Extraction and detection method of Chinese chive S-alkyl cysteine sulfoxide

Abstract

The invention relates to the technical field of extraction and detection of Chinese chive S-alkyl cysteine sulfoxide, in particular to an extraction and detection method of Chinese chive S-alkyl cysteine sulfoxide, which comprises the following steps: optimizing relevant parameters in the extraction process, and finally determining that the whole extraction process is as follows: taking a fresh Chinese chive sample, adding a 0.1% phosphoric acid aqueous solution as an extracting agent, and uniformly stirring; the mass volume ratio (g: mL) of the leek sample to the extraction solution is 1: 10; putting the Chinese chive into a centrifugal tube, performing high-temperature digestion for 20 minutes, grinding a Chinese chive sample, and performing ultrasonic-assisted extraction for 30 minutes at the ultrasonic temperature of 50 DEG C; and filtering to remove filter residues, sucking 2mL of clear liquid, adding 2mL of dichloromethane, fully shaking up, centrifuging in a centrifugal machine for 10 minutes at the centrifugal rotating speed of 10000 rpm, sucking the clear liquid after centrifuging, passing through a 0.22 mu m filter membrane, filling into a small chromatographic sample injection bottle, and measuring. A Waters high performance liquid chromatograph is used, and a 2998 detector is equipped for performing HPLC detection on the Chinese chive S-alkyl cysteine sulfoxide. The method is high in precision and good in stability, and three S-alkyl cysteine sulfoxide components can be measured at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of extraction and detection of leek S-alkyl cysteine ​​sulfoxide, and specifically to an extraction and detection method of leek S-alkyl cysteine ​​sulfoxide. Background Art

[0002] Leek (Allium tuberosum Rottler ex Sprengle) originated in my country. It is a typical vegetable with both medicinal and edible properties. It is rich in various nutrients and active substances. At the same time, its planting technology is relatively simple and convenient, and the planting cost is low. It is planted all over my country. There are many varieties of leeks in my country, and the germplasm resources are rich. However, the collection and protection of leek germplasm resources are still far behind those of other bulk vegetables. At present, there are few reports on flavor quality in the excavation and evaluation of leek germplasm resources. Among them, leek flavor precursors and volatile odors are important components of leek flavor quality.

[0003] S-alkylcysteine ​​sulfoxide, the main flavor precursor in leek, is a non-protein sulfur-containing compound with certain active functions. The main component of the special volatile smell of leek is generated by it through a series of reactions catalyzed by alliinase. At present, there are few reports on the determination of S-alkylcysteine ​​sulfoxide in leek by high performance liquid chromatography, especially the extraction and detection methods of each component are relatively insufficient. Therefore, it is urgent to optimize the extraction and detection methods of leek flavor precursors. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting and detecting S-alkylcysteine ​​sulfoxide from leek in view of the defects in the above-mentioned prior art, which is simple to operate, safe, low in cost, and high in effective ingredient content and yield.

[0005] In order to achieve the above purpose, the technical solutions provided are as follows:

[0006] The extraction and detection method of leek S-alkylcysteine ​​sulfoxide is characterized by comprising the following steps:

[0007] Step 1: Select leek samples;

[0008] Step 2: Inactivation of allicinase: inactivation of allicinase in leek samples;

[0009] Step 3, adding the extract: adding the extract to the leek sample after the alliinase is inactivated;

[0010] Step 4, grinding and ultrasonic treatment: grinding the leek sample added with the extract and then performing ultrasonic-assisted extraction to obtain an S-alkylcysteine ​​sulfoxide extract;

[0011] Step 5, filtration and centrifugation: Filter the S-alkylcysteine ​​sulfoxide extract with filter paper, absorb 2 mL of S-alkylcysteine ​​sulfoxide clear solution and centrifuge;

[0012] Step 6: Purification: After centrifugation, the S-alkylcysteine ​​sulfoxide clear solution is taken out and filtered through a membrane;

[0013] Step 7, on-machine detection: put the purified S-alkylcysteine ​​sulfoxide clear solution into a chromatographic injection vial for detection.

[0014] Preferably, in step 2, the alliinase is inactivated by high-temperature boiling, and the high-temperature boiling time is 20 minutes.

[0015] Preferably, in step three, the extract is a 0.1% phosphoric acid aqueous solution, and the solid-liquid ratio (g:mL) is maintained at 1:10.

[0016] Preferably, in step 4, the ultrasonic extraction time is 30 min and the ultrasonic temperature is 50°C.

[0017] Preferably, in step five, the centrifugation is performed at room temperature for 10 minutes, with a centrifuge speed of 10,000 rpm.

[0018] Preferably, in step six, the pore size of the filter membrane is 0.22 μm.

[0019] Preferably, in step seven, the detection includes the following parameters: using Waters e2695 high performance liquid chromatograph, XSelect HSS T3 chromatographic column 150mm×4.6mm, 5μm, Waters, with 0.1% phosphoric acid aqueous solution and methanol as mobile phase, isocratic elution (v:v) 95:5, flow rate 0.5mL / min, column temperature: 30°C, injection volume 15μL, detection wavelength 200nm.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present invention optimizes the extraction conditions and chromatographic detection conditions of the three components of leek S-alkylcysteine ​​sulfoxide, establishes the best determination method suitable for leek, and provides method support for the study of leek flavor quality. Then, the optimized method is used to determine the components and contents of S-alkylcysteine ​​sulfoxide in different leek germplasm materials, and the flavor quality of leek germplasm resources is evaluated in combination with electronic nose technology, and the volatile flavor characteristics of leek germplasm resources are clarified, providing a reference for high-quality leek germplasm innovation and variety breeding. The method has little loss of S-alkylcysteine ​​sulfoxide during the extraction process, and can simultaneously determine the three types of S-alkylcysteine ​​sulfoxides in leek. The relative standard deviation of the method precision and stability test is less than 5%, and the accuracy is high.

[0022] The coefficient of variation of flavor substances in 2.50 different varieties of leek was large, and the germplasm resources were rich. Among the three types of S-alkylcysteine ​​sulfoxides in leek, S-methylcysteine ​​sulfoxide had the highest average content, followed by S-allylcysteine ​​sulfoxide, and S-propenylcysteine ​​sulfoxide had the lowest content. The differences in volatile odor characteristics were mainly reflected in gas types such as aromatic benzene, nitrogen oxides and organic sulfides. The average content of S-alkylcysteine ​​sulfoxide in different parts of leek was in the order of flower, seed, root, pseudostem, stalk and leaf, and the size of volatile odor characteristics was basically consistent with the distribution of flavor precursor substances.

[0023] 3. Cluster analysis was used to classify 50 leek germplasm resources and combined with the entropy weight TOPSIS method to screen out five leek varieties with obvious volatile odor characteristics and relatively high S-alkylcysteine ​​sulfoxide content, namely, Budaoshi Leek, Baihua Wild Leek, Hanyu Purple Root, Jiuxing No. 20, and Baofu Leek Red. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the CSOs content measured under different enzyme inactivation methods in the embodiments of the present invention;

[0025] Figure 2 This is a graph showing the CSOs content of samples measured at different enzyme inactivation and boiling times in the examples of the present invention;

[0026] Figure 3 This is a graph showing the CSOs content measured under different auxiliary extraction methods in the embodiments of the present invention;

[0027] Figure 4 The graphs are the CSOs contents measured at different ultrasonic times (A) and ultrasonic temperatures (B) in the embodiments of the present invention;

[0028] Figure 5 This is a graph showing the CSOs content measured under different extraction solvents in the embodiments of the present invention;

[0029] Figure 6 This is a graph showing the CSOs content measured at different material-liquid ratios in the embodiment of the present invention;

[0030] Figure 7 The graph is a graph of CSOs content measured at different centrifuge speeds (A) and centrifugation times (B) in the embodiment of the present invention;

[0031] Figure 8 The peak separation of standard products under different proportions of mobile phase in the embodiment of the present invention; wherein: 1: S-methylcysteine ​​sulfoxide, 2: S-allylcysteine ​​sulfoxide, 3: S-propenylcysteine ​​sulfoxide;

[0032] Fig. 9 This is a Loadings analysis diagram of different varieties of leeks in an embodiment of the present invention;

[0033] Fig.10 The whole plant pictures of 5 leek germplasm materials in the embodiment of the present invention;

[0034] Fig.11 The CSO content in different parts of leek in the embodiment of the present invention;

[0035] Fig.12 The CSO distribution of different parts of leek in the embodiment of the present invention;

[0036] Fig.13 This is a cluster analysis of 50 leek germplasm resources in the embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Embodiment 1

[0039] 1. Experimental Materials and Reagents

[0040] 1.1 Test materials

[0041] The experimental leek variety is "Jixing No. 18", which is planted in the modern glass greenhouse of Gansu Agricultural University.

[0042] 1.2 Test reagents

[0043] Main chemicals: chromatographic methanol, acetonitrile (Sigma); chromatographic phosphoric acid, dichloromethane (Tianjin Komiou Chemical Reagent Co., Ltd.); potassium dihydrogen phosphate (Tianjin Beichen Fangzheng Reagent Factory).

[0044] Reference standards: S-methylcysteine ​​sulfoxide (MCSO), S-allylcysteine ​​sulfoxide (ACSO), and S-propenylcysteine ​​sulfoxide (PCSO) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0045] Main instruments: high performance liquid chromatograph (Waters e2695, equipped with 2998PDA detector); ultrasonic cleaner (SB-800DT; Ningbo Xinzhi Biotechnology Co., Ltd.); chromatographic column XSelect HSS T3 (4.6×150mm, 3.5μm), equipped with XSelect HSS T3 VanGuard filter protection column (3.9nm×5mm, 5μm), ultrapure water preparation instrument (WP-UP-YZ-80; Sichuan Water Treatment Equipment Co., Ltd.), vacuum freeze dryer (Shanghai Huaiyu Instrument Equipment Co., Ltd.), balance (FA2004N; Shanghai Precision Scientific Instrument Co., Ltd.); high-speed refrigerated centrifuge (3-18KS, Sigma).

[0046] 1.3 Preparation of standard solution

[0047] Preparation of standard solutions: Use ultrapure water to prepare S-methylcysteine ​​sulfoxide, S-allylcysteine ​​sulfoxide, and S-propylenecysteine ​​sulfoxide into 2000μg / mL, 2000μg / mL, and 500μg / mL stock solutions, respectively, and store them in a -20℃ refrigerator until use.

[0048] 2. Sample Pretreatment

[0049] 2.1 Leek sample processing

[0050] After cell rupture, alliinase (CS lyase) present in the vacuole contacts with S-alkylcysteine ​​sulfoxide in the cytoplasm and rapidly catalyzes the reaction of S-alkylcysteine ​​sulfoxide, thereby producing a series of pungent odors. It is very important to inactivate the enzyme during the extraction process.

[0051] 2.2 Selection of leek enzyme inactivation method

[0052] Freeze-dried sample: Take the whole leek leaves and freeze them in liquid nitrogen, then use a vacuum freeze dryer to vacuum dry for 48 hours, then take them out and seal them in a -80℃ refrigerator for later use. Weigh 0.2g of leek freeze-dried sample, add 20mL of ultrapure water, shake well and let stand for 30min, filter with filter paper to absorb 2mL of clear liquid, add 2mL of dichloromethane, centrifuge at 8000rpm, 25℃ for 10min, absorb the supernatant and pass it through a 0.22μm filter membrane, and then measure it on the machine.

[0053] Drying sample: Take the whole leek leaves and put them in an oven at 100℃ for 15min, then dry them at 80℃ to constant weight. Weigh 0.2g of dried leek sample, add 20mL of ultrapure water, shake well and let stand for 30min, filter with filter paper to absorb 2mL of supernatant, add 2mL of dichloromethane, centrifuge at 8000rpm, 25℃ for 10min, absorb the supernatant and pass it through a 0.22μm filter membrane, and then measure it on the machine.

[0054] Microwave inactivation of enzyme: take whole leek leaves and place them in a microwave oven at 700w for 1min to inactivate enzyme. Weigh 0.2g sample, add 20mL ultrapure water, shake well and let stand for 30min. Filter 2mL of supernatant with filter paper and add 2mL of dichloromethane. Centrifuge at 8000rpm and 25℃ for 10min. Aspirate the supernatant and pass it through a 0.22μm filter membrane before measuring on the machine.

[0055] High-temperature boiling to inactivate enzymes: Take whole leek leaves, cut them into 5 cm uniform small segments, weigh 2 g and put them into a 20 mL centrifuge tube, add ultrapure water, and boil them at high temperature for 20 min. Grind the boiled leek sample and put it into a 15 mL centrifuge tube together with the boiling liquid. Shake well and let it stand for 30 min. Filter with filter paper to absorb 2 mL of clear liquid, then add 2 mL of dichloromethane, centrifuge at 8000 rpm, 25 ° C for 10 min, absorb the supernatant and pass it through a 0.22 μm filter membrane, and then measure it on the machine.

[0056] Calculation of S-alkylcysteine ​​sulfoxide content under different enzyme inactivation methods:

[0057]

[0058] Where: 100 is 100 μg / mL mixed standard;

[0059] V is the product peak area of ​​each S-alkylcysteine ​​sulfoxide in the 100 μg / mL mixed standard;

[0060] Y is the peak area of ​​S-alkylcysteine ​​sulfoxide in samples measured under different enzyme inactivation methods;

[0061] M is the sample weight;

[0062] U is the water loss rate of leek under different enzyme inactivation methods.

[0063] After determining the appropriate enzyme inactivation method, different horizontal gradients are set for the parameters related to the enzyme inactivation method for optimization; different freeze-drying times are set in the freeze-dried samples, and different killing times and drying times are set in the dried samples; different enzyme inactivation times and powers are set for microwave enzyme inactivation; and different boiling times are set for high-temperature boiling enzyme inactivation.

[0064] like Figure 1 As shown in the figure, among the four enzyme inactivation methods, high temperature boiling is the best, and the MCSO and ACSO contents are higher than those in samples inactivated by other methods. The freeze-dried samples and microwave enzyme inactivation samples are the second, and the drying samples have the worst effect. The S-alkyl cysteine ​​sulfoxide content in the drying samples is the lowest, among which S-propylene cysteine ​​sulfoxide (PCSO) does not reach the detection limit, which may be related to the long-term high temperature environment during the drying process of leeks, resulting in the greatest loss.

[0065] like Figure 2As shown in the figure, by setting different soaking times of 5min, 10min, 15min, 20min, 25min, 30min, 40min, and 50min, when the high-temperature soaking is used to kill the enzyme, the soaking time of 20min has the best effect; when the time is short, the temperature of the extractant has not risen, and the garlic enzyme has some activity to catalyze the reaction of part of the S-alkyl cysteine ​​sulfoxide; as the soaking time increases, the activity of the garlic enzyme is lost, and the S-alkyl cysteine ​​sulfoxide will not be catalytically reacted. At the same time, when the high-temperature soaking is used to kill the enzyme, the S-alkyl cysteine ​​sulfoxide will also dissolve in the extract to avoid loss; but the volatilization speed of the extract is accelerated, taking away part of the S-alkyl cysteine ​​sulfoxide. Therefore, there is a trend from low to high and then to low, so the soaking time for killing the enzyme is selected as 20min.

[0066] 2.3 Selection of auxiliary extraction method

[0067] The enzyme-inactivated leek samples were used to extract S-alkylcysteine ​​sulfoxide using different auxiliary extraction methods. Ultrasonic assisted extraction was set to ultrasonic at room temperature for 30 minutes; oscillation extraction was set to oscillate at 120r / min for 30 minutes; and static extraction was set to stand for 30 minutes.

[0068] After determining the auxiliary extraction method, set different parameters to determine the optimal level. The preset parameter levels are as follows: set time ultrasound (10, 15, 20, 30, 40, 50min) and ultrasound temperature (25, 30, 40, 50, 60, 70℃) in ultrasound-assisted extraction; set oscillation time (10, 15, 20, 30, 40, 50min) and speed (50, 80, 100, 120, 150r / min) in oscillation-assisted extraction; set different times (10, 15, 20, 30, 40, 50min) during static extraction.

[0069] like Figure 3 As shown in the figure, among the three different extraction methods, ultrasound-assisted extraction had the best effect, followed by oscillation extraction, and static extraction had the worst effect. At the same time, during the extraction of the three flavor precursors, the measured contents of S-allylcysteine ​​sulfoxide and S-propylenecysteine ​​sulfoxide in the three extraction methods were slightly different. The reason may be that the contents of these two substances in leeks are relatively low, and the existing extraction conditions can already extract them completely, while the content of S-methylcysteine ​​sulfoxide is relatively high, and some of it is still attached to the leeks. Overall, ultrasound-assisted extraction has the best effect, and the parameters involved in ultrasonic extraction, such as ultrasonic time and ultrasonic temperature, need to be further screened.

[0070] like Figure 4As shown in the figure, in the extraction method, as the ultrasonic time and ultrasonic temperature increase, the more S-alkylcysteine ​​sulfoxide content measured by the high performance liquid chromatography, the more complete the extraction. After the leek sample is soaked, boiled and ground, the use of ultrasound-assisted extraction helps to dissolve the S-alkylcysteine ​​sulfoxide attached to the leek grinding residue in water, and by increasing the temperature, the molecular movement rate is accelerated, thereby accelerating the dissolution and extracting more completely. After the ultrasonic time exceeds 30 minutes, the S-alkylcysteine ​​sulfoxide content is basically unchanged ( Figure 4 A); When the ultrasonic extraction temperature is different, the content measured at different temperatures is generally very small, but through observation, it can be found that the content measured at 50℃ is relatively the highest, and there is a trend of decreasing after exceeding 50℃ ( Figure 4 B). In order to make the extraction more complete, the ultrasonic time of 30 min and the ultrasonic temperature of 50°C were selected as the extraction conditions.

[0071] 2.4 Selection of extraction agent

[0072] According to literature reports and the properties of S-alkylcysteine, the extraction solvents were ultrapure water, mobile phase, 80% ethanol, 60% ethanol, 40% ethanol, 20% ethanol, and 40% methanol.

[0073] like Figure 5 As shown, when different extraction solvents are used, taking S-methylcysteine ​​sulfoxide as an example, as the proportion of organic solvent increases, the measured content decreases; and by comparison, it is found that the extraction effect of ethanol in the same proportion is better than that of methanol in the same proportion; but 0.1% phosphoric acid aqueous solution and pure water are the best extractants, and different proportions of alcohol solutions are second. This is mainly related to the water-soluble properties of S-alkylcysteine ​​sulfoxide. There is no obvious difference in the effect when 0.1% phosphoric acid aqueous solution and pure water are used as extractants. Considering that the mobile phase in the chromatographic conditions is 0.1% phosphoric acid aqueous solution, the solvent effect can be avoided to a great extent when the extractant is the same as the mobile phase. At the same time, the pH value of 0.1% phosphoric acid aqueous solution is 2.9. Under acidic environmental conditions, most of the S-alkylcysteine ​​sulfoxide substances exist in one form, and the detection is more complete. Therefore, 0.1% phosphoric acid aqueous solution is selected as the extraction solvent for extracting S-alkylcysteine ​​sulfoxide in leek.

[0074] 2.5 Selection of material-liquid ratio

[0075] After screening out the optimal extraction solvent, different sample amount: extraction solvent mass volume ratios (g:mL) were set to 1:6, 1:8, 1:10, 1:12, 1:15, and 1:20, and the optimal solid-liquid ratio was determined according to the S-alkylcysteine ​​content.

[0076] like Figure 6As shown in the figure, with the change of the material-liquid ratio, the measured S-alkylcysteine ​​content is the highest when the material-liquid ratio is 1:10, and then decreases; it begins to increase after the material-liquid ratio is 1:12; but as the extraction solvent increases, the concentration of the sample solution becomes smaller, and if it is lower than the detection limit, the instrument will not be able to detect it, so the material-liquid ratio is selected as 1:10.

[0077] 2.6 Centrifugal speed

[0078] Different centrifuge speeds were set at 4000, 6000, 8000, 10000, and 12000 rpm, and the optimal conditions were determined based on the measured S-alkylcysteine ​​sulfoxide content and the instrument loss in actual conditions.

[0079] 2.7 Centrifugation time

[0080] Different centrifugation times were set at 5, 8, 10, 15, 20, and 30 min, and the optimal conditions were determined based on the three S-alkylcysteine ​​sulfoxide contents obtained and the instrument operation conditions in actual situations.

[0081] like Figure 7 As shown, the centrifuge speed and centrifuge time have no significant effect on the S-alkylcysteine ​​sulfoxide content measured in the extract, but through careful analysis, it can be found that with the continuous increase of centrifugation time and centrifuge speed, the measured total content has a decreasing trend; according to the high performance liquid chromatography's high requirements for sample injection, once impurities exist, it will cause pipeline blockage, and seriously contaminate the detector; at the same time, combined with the centrifuge operation loss, the centrifuge time is selected as 10min and the centrifuge speed is 10000rpm to precipitate impurities in the extract.

[0082] 3. Selection of chromatographic conditions

[0083] 3.1 Mobile phase screening and elution ratio selection

[0084] The mobile phase was selected according to literature reports and the properties of S-alkylcysteine ​​sulfoxide; organic phase A: methanol or acetonitrile, aqueous phase B: phosphoric acid aqueous solution, ultrapure water, potassium dihydrogen phosphate aqueous solution.

[0085] In the elution ratio selection, 50 μg / mL mixed standard solution was injected. The chromatographic conditions for injection and measurement are shown in Table 1-1. The best elution mobile phase ratio was selected based on the peak elution of the standard and the separation between the components.

[0086] Table 1-1 Combinations of different mobile phase ratios

[0087]

[0088] After determining the mobile phase ratio, keep the organic phase A unchanged, replace the aqueous phase B to determine the appropriate concentration (Table 1-2), and screen the best elution mobile phase ratio based on the peak elution of the standard and the separation between the components.

[0089] Table 1-2 Different water phase combinations

[0090]

[0091] After determining the proportion of mobile phase and aqueous phase (B), replace methanol with acetonitrile, keep other chromatographic conditions unchanged, and select acetonitrile or methanol as the organic phase in the mobile phase based on the peak elution of the standard and the separation between the components.

[0092] Separation calculation method:

[0093] R = (T2-T1) / [(W1+W2)×0.5]

[0094] Where T2 and T1 represent the retention time of peak 1 and peak 2, respectively;

[0095] W1 and W2 represent the peak widths of peak 1 and peak 2, respectively.

[0096] like Figure 8 As shown in A, by adjusting the ratio of 0.1% phosphoric acid aqueous solution and methanol, it was found that when the ratio was 80:20, the peaks of S-allylcysteine ​​sulfoxide and S-propylenecysteine ​​sulfoxide were at the same time, and the peaks of the two substances overlapped, making it impossible to conduct qualitative and quantitative analysis; Figure 8 As shown in Figure C, when the ratio is 95:5, the separation degree of S-allylcysteine ​​sulfoxide and S-propylenecysteine ​​sulfoxide reaches 1.5, which meets the determination requirements; it is also found that the separation degree of the three substances is greater in a high ratio or even pure aqueous solution mobile phase system.

[0097] like Figure 8 As shown in D, after the ratio of the organic phase to the aqueous phase in the mobile phase was preliminarily determined, methanol was replaced with acetonitrile while other chromatographic conditions remained unchanged. It was found that the combination of acetonitrile and phosphoric acid water had a weaker separation effect on the three CSOs than the combination of methanol and phosphoric acid aqueous solution. At the same time, the reagents used in liquid chromatography analysis are basically required to be chromatographic grade. The price of acetonitrile in chromatographic reagents is much higher than that of methanol, so methanol was selected as the organic phase in subsequent experiments.

[0098] After determining the ratio of the organic phase to the aqueous phase, different aqueous solutions were replaced. As shown in Table 1-3, there was no significant difference in the peak area of ​​the standard solution determined by combining water, 0.1% phosphoric acid aqueous solution, and 0.5mmol potassium dihydrogen phosphate aqueous solution with methanol. However, the substance to be determined, S-alkylcysteine ​​sulfoxide, is a zwitterionic compound. The pH of 0.1% phosphoric acid aqueous solution is 2.9. In an acidic environment, S-alkylcysteine ​​sulfoxide mostly exists in an ionic form, which is more conducive to instrument determination. In contrast, 0.5mmol potassium dihydrogen phosphate is used as a salt solution as the mobile phase in a high performance liquid chromatograph. If the operation is not performed properly, salting out may occur, causing blockage of the instrument pipeline.

[0099] Table 1-3 Different aqueous solutions

[0100]

[0101] 3.2 Selection of mobile phase flow rate

[0102] The flow rates were set to 0.3 mL / min, 0.5 mL / min, 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively. Other chromatographic conditions remained unchanged, and 50 μg / mL of mixed standard was injected. The flow rate was selected according to the peak area, peak shape, and separation degree of the standard.

[0103] As shown in Tables 1-4, when the mobile phase flow rates were 0.3 and 0.5 mL / min, the peak areas of the three S-alkylcysteine ​​sulfoxides were close, among which the peak areas of ACSO and MCSO were the largest when the flow rate was 0.5 mL / min. Then, with the increase of the mobile phase flow rate, the peak areas of the three S-alkylcysteine ​​sulfoxides decreased. The high flow rate was not conducive to the retention of the measured samples in the chromatographic column. If the content of the measured target substance was high in the sample determination, 0.5 mL / min was selected as the flow rate for subsequent determinations.

[0104] Table 1-4 Peak area at different flow rates

[0105]

[0106] 3.3 Column temperature selection

[0107] Set different column temperatures to 25°C, 30°C, 35°C, and 40°C, set the flow rate to 0.5mL / min, set the injection volume to 15μL, and keep other chromatographic conditions unchanged. Select the flow rate according to the peak area and separation degree of the mixed standard.

[0108] The results showed that the change of chromatographic column temperature had no significant effect on the measured S-alkylcysteine ​​sulfoxide. At the same time, it was found that due to the influence of the instrument's own heat and changes in ambient temperature, the column temperature would exceed 25°C at the beginning and approach 30°C. Therefore, choosing 30°C as the chromatographic column temperature is beneficial to reduce the temperature fluctuation of the chromatographic column and is more conducive to the stability of the test results.

[0109] 3.4 Optimization of injection volume

[0110] The injection volume was set to 5 μL, 10 μL, 15 μL, and 20 μL, respectively, and the flow rate was set to 0.5 mL / min. Other chromatographic conditions remained unchanged. The injection volume was selected according to the peak area and separation degree of the mixed standard.

[0111] As shown in Table 1-5, as the injection volume increases, the peak areas of the three standard products measured by the instrument increase accordingly. However, after 15 μL, the peak area increases and decreases. When the injection volume is 15 μL, the peak shape of the standard product is the best. At the same time, the problem of whether the chromatographic column capacity is overloaded should also be considered. Therefore, the optimal injection volume is selected as 15 μL.

[0112] Table 1-5 Peak area at different injection volumes

[0113]

[0114] 3.5 Selection of the optimal detection wavelength

[0115] The 3D channel full wavelength scan at 190-400 nm was performed on each standard solution using a high performance liquid chromatograph PDA detector, and the appropriate detection wavelength was selected within ±20 nm near the maximum wavelength according to the peak of the sample.

[0116] When exploring the optimal detection wavelength of S-alkylcysteine ​​sulfoxide, the chromatographic conditions are: chromatographic column C18 (150nm×4.6mm, 5μm, waters), mobile phase is methanol and 0.1% phosphoric acid aqueous solution, isocratic elution ratio is 5:95, flow rate is 0.5mL / min, injection volume is 15μL, column temperature is 30℃, detection wavelength is 214nm, and 3D channel is opened for full wavelength scanning. As shown in Table 1-6, after the determination, the wavelength with the highest peak is selected as the maximum wavelength in the 3D channel. It is found that the wavelengths of the three standards are relatively low. If the maximum wavelength is directly selected as the detection wavelength, the peak of impurities will also appear when the leek sample is determined, so the peak at 200nm is selected for quantitative use.

[0117] Table 1-6 Maximum absorption wavelength of each standard substance

[0118]

[0119] Example 2 Methodology Verification

[0120] 1. Precision

[0121] Intra-day precision: within 24 hours, take a standard sample of a certain concentration (200 μg / mL) and measure it using the optimized determination method. Inject the sample five times in succession and calculate the intra-day relative standard deviation (RSD) based on the peak areas of the three standards.

[0122] Inter-day precision: The above method was used for determination on 3 consecutive days. The 200 μg / mL S-alkylcysteine ​​sulfoxide mixed standard was determined using the optimized method, and the inter-day relative standard deviation (RSD) was calculated based on the product peak area of ​​the three standards.

[0123] As shown in Table 2-1, the mixed standard was measured within one day and for three consecutive days, and the relative standard deviation was calculated based on the peak area of ​​each standard. The results showed that both the intra-day precision and the inter-day precision were less than 5%, indicating that the precision was good and met the measurement requirements.

[0124] Table 2-1 Intra-day and inter-day precision (RSD%) of mixed standard solution

[0125]

[0126] 2. Repeatability test

[0127] The 100 μg / mL mixed standard solution was subjected to HPLC determination using the determined chromatographic conditions, and the injection was repeated 6 times. The relative standard deviation (RSD) was calculated based on the peak area of ​​each substance.

[0128] The mixed standard (100 μg / mL) was tested by six consecutive injections, as shown in Table 2-2: the relative standard deviations of the peak areas of the three leek flavor precursors were all less than 5%, among which the maximum RSD of S-methylcysteine ​​sulfoxide was 4.636%, the RSD of S-allylcysteine ​​sulfoxide was 1.393%, and the RSD of S-propylenecysteine ​​sulfoxide was 1.505%, indicating that the determination method has good repeatability.

[0129] Table 2-2 Peak area of ​​continuous injection of mixed standard solution

[0130]

[0131]

[0132] 3. Stability test

[0133] Take a standard sample of a certain concentration, place it at room temperature for 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h, perform HPLC determination, and calculate the relative standard deviation (RSD) based on the peak area of ​​each substance.

[0134] It can be seen from Table 2-3 that the maximum relative standard deviation of the peak area of ​​S-methylcysteine ​​sulfoxide within 48 hours is 5.974%, and the relative standard deviations of the peak areas of the three substances are all less than 10%, indicating that the sample is stable in nature within 48 hours, and also indicating that the determination method has good stability.

[0135] Table 2-348h S-alkylcysteine ​​sulfoxide peak area

[0136]

[0137] 4. Recovery rate

[0138] A known amount of mixed standard was added to the leek sample, extracted according to the optimized sample pretreatment method, and HPLC determination was performed under the determined chromatographic conditions to calculate the spike recovery rate of each substance.

[0139] Spiked recovery rate (%) = [measured value (mg) - content in leek sample (mg)] / added value (mg) × 100%.

[0140] Before extracting the content of S-alkylcysteine ​​sulfoxide in leek samples, a known amount of standard was added, and the sample was extracted according to the optimized sample extraction method. After the determination was completed, the amount of standard added was calculated to obtain the recovery rate. The recovery rates of S-methylcysteine ​​sulfoxide, S-allylcysteine ​​sulfoxide, and S-propylenecysteine ​​sulfoxide in the three CSOs were 93.18%, 97.63%, and 95.57%, respectively, all greater than 90%, indicating that the loss of CSOs caused by the extraction method during the extraction process was small, and the reliability of the entire extraction process was high.

[0141] 5. Linear Relationship

[0142] Prepare the standard solution with different concentrations and measure it according to the optimized chromatographic conditions. Inject each concentration 3 times and take the average value. Draw a standard curve with the concentration of the standard as the horizontal axis and the peak area as the vertical axis to obtain the linear equation. 2 >0.99 indicates a good linear relationship between peak area and concentration.

[0143] Regression analysis was performed with the concentration of each standard as the horizontal axis and the peak area as the vertical axis to obtain the linear equation and correlation coefficient of each standard. As shown in Table 2-4, there is a good linear relationship between the concentration and peak area of ​​each standard. When the sample is measured later, the peak area of ​​the sample is substituted into the equation to calculate the concentration of each S-alkylcysteine ​​sulfoxide in the sample, thereby calculating the content.

[0144] Table 2-4 Regression equation and correlation coefficient of standard curve

[0145]

[0146] 6. Limit of detection and limit of quantification

[0147] It is determined according to the signal-to-noise ratio (SNR); SNR = S / N, S is the peak height when measuring a certain concentration of standard, and N is the baseline peak height; when SNR = 3, the concentration of the standard is the detection limit; when SNR = 10, the concentration of the standard is the quantification limit.

[0148] The detection limits and quantification limits of the three S-alkylcysteine ​​sulfoxides are shown in Table 2-5. By continuously diluting the 50μg / mL concentration of the standard solution, when the standard concentration is about 0.04μg / mL, the ratio of the signal measured by the three S-alkylcysteine ​​sulfoxide standards with known concentrations to the signal measured by the blank sample is about 3, which is the detection limit; when the standard concentration is about 0.1μg / mL, the ratio is about 10, and this concentration is the quantification limit.

[0149] Table 2-5 Detection limits and quantification limits of three S-alkylcysteine ​​sulfoxides

[0150]

[0151] Example 3 Determination of S-alkylcysteine ​​sulfoxide and volatile odor characteristics of different leek germplasm resources

[0152] 1. Materials and Methods

[0153] 1.1 Test materials

[0154] The test materials are 50 different varieties of leeks, as shown in Table 3-1. The 50 leek germplasm materials were planted in the leek germplasm resource garden of Kangyuan Modern Agriculture Demonstration Park in Yuzhong County, with a row spacing of 40 cm and a hole spacing of 15 cm. 4 to 5 plants were planted in each hole, with deep planting and shallow soil covering, and a furrow depth of 20 cm. They were uniformly harvested before the measurement and cut off at 5 cm from the root; after the upper part of the leek grew again, sampling and measurement were carried out.

[0155] Table 3-150 leek germplasm resources information

[0156]

[0157] 2. Experimental Design

[0158] 2.1 Determination of S-alkylcysteine ​​sulfoxide content in 50 leek germplasm resources

[0159] The optimized detection method was used to determine the content of S-alkylcysteine ​​sulfoxide in different leek materials. Overall, as shown in Tables 3-2 and 3-3, the average content of S-methylcysteine ​​sulfoxide in 50 leeks was the highest, followed by S-allylcysteine ​​sulfoxide, and the content of S-propylenecysteine ​​sulfoxide was the lowest; the total content of S-alkylcysteine ​​sulfoxide in 50 leeks was between 6.06 and 11.88 mg / g, with an average content of 8.76 mg / g, of which the average content of S-methylcysteine ​​sulfoxide was 5.22 mg / g, the maximum was 7.94 mg / g, and the minimum was 2.76 mg / g. , the coefficient of variation was 24.49%, and the diversity index was 1.95; the average content of S-allylcysteine ​​sulfoxide was 2.98 mg / g, the maximum value was 5.26 mg / g, the minimum value was 1.15 mg / g, the coefficient of variation was 26.11%, and the diversity index was 1.91; the average content of S-propenylcysteine ​​sulfoxide was 0.56 mg / g, the maximum value was 1.73 mg / g, the minimum value was 0.24 mg / g, the coefficient of variation was 40.96%, and the diversity index was 1.66.

[0160] The contents of the three S-alkylcysteine ​​sulfoxides in different varieties of leeks vary greatly (Table 3-3). Among the 50 leeks, the variety with the highest total content of S-alkylcysteine ​​sulfoxides is the yellow grid leek, and the variety with the lowest content is the Hami hook leek; the leek variety with the highest content of S-methylcysteine ​​sulfoxide is Pengjiu No. 8, and the content of S-allylcysteine ​​sulfoxide in Pengjiu No. 8 is 2.91 mg / g, and the content of S-allylcysteine ​​sulfoxide is 0.60 mg / g, which is not the highest content, but close to the average content; The leek variety with the highest content of S-allylcysteine ​​sulfoxide is Changfeng 791 leek, while the content of S-methylcysteine ​​sulfoxide in Changfeng 791 leek is 5.26 mg / g, and the content of S-propylene cysteine ​​sulfoxide is 0.71 mg / g; the variety with the highest content of S-propylene cysteine ​​sulfoxide is white flower wild leek, while the content of S-methylcysteine ​​sulfoxide in white flower wild leek is 4.02 mg / g, and the content of S-allylcysteine ​​sulfoxide is 1.73 mg / g, both of which are lower than the average content.

[0161] Table 3-2 Descriptive analysis of S-alkylcysteine ​​sulfoxide in different varieties of leeks

[0162]

[0163] Table 3-3 S-alkylcysteine ​​sulfoxide content in different varieties of leeks

[0164]

[0165]

[0166] 2.2 Determination of volatile odor characteristics of 50 leek germplasm resources

[0167] The electronic nose technology (electronic nose: PEN-3; Airsense Analytical Instrument Co., Ltd., Germany) was used to determine the volatile odor characteristics in the leek material. The determination method is as follows: take the leek sample, mix it, add liquid nitrogen and grind it, weigh 1±0.1g of the sample and place it in a 50mL conical flask, add 2mL of deionized water and a magnetic stirring rotor, quickly seal the bottle mouth with a sealing film, place it on a constant temperature magnetic stirrer, rotate at 1000rpm, and balance at room temperature for 30min. After the volatile components of the sample reach equilibrium, insert the electronic nose probe (about 3cm) to absorb the top air for analysis and determination of its volatile odor. Each sample was tested in parallel 4 times.

[0168] The electronic nose measurement parameters were set as follows: sample interval 1s, sensor cleaning time 120s, zeroing time 10s, sample preparation time 5s, measurement time 150s, clean dry air as carrier gas, carrier gas flow rate 400mL / min, injection flow rate 400mL / min. Sensor information is shown in the table below (Table 3-4). The stable signal obtained by the sensor at the 120th second was analyzed.

[0169] Table 3-4 Electronic nose sensor names and performance descriptions

[0170]

[0171] The volatile odor characteristics of leaves of 50 leek germplasm resources were measured by electronic nose, with clean air as carrier gas and blank control. The odor concentration was expressed according to the size of the instrument sensor response value (G / GO). The sensor response values ​​were all around 1 during blank injection. As shown in Tables 3-5, it was found through measurement that the sensors that responded to the volatile odor profile of leek were W1C, W5S, W3C, W6S, W5C, and W2W sensors, and their response values ​​were all greater than 1. The corresponding sensitive odors were aromatic benzene, nitrogen oxides, ammonia, hydride, short-chain alkane, and organic sulfide; the odor differences of different varieties of leek were mainly manifested in the W1C (aromatic benzene), W5S (nitrogen oxides), W3C (ammonia), W2W (organic sulfide), and W5C (short-chain alkane) sensors. According to the electronic nose response values ​​of different leek volatile odors, the Loadings pattern diagram ( Fig. 9 ), it can also be found that sensor W3C contributes the most to the first principal component, followed by sensors W1C, W5C, W2W, and W5S. From Table 3-6, it can be clearly seen that the variation of the response values ​​of these five electronic nose sensors for 50 kinds of leeks, and their corresponding coefficients of variation are 13.2%, 13.61%, 10.77%, 17.31%, and 49.81%, respectively. According to the analysis, it was also found that there was no obvious difference in the sensors W6S, W5C, W1S, W1W, W2S, and W3S when measuring different varieties of leeks.

[0172] The 50 leek germplasm resources have obvious differences in volatile odors, as shown in Tables 3-5 and 3-6. The response value range of sensor W1C is 4.2780-7.9625; the leek with the most obvious response to sensor W1C is the leek with the weakest response is the snow leek king; the sensor W5S response value is the largest overall, ranging from 14.8078 to 213.0273, with an average of 85.4084, among which the leek with the lowest response value is the snow leek king, and the highest The sensor W3C response value range is 2.4560~4.4028, with an average of 3.4103. The leek variety with the largest sensor W3C response value is the leek variety with the largest leek, and the leek variety with the smallest sensor W3C response value is the snow leek king. The sensor W2W response value range for different leek odors is 6.6283~15.0777, with an average response value of 11.0545. The leek variety with the largest response value is the pengbao, and the leek variety with the smallest response value is the snow leek king. The difference in response values ​​shows that different leek varieties have obvious differences in odor. At the same time, the diversity indexes of the response values ​​of the electronic nose sensors for the volatile odors of leeks were 2.0627, 1.9584, 1.9564, 1.5354, 2.0323, 1.6736, 1.8592, 1.4048, 2.0146, and 1.8189, respectively, indicating that leeks have rich diversity in odor and have great potential for improvement.

[0173] Table 3-5 Differences in electronic nose response values ​​of different varieties of leeks

[0174]

[0175] Table 3-6 Response values ​​of electronic nose sensors for different varieties of leeks

[0176]

[0177]

[0179]

[0180] 2.3 Determination of S-alkylcysteine ​​sulfoxide in different parts of leek

[0181] Five types and sources of leek germplasms with the same growth were selected, namely commercial varieties: Budaoshi leek and Jiuxing No. 20; local varieties: Changge Dazhao leek and Chinese leek king; wild variety: white wild leek; after the first cut of leek, no more harvesting, after the leek blooms, the whole leek is dug out, and the roots, pseudostems, leaves, stalks, and flowers are taken. After the seeds are mature, the seeds are collected and the content of S-alkylcysteine ​​sulfoxide in the six parts is determined. The determination method adopts the optimized HPLC method.

[0182] like Fig.10 As shown in Figure 1, S-alkylcysteine ​​sulfoxide was determined in different parts of leek from five different germplasm resources. Fig.11 and Fig.12 As shown in the figure, the average content of CSOs in all parts of leek is S-methylcysteine ​​sulfoxide, followed by S-allylcysteine ​​sulfoxide, and S-propenylcysteine ​​sulfoxide. The content of S-methylcysteine ​​sulfoxide in leek flowers is higher than that in other parts. At the same time, the average content of S-allylcysteine ​​sulfoxide and S-propenylcysteine ​​sulfoxide in the five types of leek is higher in flowers than in other parts. Among them, the content of Changge Dazhao leek in the flower part is the highest at 31.75 mg / g; the content in white wild leek is the lowest at 15.5 mg / g; it is worth noting that the content of S-methylcysteine ​​sulfoxide in white wild leek in the root part is the highest at 15.19 mg / g.

[0183] The content of S-allylcysteine ​​sulfoxide is the highest in the flower part, followed by the root and seed, while the content of S-allylcysteine ​​sulfoxide is the lowest in the leaf. The content of S-propylenecysteine ​​sulfoxide is also the highest in the flower part, followed by the seed, and the content difference in the leek stalk, leaf, pseudostem, and root part is small. According to the total content, the content of S-alkylcysteine ​​sulfoxide in leek is in the order of flower, seed, root, pseudostem, stalk, and leaf.

[0184] 2.4 Determination of volatile odor characteristics in different parts of leek

[0185] The test materials were the same as those in 2.3, and the electronic nose instrument was used for determination, with the instrument parameter settings being the same as those in 2.2.

[0186] Table 3-7 Average response of electronic nose sensors in different parts of leek

[0187]

[0188]

[0189] As shown in Table 3-7, the response values ​​of the volatile odors in different parts of the five leeks to the electronic nose sensors W1C, W5S, W3C, and W2W are still the largest, and the differences in different parts are also mainly reflected in these four sensors. By comparison, it was found that the average response value of the W5S sensor in the leek flower was the largest, among which the value of the corpse-free leek was the largest at 132.96 (Table 3-8); followed by leek seeds and roots, and the differences in pseudostems, leaves and leek stalks were relatively small. Sensor W2W also had the largest response value to the flower part, followed by seeds, and the smallest response value in leaves. Overall, the electronic nose response values ​​of volatile odors in various parts of leeks are in the order of flowers, seeds, roots, pseudostems, leaves, and leek stalks.

[0190] Table 3-8 Electronic nose response values ​​of different parts of leek

[0191]

[0192]

[0193] 3. Comprehensive analysis of leek germplasm resources

[0194] The cluster analysis method was used to classify leeks according to the S-alkylcysteine ​​sulfoxide and volatile odor characteristics in leeks. The entropy weight TOPSIS method was used to rank the comprehensive scores of 50 leek germplasm resources based on the index values ​​of S-alkylcysteine ​​sulfoxide and volatile odor characteristics in leek.

[0195] 3.1 Cluster analysis of leek germplasm

[0196] Cluster analysis was used to analyze the volatile odor characteristics and S-alkylcysteine ​​sulfoxide of 50 leek germplasm resources. Fig.13 As shown, when the Euclidean square distance is 10, the germplasm resources can be divided into three groups, and the specific members of the groups are shown in Table 3-9. There are 22 leek varieties in Category I, which are characterized by low volatile odor electronic nose response values ​​and high S-alkylcysteine ​​sulfoxide content; there are 26 leek varieties in Category II, which are characterized by high volatile odor electronic nose response values, high S-alkylcysteine ​​sulfoxide content and no significant difference in Category II. There are two main members in Category III, Baofu Chive Red and Budaoshi Chive, whose main characteristics are that the volatile odor electronic nose response values ​​and S-alkylcysteine ​​sulfoxide content are higher than other varieties.

[0197] Table 3-9 Members of different leek cluster analysis categories

[0198]

[0199] 3.2 Analysis of leek germplasm resources based on entropy weight TOPSIS method

[0200] The different leek germplasm resources were comprehensively evaluated based on the contents of three S-alkylcysteine ​​sulfoxides in 50 different varieties of leek and the response values ​​of the electronic nose sensor. The entropy weight TOPSIS method was used to comprehensively score the 50 different leek germplasm resources, and the comprehensive ranking is shown in Table 3-10. The top ten leek varieties are white wild leek, Budaoshi leek, Hanyu purple root, Jiuxing No. 20, Baofu leek red, Pingjiu No. 6, Yuzhong leek, Fujiu Hansheng, Xuelihuang, and Changjingpengjiuwang, with scores of 0.6467, 0.4608, 0.4508, 0.4467, 0.4176, 0.409, 0.3679, 0.3676, 0.3671, and 0.3579, respectively. The top ten leeks in the comprehensive ranking are Snow Leek King, Malian Big Leaf White Root, Jiuxing 23, Jiuxing 21, Fusheng 2, Broad Leaf Leek, Alpine Leek, Narrow Leaf Leek, Pingjiu 2, Jiuxing 15, and Jiushen; the scores are 0.1529, 0.1633, 0.1650, 0.1756, 0.1795, 0.1987, 0.2182, 0.224, 0.2251, 0.2316, and 0.2362 respectively. By comparing (Table 3-4, Table 3-6), the results of the electronic nose sensor response values ​​and S-alkylcysteine ​​sulfoxide content of the top ten leek varieties are consistent.

[0201] Table 3-10 Comprehensive scores of different varieties of leeks

[0202]

[0203]

[0204] in conclusion

[0205] 1. Determination of S-alkylcysteine ​​sulfoxide by high performance liquid chromatography

[0206] High performance liquid chromatography is widely used in the detection and analysis of substances. When using high performance liquid chromatography to detect substances, the properties of the substances to be measured must be clear. For example, the S-alkyl cysteine ​​sulfoxide substances to be measured in this experiment are the main flavor precursors in Allium plants. These substances are medium polar substances with strong hydrophilicity. They must be measured using high performance liquid chromatography. Ideally, a forward high performance liquid chromatography must be used. The difference between a forward high performance liquid chromatography and a reverse high performance liquid chromatography is that the polarity of the mobile phase and the stationary phase (chromatographic column) is different. The stationary phase (chromatographic column) of the reverse high performance liquid chromatography has a large polarity and a small polarity of the mobile phase, while the forward high performance liquid chromatography is the opposite. According to the characteristics of different types of high performance liquid chromatography, there will be a focus when measuring different substances. Generally, a forward high performance liquid chromatography is suitable for measuring substances with greater polarity, while a reverse high performance liquid chromatography is suitable for measuring non-polar or medium polar substances. In the present invention, Waterse2695 high performance liquid chromatograph is selected for the test. This is a reverse high performance liquid chromatograph. The main reasons are: first, the current market share of reverse high performance liquid chromatograph is much larger than that of the forward type; second, the chromatographic column is upgraded. The chromatographic column is an important component in the high performance liquid chromatograph. When the measured substance passes through the chromatographic column with the mobile phase, it is continuously adsorbed and analyzed to separate the required substances to achieve the purpose of measurement. The role of the chromatographic column is irreplaceable. With the advancement of science and technology, corresponding chromatographic columns are developed according to the different properties of the substances to be measured. The Waters T3 chromatographic column selected in this experiment has strong retention for polar compounds and can use a 100% aqueous phase system. Its performance meets the test requirements.

[0207] When using high performance liquid chromatography to determine S-alkyl cysteine ​​sulfoxide, instrument-related parameters such as mobile phase type, mobile phase ratio, detection wavelength, column temperature, etc. will affect the detection results. Xu Yang used a mobile phase of V (methanol): V (water) of 80:20 and a detection wavelength of 214nm when determining alliin in onions. Zhang Min et al. used the same method when determining alliin in garlic. However, when determining the mobile ratio in this experiment, it was found that when the aqueous solution and methanol (v:v) were 95:5 or even greater, the peaks of the S-alkyl cysteine ​​sulfoxide mixed solution could be separated. When the ratio was 80:20, the retention times of the three CSOs were the same and the peaks overlapped. By consulting the literature and combining the characteristics of high performance liquid chromatography, it is known that in general, a higher proportion of water phase is suitable for the separation of S-alkyl cysteine ​​sulfoxide substances. The problem brought about by this is that it is necessary to pay attention to whether the chromatographic column used can use a high proportion of water as the mobile phase.

[0208] In the optimal wavelength detection, the full wavelength scanning function of the instrument is used to determine the maximum absorption wavelength of S-alkylcysteine ​​sulfoxide, which is a function of the PDA detection liquid phase; from the results, the maximum detection wavelengths of the three CSOs are relatively low, which leads to the mixing of impurity peaks and sample peaks when measuring samples. At this time, the maximum detection wavelength cannot be used as the optimal detection wavelength. In this experiment, 200nm is selected as the optimal detection wavelength of CSO to reduce impurity peaks. Wang Minglu set the detection wavelength to 195nm when measuring alliin in garlic, and ICHIKAWA used a detection wavelength of 210nm when measuring alliin and isoalliin; it can be noticed that the detection wavelength of the entire S-alkylcysteine ​​sulfoxide is still relatively low. To solve similar problems, ion pair reagents can be used or the samples can be derivatized, but after using ion pair reagents, the chromatographic column can only detect this type of substance, resulting in waste, and when the samples are derivatized, attention should be paid to the incomplete reaction between the derivatization reagent and the substance to be measured.

[0209] The properties of S-alkylcysteine ​​sulfoxide substances are relatively stable. When measuring the stability of the samples, after being placed at room temperature for 48 hours, the relative standard deviation was calculated based on the measured peak surface area. The RSDs of the three substances were all lower than 6%, and the spike recovery rate was over 95%, indicating that the operation during the extraction process had little effect on S-alkylcysteine ​​sulfoxide, proving that its properties are relatively stable. S-alkylcysteine ​​sulfoxide exists in the cytoplasm, and only reacts when the cells are ruptured and come into contact with allinase under the catalysis of allinase. During the determination, allinase is inactivated while ensuring that the loss of S-alkylcysteine ​​sulfoxide is small. In previous related reports, Xu Keke et al. used microwave inactivation to determine the alliin content in garlic, and Dong Yuhui

[41] When extracting and separating isoalliin from onions, the onions were placed in a microwave oven for 5 minutes at a power of 700 W to inactivate the enzyme. In this experiment, it was found through comparison that high-temperature boiling was the best method for inactivating alliinase in leeks and determining S-alkylcysteine ​​sulfoxide. The reason may be related to the texture and water content of leeks. Compared with garlic and onions, leeks have a soft texture and a water content of up to 95%. When microwaved, leeks will char in a very short time, while high-temperature boiling has a better effect of inactivating enzymes. During boiling, S-alkylcysteine ​​sulfoxide is dissolved in the extract to avoid loss.

[0210] 2. Flavor and quality characteristics of different leek germplasm resources

[0211] S-alkylcysteine ​​sulfoxide and volatile odor in leek are both important flavor qualities of leek. In the identification of leek germplasm resources, the two are rarely measured. In the previous studies, the evaluation of leek germplasm resources focused on agronomic traits, quality and other aspects. In the present invention, by measuring the three main S-alkylcysteine ​​sulfoxides in the leaf parts of 50 different germplasm resources of leek, it is found that the content of S-methylcysteine ​​sulfoxide in most leeks is the highest, followed by S-allylcysteine ​​sulfoxide and S-propylene cysteine ​​sulfoxide; this is consistent with the results reported by some scholars that the content of S-methylcysteine ​​sulfoxide in leek is the highest. There are also studies that point out that each Allium plant has 2 to 3 main flavor precursors. In the experimental process, it is also found that in addition to the large peaks of the three substances to be measured in the experiment, there are other unknown substances with very small peak areas. Due to the lack of corresponding standard products, qualitative and quantitative analysis cannot be performed.

[0212] Similarly, the average content of S-methylcysteine ​​sulfoxide in the roots, pseudostems, flowers, stalks, and seeds of leek is the highest, while S-allylcysteine ​​sulfoxide and S-propylenecysteine ​​sulfoxide are relatively low. At the same time, we also noticed that the content of S-methylcysteine ​​sulfoxide is much higher than the other two S-alkylcysteine ​​sulfoxides. This corresponds to the existing reports that the main components of the volatile sulfide odor of leek are dimethyl disulfide and dimethyl trisulfide. When S-alkylcysteine ​​sulfoxide is catalyzed by alliinase, the CS bond will break, so the volatile sulfides are mainly methyl sulfide. In terms of the total content of the three main S-alkylcysteine ​​sulfoxides, the flowers have the highest content, followed by seeds, roots, pseudostems, stalks, and leaves; the leaves have the lowest content, which is similar to the reports of LACASTER et al.

[0213] In addition to the appearance characteristics, the special flavor of leek can also leave a deep impression on people. At present, there are many studies on the volatile flavor of leek and Allium plants. The composition of volatile odors has been clearly understood using gas chromatography GC-MS technology. Unlike gas chromatography technology, electronic nose technology cannot measure the specific components of volatile odors, and the overall odor profile of volatile odors is detected. In the present invention, by measuring the volatile odors in different leek germplasm resources, it is found that the sensors W1C, W5S, W3C, and W2W that the electronic nose responds to the most obvious volatile odors of leek are aromatic benzene, nitrogen oxides, ammonia, and organic sulfides, which are consistent with the leek flavor trend measured by Li Wei and Wang Cheng.

[0214] Among the different parts of leek, the volatile odor of leek flowers responded most significantly to the sensors W1C (aromatic benzene), W5S (nitrogen oxides), W3C (ammonia), and W2W (organic sulfide), followed by leek seeds, roots, and pseudostems, which is consistent with Wang Hongmei et al.

[115] The reported content of disulfide in the volatile oils of the three parts of leek rhizomes, flowers and leaves is consistent with that of leek flowers, leek leaves and leek rhizomes. [ When reporting on sulfur compounds in leek, it was also mentioned that the content and types of sulfides in leek flowers are higher than those in leek leaves.

[0215] The volatile flavor in leek was measured using electronic nose technology. The size of the electronic nose sensor response value represents the odor profile. From the overall results, the trends of S-alkylcysteine ​​sulfoxide in leek and the electronic nose sensor response value in different parts of leek are somewhat similar, but the correlation is not obvious in each leek variety. The reason may be that S-alkylcysteine ​​sulfoxide does not directly generate volatile gas under the catalysis of alliinase. During the entire reaction, sulfonic acid will be generated, which will polymerize to form thiosulfonic acid lipids, and then further react to generate volatile gas.

[0216] 3. Analysis of the diversity of leek germplasm resources

[0217] my country has many varieties of leeks and rich germplasm resources. In the present invention, 50 leek germplasm resources were selected to measure and analyze the flavor-related qualities. Many scholars have studied other aspects of leek germplasm resources. Li Yixiao selected 186 germplasm resources from 24 regions when studying the diversity of agronomic traits of different leeks. In 2011, Wang Zhen used 174 leek germplasm resources based on 15 phenotypic traits to screen out 69 for the establishment of the primary core library of the leek germplasm resource bank. Zhang Ming et al. used ISSR markers to explore the heredity of 36 leek germplasm resources and established a DNA fingerprint map. Jiao Ziyuan et al. conducted a diversity study on the phenotypic traits of flower organs of 56 leek germplasm resources.

[0218] In describing the diversity of germplasm resources, the coefficient of variation and genetic diversity index are often used. Wu Zhen et al. measured the quantitative traits of 11 garlic samples and found that the coefficient of variation of the longitudinal diameter of the bulb was as low as 11%, and the coefficient of variation of the transverse stem of garlic cloves was as high as 36%. When comparing the flower organs of leek, Jiao Ziyuan found that the coefficient of variation of calyx length / calyx width was larger than that of petal length / petal width, indicating that the degree of variation of calyx in leek germplasm resources was large. In the present invention, the coefficients of variation of S-methylcysteine ​​sulfoxide, S-allylcysteine ​​sulfoxide, and S-propylenecysteine ​​sulfoxide in 50 leek germplasms were 24.49%, 26.11%, and 40.96%, respectively, indicating that there were differences in the content of flavor precursors in the 50 leeks, the germplasm resources were rich, the potential for variation was large, and the probability of selecting high-quality individuals was high.

[121] ; Similarly, the coefficients of variation of the response values ​​of W1C, W5S, W3C, W6S, W5C, W1S, W1W, W2W, and W3S of the 50 leek electronic nose odor sensors were 14.1%, 52.72%, 13.2%, 5.61%, 10.78%, 2.66%, 2.10%, 4.24%, 17.31%, and 11.83%, respectively. Among them, the coefficients of variation of the response values ​​of 6 sensors were greater than 10%, indicating that there are differences between different varieties of leek and that leek resources are rich in types.

[0219] Cluster analysis, principal component analysis, and membership function method are commonly used in comprehensive analysis methods of germplasm resources; Li Yixiao used cluster analysis to divide 186 leek germplasm resources into four major groups, and used the principal component analysis method to perform principal component analysis on 11 traits, extracted 3 principal components, and concluded that the first principal component had the highest contribution rate. The main indicators were single plant weight, plant height, leaf width, pseudostem length, and pseudostem thickness, and these high-indicator varieties could be preferred during variety selection. Xu Yanxia used the TOPSIS method to evaluate the salt tolerance of corn germplasm based on four indicators: corn germination rate, radicle length, seedling survival rate, and plant height. In the present invention, cluster analysis was used to classify 50 leeks using flavor indicators, and they were divided into 3 categories according to the characteristics of leek samples. At the same time, the entropy weight TOPSIS method was used to comprehensively rank 50 leek germplasms. The use of the entropy weight TOPSIS method can avoid human subjective factors. The results show that the top-ranked leek varieties have large electronic nose response values ​​for volatile odors and high relative content of S-alkyl cysteine ​​sulfoxide. But it is also worth noting that white-flowered wild leek ranks first. The main reason may be related to its S-propylene cysteine ​​sulfoxide content which is much higher than other varieties. White-flowered wild leek is a wild variety, which may be its characteristic. However, this may also be related to a shortcoming of the TOPSIS analysis method. If some indicators have a large degree of dispersion during the analysis, the results will be unstable due to the influence of this value, thus affecting the entire ranking results.

[0220] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for extracting and detecting S-alkylcysteine ​​sulfoxide from leek, characterized in that: The steps include: Step 1: Select leek samples; Step 2: Inactivation of allicinase: inactivation of allicinase in leek samples; Step 3, adding the extract: adding the extract to the leek sample after the alliinase is inactivated; Step 4, grinding and ultrasonic treatment: grinding the leek sample added with the extract and then performing ultrasonic-assisted extraction to obtain an S-alkylcysteine ​​sulfoxide extract; Step 5, filtration and centrifugation: Filter the S-alkylcysteine ​​sulfoxide extract with filter paper, take 2 mL of S-alkylcysteine ​​sulfoxide clear solution and centrifuge; Step 6: Purification: After centrifugation, the S-alkylcysteine ​​sulfoxide clear solution is taken out and filtered through a membrane; Step 7, on-machine detection: put the purified S-alkylcysteine ​​sulfoxide clear solution into a chromatographic injection vial for detection.

2. The extraction and detection method of leek S-alkylcysteine ​​sulfoxide according to claim 1, characterized in that: In step 2, the garlic enzyme is inactivated by high-temperature boiling, and the high-temperature boiling time is 20 minutes.

3. The extraction and detection method of leek S-alkylcysteine ​​sulfoxide according to claim 1, characterized in that: In step three, the extract is a 0.1% phosphoric acid aqueous solution, and the solid-liquid ratio (g:mL) is maintained at 1:

10.

4. The extraction and detection method of leek S-alkylcysteine ​​sulfoxide according to claim 1, characterized in that: In step 4, the ultrasonic extraction process is performed for 30 minutes at a temperature of 50°C.

5. The extraction and detection method of leek S-alkylcysteine ​​sulfoxide according to claim 1, characterized in that: In step five, the centrifugation is performed at room temperature for 10 minutes, and the centrifuge speed is 10000 rpm.

6. The extraction and detection method of leek S-alkylcysteine ​​sulfoxide according to claim 1, characterized in that: In step six, the pore size of the filter membrane is 0.22 μm.

7. The extraction and detection method of leek S-alkylcysteine ​​sulfoxide according to claim 1, characterized in that: In step seven, the detection includes the following parameters: using Waters e2695 high performance liquid chromatograph, XSelect HSS T3 chromatographic column 150mm×4.6mm, 5μm, Waters, with 0.1% phosphoric acid aqueous solution and methanol as mobile phase, isocratic elution (v:v) 95:5, flow rate 0.5mL / min, column temperature: 30°C, injection volume 15μL, detection wavelength 200nm.

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