Method for identifying producing area of asparagus cochinchinensis

Abstract

The invention provides a method for identifying the producing area of radix asparagi, which is characterized in that hexanal, 1-octylene-3-ketone, 2-isopropyl-3-methoxypyrazine and (E, E)-2, 4-nonadienal are used as key odor markers of radix asparagi, and the radix asparagi of different producing areas is identified by measuring the odor activity values (OAV) of the odor markers. According to the present invention, the volatile components of the asparagus cochinchinensis produced in Yunnan, Guizhou, Sichuan and Guangxi are systematically represented and quantified through the HS-SPME / GC-QQQ-MS / MS technology, the characteristic substances with the odor contribution are screened through the odor activity value (OAV), the asparagus cochinchinensis odor intensity characteristic spectrum is established, and the chemical component-flavor characteristic-origin identification method is constructed; the subjective limitation of traditional sensory evaluation is broken through, objectiveness and standardization of origin identification are realized, and the method has important application value in origin traceability of asparagus cochinchinensis.

Description

Technical Field

[0001] The invention relates to a method for identifying the origin of asparagus cochinchinensis. Background Art

[0002] Asparagus cochinchinensis (Lour.) Merr. is a dried tuberous root of the Liliaceae family. Due to differences in soil composition and climatic conditions, the odor and bitterness of asparagus from different origins exhibit distinct regional characteristics. As a traditional medicinal and edible plant, its quality is closely related to the ecological environment of its origin. Existing identification techniques primarily focus on morphological observation, microscopic identification, thin-layer chromatography, molecular identification, and characteristic pattern analysis.

[0003] Ding Yanxia et al., "Study on the Botanical Characteristics of Asparagus Codonopsis from Different Origins," Journal of Traditional Chinese Medicine, Vol. 18, No. 6, 2007, published a study on the botanical characteristics of Asparagus cochinchinensis from Guizhou Province, which revealed significant differences in characteristics from other origins. Microscopically, the distribution of stone cells in Shuicheng, Guizhou Province, and Shandong Province differed significantly from other origins, while the differences from other origins were almost the same. Li Min et al., "A Comparative Pharmacognosy of Asparagus Cochinchinensis from Neijiang, Sichuan Province, and Other Origins," Journal of Chengdu University of Traditional Chinese Medicine, Vol. 26, No. 3, September 2003, reported significant morphological differences between Asparagus cochinchinensis from Xishui, Guizhou Province, and Yunnan Province, and from Neijiang, Sichuan Province. The presence, shape, and distribution of stone cells, as well as the presence of calcium oxalate raphide bundles, were used as microscopic distinguishing features of Asparagus cochinchinensis from different origins. Microscopically, Asparagus cochinchinensis from Yunnan Province, Binchuan, Dali, Yunnan Province, and Xishui, Guizhou Province, differed significantly from those from Neijiang, Sichuan Province, while Asparagus cochinchinensis from Yulin, Guangxi Province, and Neijiang, Sichuan Province, were almost indistinguishable.

[0004] The above methods have the following technical bottlenecks: First, morphological observation is highly subjective and lacks standardization. It is greatly affected by the experience of the observer and the processing and storage of medicinal materials. Microscopic identification and thin-layer identification require experienced professionals to operate, and different observers may affect the consistency of results due to differences in subjective judgment. Moreover, they are mainly based on qualitative or semi-quantitative analysis and lack the support of quantitative indicators. Second, molecular identification can only identify its closely related species and cannot trace the origin. Third, although the characteristic spectrum is quantifiable and costly, the above methods are all based on qualitative or semi-quantitative analysis and lack the support of quantitative indicators, making it difficult to achieve accurate traceability and large-scale data comparison.

[0005] As a long-standing medicinal and edible herb, asparagus has been widely used in Traditional Chinese Medicine (TCM) and folk practices since ancient times. Southwest my country is the primary production area for asparagus. However, due to its complex terrain, variable climate, and vastly varying altitudes, the quality of asparagus varies depending on its habitat. The flavor composition and content of asparagus grown in Yunnan, Guizhou, Sichuan, and Guangxi vary significantly. Summary of the Invention

[0006] The invention provides a method for identifying the origin of Asparagus cochinchinensis.

[0007] The present invention provides a method for identifying the origin of asparagus cochinchinensis. The method uses n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal as key odor markers of asparagus cochinchinensis, and identifies asparagus cochinchinensis from different origins by measuring the odor activity values ​​(OAVs) of the odor markers.

[0008] It includes the following steps:

[0009] a. Weigh the asparagus sample to be tested;

[0010] b. Detect the concentrations of n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal in the sample;

[0011] c. Calculate the odor activity value:

[0012] The formula is: Where OAV is the intensity of the odor component; C represents the concentration of the volatile substance in the sample, and OA represents the odor threshold of the component (ng / g);

[0013] d. Differentiate the asparagus from different origins based on the odor activity value, among which,

[0014] The OAV values ​​of n-hexanal are 1.67-8.15, 1-octen-3-one is 4.91-9.0, 2-isopropyl-3-methoxypyrazine is 229.26-352.31, and (E,E)-2,4-nonadienal is 2.41-14.16, which is Asparagus cochinchinensis;

[0015] The OAV values ​​of n-hexanal are 30.37-33.17, 1-octen-3-one is 21.06-29.45, 2-isopropyl-3-methoxypyrazine is 460.74-558.70, and (E,E)-2,4-nonadienal is 65.98-75.31, which are Guizhou Asparagus cochinchinensis;

[0016] The OAV values ​​of n-hexanal are 84.73-90.87, 1-octen-3-one is 85.12-92.97, 2-isopropyl-3-methoxypyrazine is 0, and (E,E)-2,4-nonadienal is 254.42-274.79, which is Sichuan Asparagus cochinchinensis;

[0017] The OAV values ​​of n-hexanal are 22.40-77.60, 1-octen-3-one is 12.93-88.93, 2-isopropyl-3-methoxypyrazine is 117.69-377.13, and (E,E)-2,4-nonadienal is 31.78-265.59, which are Guangxi Asparagus cochinchinensis.

[0018] The method for detecting the concentration of n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal comprises the following steps:

[0019] a. Sample pretreatment: Dry the asparagus cochinchinensis using a vacuum freeze-drying method; then grind the dried asparagus cochinchinensis in a high-speed grinder and pass the powder through a 50-mesh sieve. Take about 0.5 g of asparagus cochinchinensis powder, accurately weigh it, and place the sample to be tested in a 20 mL inerted headspace bottle for later use.

[0020] b. Detection method: Headspace solid phase microextraction-gas chromatography triple quadrupole tandem mass spectrometry was used to detect the concentrations of hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal.

[0021] The gas chromatography conditions are:

[0022] The chromatographic column was an InertCap Pure-WAX capillary column (30 m × 0.25 mm × 0.25 μm); the inlet temperature was 250°C, the injection mode was split injection with a split ratio of 5:1, and the inlet pressure was 83.5 kPa. The carrier gas was helium (99.999%), and the carrier gas control mode was constant pressure mode. The purge volume flow rate was set at 3.0 mL / min. The column temperature was initially 40°C, and the temperature program was as follows: hold at 40°C for 5 min, increase at 3°C / min to 250°C, and hold for 15 min. The column equilibration time was 3 min.

[0023] Mass spectrometry conditions are:

[0024] The electron impact ion source (EI) was used, the acquisition mode was full scan mode (SCAN), the ion source temperature was 200°C, the interface temperature was 250°C, the collider was argon, the ionization energy was 70 eV, the mass range was 35-500, the detector voltage was +0.1 kV relative to the tuning result, and the solvent delay time was 1 min.

[0025] The HS-SPME method is:

[0026] The sample x in the x headspace bottle was equilibrated at 50°C for 10 minutes; it was first pretreated in a 250°C thermal desorption unit for 3 minutes to eliminate residues, and then penetrated the PTFE sealing septum and inserted into the upper space of the headspace bottle (non-contact adsorption mode), and the volatile component enrichment was completed for 15 minutes at a constant temperature of 50°C; after the extraction was completed, the fiber head was immediately transferred to the GC-MS / MS injection port, and subjected to high-temperature thermal desorption at 250°C for 2 minutes, and the analyte entered the chromatographic separation system with the carrier gas.

[0027] Qualitative and quantitative methods

[0028] Data were analyzed using a Shimadzu gas chromatography-mass spectrometry workstation (GC-MS Solution Version 4.54). The samples were compared with the Smart aroma database (a database covering 519 odorous substances established by adjusting the retention time of target components based on n-alkanes C9-C30, calculating relative response factors (RRFs) using three internal standards, and combining them with reference substances). The odorous components in the samples were screened based on retention indices (RIs) and standard mass spectra, and quantified using RRF values ​​and internal standards. The calculation formula is as follows:

[0029]

[0030] Where RRF is the relative response factor, m is the sample weight (g), V is the volume of the internal standard (mL), X is the mass fraction of the aroma component in the sample (μg / g), A is the peak area value, C is the mass concentration (mg / L), i is the internal standard, and t is the aroma component. The three internal standards are: 4-bromofluorobenzene, 1,2-dichlorobenzene-d4, and acenaphthene-d10.

[0031] The freeze-drying conditions in step a are as follows: pre-freeze the fresh asparagus sample at -80°C for 4 hours, vacuum dry for 72 hours, and control the moisture content of the raw material at 5±10% after drying.

[0032] This study systematically characterizes and quantifies the volatile components of Asparagus cochinchinensis (A. cochinchinensis) from Yunnan, Guizhou, Sichuan, and Guangxi provinces using HS-SPME / GC-QQQ-MS / MS. Furthermore, the method uses odor activity value (OAV) to screen for characteristic substances contributing to the aroma, establishing an odor intensity profile for Asparagus cochinchinensis. This method, based on a "chemical composition-flavor profile-origin identification" methodology, overcomes the subjective limitations of traditional sensory evaluation and achieves objectivity and standardization in origin identification, providing valuable application for traceability of Asparagus cochinchinensis' origin.

[0033] Figures in the specification

[0034] Figure 1Volatile substance analysis results, (A) the number of each compound in each sample, (B) the content of each compound in each sample, (C) Venn diagram of common and unique compounds in each origin;

[0035] Figure 2 (A) PCA score plot of 109 volatile compounds, (B) PLS-DA score plot, (C) model cross-validation experiment, (D) variable projection importance (VIP) dot-stick heat map;

[0036] Figure 3 Odor characteristics spectrum of Asparagus cochinchinensis (OICS). DETAILED DESCRIPTION

[0037] Example 1 Establishment of the method for identifying the origin of Asparagus cochinchinensis of the present invention

[0038] 1. Instruments and Materials

[0039] 1.1 Instrument

[0040]

[0041] 1.2 Materials

[0042] The samples used in this study were collected from Yunnan, Guizhou, Sichuan, and Guangxi provinces and identified by Professor Wang Guangzhi of Chengdu University of Traditional Chinese Medicine as tuberous roots of Asparagus cochinchinensis (Lour.) Merr. Detailed information is shown in Table 1.

[0043] Table 1 Collection information of Asparagus cochinchinensis samples

[0044]

[0045] 1.3 Reagents

[0046] Composite standard working solution of 4-bromofluorobenzene and deuterated 1,2-dichlorobenzene (d4), deuterated acenaphthene (d10) standard working solution (O2SI, USA, mass concentration of both is 100 μg / L)

[0047] 2. Experimental Methods

[0048] 2.1 Gas chromatography conditions

[0049] The chromatographic column was an InertCap Pure-WAX capillary column (30 m × 0.25 mm × 0.25 μm); the inlet temperature was 250°C, the injection mode was split injection, the split ratio was 5:1, and the inlet pressure was 83.5 kPa; the carrier gas was helium (99.999%), and the carrier gas control mode was constant pressure mode; the purge volume flow rate was set to 3.0 mL / min; the initial column temperature was 40°C, and the temperature ramp was as follows: maintain at 40°C for 5 min, increase to 250°C at 3°C / min, and maintain for 15 min; the column equilibration time was 3 min.

[0050] 2.2 Mass spectrometry conditions

[0051] The electron impact ion source (EI) was used, the acquisition mode was full scan mode (SCAN), the ion source temperature was 200°C, the interface temperature was 250°C, the collider was argon, the ionization energy was 70 eV, the mass range was 35-500, the detector voltage was +0.1 kV relative to the tuning result, and the solvent delay time was 1 min.

[0052] 2.3 HS-SPME conditions

[0053] Asparagus cochinchinensis was dried using a vacuum freeze-drying method. The entire fresh asparagus sample was pre-frozen at -80°C for 4 hours and vacuum-dried for 72 hours. After drying, the moisture content of the raw material was controlled at 5±10%. The dried asparagus was then pulverized in a high-speed grinder and passed through a 50-mesh sieve. Approximately 0.5 g of asparagus cochinchinensis powder was accurately weighed and placed in a 20 mL inerted headspace vial. The sample was equilibrated at 50°C for 10 minutes. After pretreatment in a 250°C thermal desorption unit for 3 minutes to eliminate residual residues, the sample was then inserted into the headspace of the headspace vial (non-contact adsorption mode) through the PTFE septum. Volatile components were enriched at 50°C for 15 minutes. After extraction, the fiber tip was immediately transferred to the GC-MS / MS injection port and subjected to thermal desorption at 250°C for 2 minutes. The analytes were then introduced into the chromatographic separation system along with the carrier gas.

[0054] 2.4 Qualitative and quantitative methods

[0055] Data were analyzed using a Shimadzu gas chromatography-mass spectrometry workstation (GC-MS Solution Version 4.54). The samples were compared with the Smart aroma database (a database covering 519 odorous substances established by adjusting the retention time of target components based on n-alkanes C9-C30, calculating relative response factors (RRFs) using three internal standards, and combining them with reference substances). The odorous components in the samples were screened based on retention indices (RIs) and standard mass spectra, and quantified using RRF values ​​and internal standards. The calculation formula is as follows:

[0056]

[0057] Where RRF is the relative response factor, m is the sample weight (g), V is the volume of the internal standard (mL), X is the mass fraction of the aroma component in the sample (μg / g), A is the peak area value, C is the mass concentration (mg / L), i is the internal standard, and t is the aroma component. The three internal standards are: 4-bromofluorobenzene, 1,2-dichlorobenzene-d4, and acenaphthene-d10.

[0058] The headspace solid-phase microextraction (HS-SPME) testing process is as follows: The HS-SPME testing process primarily includes sample preparation, heating equilibrium, extraction, desorption and injection, chromatographic analysis, and cleaning and regeneration. First, the sample to be tested is placed in a sealed headspace vial. The vial is then heated in an oven at a set temperature, allowing volatile components in the sample to diffuse into the headspace and reach equilibrium. Next, an extraction tip coated with a suitable stationary phase is inserted into the headspace of the vial for extraction, where it adsorbs the volatile components. After extraction, the tip is quickly inserted into the chromatographic inlet, where it is desorbed under high temperature or solvent elution conditions and enters the chromatographic system for separation and detection. The chromatograph collects data and generates a chromatogram. The target analyte content is calculated by comparison with a standard curve. Finally, the tip is cleaned and regenerated to remove residues and restore performance. The entire process is simple, solvent-free, rapid, and efficient, making it suitable for the detection of volatile components in a variety of fields, effectively improving analytical efficiency and accuracy.

[0059] 2.5 Statistical analysis

[0060] All samples were assayed in triplicate. Principal component analysis (PCA) and orthogonal projection to partial least squares discriminant analysis (PLS-DA) were performed and visualized using SIMCA 14.1 (Umetrics AB, Umea, Sweden). Data were statistically analyzed and images were created using OriginPro 2024 (OriginLab, Hampton, Massachusetts, USA) and Omicshare (https: / / www.omicshare.com / ).

[0061] 3. Experimental Results

[0062] 3.1 Volatile component research results

[0063] Variations in the types and concentrations of volatile organic compounds (VOCs) cause asparagus from different origins to exhibit different intensities and types of aroma. A total of 109 VOCs were detected and identified in asparagus from Yunnan, Guizhou, Sichuan, and Guangxi, which can be divided into 10 categories. The number of each type of compound varies in a gradient ( Figure 1A), of which alcohols were the most abundant, with a total of 30 detected, followed by aldehydes (22), acids (13), ketones (13), esters (12), heterocyclics (7), phenols (5), sulfurs (3), benzenes (3), and alkenes (1). Alcohols and aldehydes accounted for 27.52% and 20.18% of the total, respectively. The total volatile content of each sample ranged from 923.4873 to 6730.378 ng / g ( Figure 1 B); Among various volatile compounds, the contents of aldehydes, acids and alcohols were relatively high, which were 20751.202ng / g, 10273.047ng / g and 5584ng / g respectively.

[0064] Common and unique volatile components in each origin, such as Figure 1 As shown in Figure C, 40 compounds are common across the four production areas, with n-hexanal, (E)-2-heptenal, acetic acid, and 1-octen-3-ol being particularly abundant. Compounds unique to Sichuan are all esters, namely ethyl acetate (banana or apple aroma, with a slightly fruity wine aroma), amyl acetate (banana), and amyl butyrate (banana). Compounds unique to Guizhou are all alcohols, namely 1-tetradecanol (waxy), 1-hexadecanol, and 2,3-butanediol (fruit, onion).

[0065] 3.2 Multivariate statistical analysis of volatile components

[0066] Based on the content of volatile compounds in each Asparagus cochinchinensis sample, PCA analysis was first performed ( Figure 2 A), the results showed that samples from the same geographical origin clustered closely, while samples from different production areas showed better separation, indicating that the volatile components of Asparagus cochinchinensis from the same production area were relatively small, while the odor components of Asparagus cochinchinensis from different production areas were relatively different. However, the two samples from Nanning, Guangxi and the sample from Guizhou were relatively close, suggesting that the Asparagus cochinchinensis from the two areas had similar genetic backgrounds or were cultivated in similar ways. In order to further identify the influencing variables that led to the differences between the groups, partial least squares discriminant analysis was used to establish a relationship model between the expression levels of volatile compounds and sample categories, and to clarify the differential odor components between Asparagus cochinchinensis from different production areas. Similar to the results of PCA, in PLS-DA ( Figure 2 B) Samples from the same geographical origin cluster together, while samples from different production areas show good separation. Model cross-validation experiment (n = 200), such as Figure 2C, the intersection of Q2 and the Y axis is negative, which proves that the model is reliable and there is no overfitting. The variable projection importance (VIP) can measure the influence and explanatory power of the response characteristics of each compound on the classification and discrimination between each group of samples. Generally, a VIP value > 1.0 can provide a statistical basis for the screening of characteristic metabolites. A total of 21 key differential compounds were screened out from 109 compounds (p < 0.05). Therefore, these 21 compounds are the main differential markers of volatile components in the samples ( Figure 2 D). The larger the VIP value, the greater the contribution of the substance to distinguishing between the groups of samples. 2,3-Butanediol has a fruity, creamy, and oily flavor and was only detected in samples from Guizhou. (E)-2-penten-1-ol was only detected in Guizhou and Guangxi, and the content of (E)-2-penten-1-ol in Guizhou (78.28 ng / g) was significantly higher than that in Guangxi (p<0.001). The content of nonanal in samples from Yunnan and Guangxi was 10.00 ng / g and 11.01 ng / g, respectively, significantly higher than the 0.91 ng / g in Guizhou (p<0.05). No samples from Sichuan were detected. The 21 components are mainly alcohols and ketones. Phenylethanol has a rose aroma and accounts for a high proportion among alcohols. 3-Hydroxy-2-butanone has a buttery and creamy flavor and accounts for a high proportion among ketones. However, this is only a difference caused by the content and cannot explain the impact on the overall aroma. To this end, we will further explore the components that contribute to the overall aroma of Asparagus cochinchinensis.

[0067] 3.3 Analysis of Odor Substances in Asparagus cochinchinensis

[0068] The odor threshold refers to the lowest concentration of a substance that triggers human smell (ng / g). The performance of aroma is not entirely determined by the content, but also depends on the threshold of the component. The ratio of concentration to threshold is called the odor activity value (OAV):

[0069]

[0070] In formulas 1-3, OAV represents the intensity of the odor component; C represents the concentration of the volatile compound in the sample; and OA represents the odor threshold (ng / g) of that component. Generally, components with OAV values ​​greater than 1 play a crucial role in shaping the unique aroma profiles of different regions. There are 22 odor components with OAV values ​​greater than 1 in the Asparagus cochinchinensis samples, as shown in Table 2.

[0071]

[0072] 3.4 Odor intensity profile

[0073] The odor intensity signature spectrum (OICS) can provide a more accurate and intuitive description of the concentration and odor contribution of aroma components. Volatile compounds with OAV>1 were screened and serial numbers from 1 to 22 were compiled as the horizontal axis, and the OAV value was used as the vertical axis to plot the OICS of Asparagus cochinchinensis. Figure 3 The results show that the key odorants of various asparagus samples from the same origin show significant convergence, exhibiting a unique odor profile. Hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal are the key odor markers of asparagus. The asparagus from Yunnan has a single odor profile, and 2-isopropyl-3-methoxypyrazine significantly affects its flavor, giving it an unpleasant pea and earthy flavor. The asparagus from Guizhou and Guangxi regions have similar odor profiles, with the key odorants being hexanal, 1-octen-3-one, and (E,E)-2,4-nonadienal, exhibiting a grassy and fatty aroma, but is also negatively affected by 2-isopropyl-3-methoxypyrazine. The OAV values ​​of n-hexanal and 1-octen-3-one in Sichuan are relatively high, and (E,E)-2,4-nonadienal is more prominent, bringing the aroma of flowers, fruits, oil, and chicken soup. At the same time, 2-isopropyl-3-methoxypyrazine was not detected, avoiding interference with the overall aroma.

[0074] This study systematically analyzed the compositional characteristics of volatile components in Asparagus cochinchinensis and their origin differences using HS-SPME / GC-QQQ-MS / MS technology. Key odor markers were screened out using multivariate statistics and odor activity values ​​(OAV), revealing the aroma characteristics of Asparagus cochinchinensis from different origins. A total of 109 volatile organic compounds (VOCs) in 10 categories were identified in Asparagus cochinchinensis from Yunnan, Guizhou, Sichuan, and Guangxi, of which alcohols and aldehydes were the most abundant and had the highest content. Twenty-one key differential compounds were further screened out, primarily alcohols and ketones. However, these differences were based solely on content and could not fully reflect their impact on the overall aroma. Therefore, odor activity value (OAV) analysis revealed that 22 odor components with an OAV greater than 1 significantly contribute to the unique aroma characteristics of Asparagus cochinchinensis. Hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal are key odor markers of Asparagus cochinchinensis. The aroma profile of asparagus from Yunnan is monotonous, with 2-isopropyl-3-methoxypyrazine significantly impacting its flavor, resulting in unpleasant pea and earthy notes. Asparagus from Guizhou and Guangxi regions have similar aroma profiles, with key odorants including n-hexanal, 1-octen-3-one, and (E,E)-2,4-nonadienal, imparting grassy, ​​fatty notes. This is also negatively impacted by 2-isopropyl-3-methoxypyrazine. Asparagus from Sichuan, with n-hexanal and 1-octen-3-one showing high OAV values, and (E,E)-2,4-nonadienal being more prominent, imparts floral, fruity, and oily aromas, reminiscent of chicken broth.

[0075] 2-Isopropyl-3-methoxypyrazine (earthy flavor) negatively impacts the aroma of asparagus in Yunnan, Guizhou, and Guangxi. Modifying it can improve the aroma quality of asparagus. It was not detected in Sichuan, resulting in a purer floral, fruity, and oily aroma. This research, the first systematic construction of an asparagus aroma profile, clarifies the odor characteristics of asparagus from various origins, fills a gap in asparagus aroma research, and provides a scientific basis for identifying asparagus origin.

[0076] Example 2 Method for Identifying the Origin of Asparagus cochinchinensis of the Present Invention

[0077] Odor activity value (OAV) analysis revealed that n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal are key odor markers of asparagus root. Asparagus root from Yunnan Province exhibits a monotonous odor profile, with 2-isopropyl-3-methoxypyrazine significantly impacting its flavor, resulting in unpleasant pea and earthy notes. Asparagus root from Guizhou and Guangxi Provinces have similar odor profiles, with n-hexanal, 1-octen-3-one, and (E,E)-2,4-nonadienal as key odorants, exhibiting grassy, ​​fatty aromas, negatively impacted by 2-isopropyl-3-methoxypyrazine. Asparagus root from Sichuan Province exhibits higher OAV values ​​for n-hexanal and 1-octen-3-one, with (E,E)-2,4-nonadienal being more prominent, imparting floral, fruity, and oily aromas, reminiscent of chicken broth. 2-Isopropyl-3-methoxypyrazine (earthy smell) is a negative interference factor in the aroma of asparagus in Yunnan, Guizhou and Guangxi. Regulating it can improve the aroma quality of asparagus. However, it was not detected in Sichuan, and its floral, fruity and oily aroma was purer.

[0078] Table 3 OAV values ​​of odor markers of various batches of asparagus in Yunnan

[0079]

[0080] Table 4 OAV values ​​of odor markers of various batches of asparagus in Guizhou

[0081]

[0082] Table 5 OAV values ​​of odor markers of various batches of asparagus in Sichuan

[0083]

[0084] Table 6 OAV values ​​of odor markers of various batches of asparagus in Guangxi

[0085]

[0086] According to the test results in Tables 4 to 7, the OAV value ranges of the odor markers of Asparagus cochinchinensis from different origins are defined as follows:

[0087] The OAV values ​​of n-hexanal are 1.67-8.15, 1-octen-3-one is 4.91-9.0, 2-isopropyl-3-methoxypyrazine is 229.26-352.31, and (E,E)-2,4-nonadienal is 2.41-14.16, which is Asparagus cochinchinensis;

[0088] The OAV values ​​of n-hexanal are 30.37-33.17, 1-octen-3-one is 21.06-29.45, 2-isopropyl-3-methoxypyrazine is 460.74-558.70, and (E,E)-2,4-nonadienal is 65.98-75.31, which are Guizhou Asparagus cochinchinensis;

[0089] The OAV values ​​of n-hexanal are 84.73-90.87, 1-octen-3-one is 85.12-92.97, 2-isopropyl-3-methoxypyrazine is 0, and (E,E)-2,4-nonadienal is 254.42-274.79, which is Sichuan Asparagus cochinchinensis;

[0090] The OAV values ​​of n-hexanal are 22.40-77.60, 1-octen-3-one is 12.93-88.93, 2-isopropyl-3-methoxypyrazine is 117.69-377.13, and (E,E)-2,4-nonadienal is 31.78-265.59, which are Guangxi Asparagus cochinchinensis.

[0091] Example 3 Validation test of the method for identifying the origin of Asparagus cochinchinensis of the present invention

[0092] Asparagus cochinchinensis samples purchased from Yunnan, Guizhou, Sichuan and Guangxi production areas were tested and their odor activity values ​​(OAVs) were calculated. The specimens from each production area were identified as asparagus cochinchinensis from that production area by Professor Wang Guangzhi of Chengdu University of Traditional Chinese Medicine.

[0093] The samples from four production areas were tested using the unblinding method and were labeled as: Sample 1, Sample 2, Sample 3, and Sample 4. The test results are as follows:

[0094] Sample 1: The OAV value of n-hexanal is 7.13, the OAV value of 1-octen-3-one is 6.21, the OAV value of 2-isopropyl-3-methoxypyrazine is 260.28, and the OAV value of (E,E)-2,4-nonadienal is 12.16.

[0095] Sample 2: The OAV value of n-hexanal is 30.83, the OAV value of 1-octen-3-one is 21.98, the OAV value of 2-isopropyl-3-methoxypyrazine is 476.11, and the OAV value of (E,E)-2,4-nonadienal is 67.10.

[0096] Sample 3: The OAV value of n-hexanal is 88.46, the OAV value of 1-octen-3-one is 88.55, the OAV value of 2-isopropyl-3-methoxypyrazine is 0, and the OAV value of (E,E)-2,4-nonadienal is 269.32.

[0097] Sample 4: The OAV value of n-hexanal is 76.94, the OAV value of 1-octen-3-one is 77.87, the OAV value of 2-isopropyl-3-methoxypyrazine is 353.06, and the OAV value of (E,E)-2,4-nonadienal is 256.90.

[0098] Comparing the OAV value ranges of the odor markers of Asparagus cochinchinensis from different origins in Example 2, the results are as follows:

[0099] Sample 1 is the asparagus produced in Yunnan, sample 2 is the asparagus produced in Guizhou, sample 3 is the asparagus produced in Sichuan, and sample 4 is the asparagus produced in Guangxi.

[0100] Finally, the unblinded samples were consistent with the identification.

[0101] The data verified that the quality characteristics of asparagus cochinchinensis in each production area were consistent with the expectations of the theoretical model, providing a scientific basis for the identification of the origin of asparagus cochinchinensis.

Claims

1. A method for identifying the origin of Asparagus cochinchinensis, characterized in that: It uses n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal as key odor markers of asparagus, and identifies asparagus from different origins by measuring the odor activity value (OAV) of the odor markers.

2. The method for identifying the origin of Asparagus cochinchinensis according to claim 1, wherein: It includes the following steps: a. Weigh the asparagus sample to be tested; b. Detect the concentrations of n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal in the sample; c. Calculate the odor activity value: The formula is: Where OAV is the intensity of the odor component; C represents the concentration of the volatile substance in the sample, and OA represents the odor threshold of the component (ng / g); d. Differentiate the asparagus from different origins based on the odor activity value, among which, The OAV values ​​of n-hexanal are 1.67-8.15, 1-octen-3-one is 4.91-9.0, 2-isopropyl-3-methoxypyrazine is 229.26-352.31, and (E,E)-2,4-nonadienal is 2.41-14.16, which is Asparagus cochinchinensis; The OAV values ​​of n-hexanal are 30.37-33.17, 1-octen-3-one is 21.06-29.45, 2-isopropyl-3-methoxypyrazine is 460.74-558.70, and (E,E)-2,4-nonadienal is 65.98-75.31, which are Guizhou Asparagus cochinchinensis; The OAV values ​​of n-hexanal are 84.73-90.87, 1-octen-3-one is 85.12-92.97, 2-isopropyl-3-methoxypyrazine is 0, and (E,E)-2,4-nonadienal is 254.42-274.79, which is Sichuan Asparagus cochinchinensis; The OAV values ​​of n-hexanal are 22.40-77.60, 1-octen-3-one is 12.93-88.93, 2-isopropyl-3-methoxypyrazine is 117.69-377.13, and (E,E)-2,4-nonadienal is 31.78-265.59, which are Guangxi Asparagus cochinchinensis.

3. The method for identifying the origin of Asparagus cochinchinensis according to claim 2, wherein: The method for detecting the concentration of n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal comprises the following steps: a. Sample pretreatment: Dry the asparagus by vacuum freeze drying; then grind the dried asparagus in a high-speed grinder and pass the powder through a 50-mesh sieve. Take approximately 0.5 g of the asparagus powder, accurately weigh it, and place the sample to be tested in a 20 mL inerted headspace vial for later use; b. Detection method: Headspace solid phase microextraction-gas chromatography triple quadrupole tandem mass spectrometry was used to detect the concentrations of n-hexanal, 1-octen-3-one, 2-isopropyl-3-methoxypyrazine, and (E,E)-2,4-nonadienal. The gas chromatography conditions are: The chromatographic column was an InertCap Pure-WAX capillary column (30 m × 0.25 mm × 0.25 μm); the inlet temperature was 250°C, the injection mode was split injection with a split ratio of 5:1, and the inlet pressure was 83.5 kPa. The carrier gas was helium (99.999%), and the carrier gas control mode was constant pressure mode. The purge volume flow rate was set at 3.0 mL / min. The column temperature was initially 40°C, and the temperature program was as follows: hold at 40°C for 5 min, increase at 3°C / min to 250°C, and hold for 15 min. The column equilibration time was 3 min. Mass spectrometry conditions are: The electron impact ion source (EI) was used in full scan mode (SCAN), with a source temperature of 200°C, an interface temperature of 250°C, an argon collider, an ionization energy of 70 eV, a mass range of 35–500, a detector voltage of +0.1 kV relative to the tuning result, and a solvent delay time of 1 min. The HS-SPME method is: The sample x in the headspace vial was equilibrated at 50°C for 10 minutes. It was first pretreated in a thermal desorption unit at 250°C for 3 minutes to eliminate residues. Then, it was inserted into the upper space of the headspace vial through the PTFE sealing septum (non-contact adsorption mode) and the volatile components were enriched at a constant temperature of 50°C for 15 minutes. After the extraction is completed, the fiber head is immediately transferred to the GC-MS / MS injection port and subjected to thermal desorption at 250°C for 2 min. The analytes enter the chromatographic separation system along with the carrier gas. The qualitative and quantitative methods are: Data were analyzed using a Shimadzu gas chromatography-mass spectrometry workstation (GC-MS Solution Version 4.54). The samples were compared with the Smart aroma database (a database covering 519 odorous substances established by adjusting the retention time of target components based on n-alkanes C9-C30, calculating relative response factors (RRFs) using three internal standards, and combining them with reference substances). The odorous components in the samples were screened based on retention indices (RIs) and standard mass spectra, and quantified using RRF values ​​and internal standards. The calculation formula is as follows: Where RRF is the relative response factor, m is the sample weight (g), V is the volume of the internal standard (mL), X is the mass fraction of the aroma component in the sample (μg / g), A is the peak area value, C is the mass concentration (mg / L), i is the internal standard, and t is the aroma component. The three internal standards are: 4-bromofluorobenzene, 1,2-dichlorobenzene-d4, and acenaphthene-d10.

4. The method for identifying the origin of Asparagus cochinchinensis according to claim 3, wherein: The freeze-drying conditions in step a are as follows: pre-freeze the fresh asparagus sample at -80°C for 4 hours, vacuum dry for 72 hours, and control the moisture content of the raw material at 5±10% after drying.

Images