3. Analysis of volatile compounds: Comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-QTOFMS) was used to analyze the volatile compounds in apples; S1.4, qualitative analysis of volatile compounds: screen and verify the data based on retention index, matching degree, aroma description and standard information, and qualitatively analyze the volatile compounds; S1.

5. Quantification of volatile compounds: Quantification of volatile compounds is performed by constructing a standard curve of the compounds.

3. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 2, characterized in that: In S1.2, the sample extract is 5 mL, sodium chloride is 1 g, and the internal standard is 10 μL of 4-methyl-2-pentanol (324.4 μg / L); The headspace bottle containing the sample was equilibrated in a multifunctional autosampler at 250 r / min and 45 °C for 10 min, and then an aged solid phase microextraction needle (50 / 30 μm, DVB / CAR / PDMS) was inserted into the headspace bottle for extraction for 30 min. Subsequently, the solid phase microextraction needle was inserted into the gas chromatography inlet and desorbed for 3 min in the non-split injection mode.

4. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 2, characterized in that: The instrument conditions for the GC×GC-QTOFMS analysis in S1.3 are as follows: GC×GC system conditions: one-dimensional column DB-WAX (30m×0.25mm×0.25μm), two-dimensional column DB-17MS (1.85m×0.180mm×0.18μm); injection port temperature was 250℃, splitless injection mode; column oven temperature program was: initial 50℃, maintained for 1min, then increased to 230℃ at 3℃ / min and maintained for 2min; helium (99.999%) was used as carrier gas, with a constant flow rate of 1mL / min; transfer line temperature was 260℃; the two columns were connected by a SV series modulation column (1.3m×0.25mm, C6-C40), a solid-state thermal regulator was used for heating and cooling, and the modulation cycle was 4s; QTOFMS system conditions: The acquisition process adopted full scan mode, the mass scan range was 45-500 m / z, and the acquisition rate was 50 Hz; the temperatures of the ion source and quadrupole were set to 230 °C and 150 °C, respectively, the EI ionization energy was 70 eV, and the solvent delay time was 2.0 min.

5. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 2, characterized in that: In the S1.4, the Canvas software workstation is used to process the mass spectrometry information, and the minimum peak detection signal-to-noise ratio is set to 10; the mass spectrometry data is compared with the NIST20 standard library; the actual retention index (RI) value of the normal alkane standard solution is calculated; the odor and mass spectrum of the standard compound are compared to accurately identify the volatile compounds.

6. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 2, characterized in that: The S1.5 described above dissolves each standard solution in methanol to prepare a mixed standard solution, dilutes the mixed standard solution in a 2n gradient, and continuously dilutes it into 9 solutions of different concentrations, and adds 4-methyl-2-pentanol (10 μL, 324.4 μg / L) as an internal standard. Then, the standard compound is extracted under the same conditions as the sample, and the corresponding standard curve is plotted by the peak area ratio of the standard compound and the internal standard and the concentration ratio. All analyses are performed in triplicate. For compounds without a standard curve, a standard curve of compounds with similar substance types and similar carbon atom numbers is used for quantification.

7. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 1, characterized in that: The GC-O in S2 is measured by the aroma intensity method, which is composed of 6 experts with GC-O experience. When the material flows out of the olfactory detection port, the odor attribute, aroma intensity, and the start and end time are recorded. The aroma intensity is expressed from 0 (none) to 4 (very strong), and the final aroma intensity value is expressed as the average value.

8. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 1, characterized in that: The odor activity value (OAV) in S2 was calculated using the following formula: OAV=(Ci / OTi), where Ci is the concentration of compound i in apple fruit and OTi is the odor threshold of the compound in water.

9. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 1, characterized in that: The aroma reconstitution experiment in S3 is to dissolve the characteristic aroma compound standards detected by GC-O and OAV in apples in methanol, dissolve them in simulated solutions according to the corresponding measured concentrations, and perform sensory evaluation on the reconstituted samples to compare the differences between them and the original samples.

10. The method for identifying key aroma substances in apple fruit based on sensory omics according to claim 1, characterized in that: The aroma omission experiment in S3 is based on aroma recombination. By omitting an aroma-active compound from the complete recombination model, different omission models are prepared, and aroma omission experiments are performed. The differences between each omission model and the corresponding original recombination model are compared using a three-point test method.

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