Method for analyzing formula of functional flavouring base

Abstract

A method for analysing a formula of a functional flavouring base, such as a natural spice, comprises separately subjecting aqueous extracts of a plurality of natural spices to polar gradient extractio

Description

[0001] The present disclosure relates to the technical field of analysis of spice components, in particular to a method for analyzing a formula of a functional flavoring base. BACKGROUND

[0002] Natural spices have not been processed and have been extracted or refined by physical methods without changing their original components. Compared with synthetic spices, natural spices are widely used in food, medicine, tobacco, cosmetics, and luxury goods for fragrance enhancement, deodorization, sterilization, and preservation due to their advantages, such as rich and full aroma substance components, and more fragrance. In recent years, after the implementation of the "Harm Reduction and Coke Reduction" project in the tobacco industry, the smell and aroma of tobacco have been weakened, which is difficult for consumers to accept. Therefore, cigarette companies adopt flavor enhancement technology to improve the cigarette consumption experience through the interaction between the aroma components produced in the burning process of cigarettes and the aroma components in natural spices. Also, different natural spices having different fingerprint characteristics are used to form a cigarette brand with unique relish and efficacy. Therefore, the composition analysis of natural plant spice raw materials with traditional Chinese herbal medicines (such as chicory, alfalfa, angelica, wolfberry, and dandelion) as the main research target, and the application and development of functional flavoring base modules have become the hot spots in the research and development of essences and spices in the tobacco industry.

[0003] It is crucial for the traceability research of functional flavoring bases to analyze more components from compounds with complex matrices, and the analysis of more components is usually directly related to the extraction technology used in the experimental device. At present, conventional extraction methods include liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase microextraction (SPME), and liquid-phase microextraction (LPME). LLE is widely used for the separation of mixtures with complex components and large differences in substance contents due to its advantages of unchanged material structure and chemical properties, high extraction efficiency, simple operations, and low cost. However, the extraction capacity of conventional LLE is limited, and the extraction efficiency is closely related to the selection of the extraction agent. Therefore, it is impossible to obtain richer composition information for complex matrices. The functional flavoring base is a mixture containing a variety of aroma components and being of a specific aroma type. The components of the functional flavoring base are relatively complex. In addition to solvents and a small amount of volatile components, the functional flavoring base also contains a large quantity of sugars, acids, pigments, waxes, and so on. The complex matrices will lead to the information of low-content aroma components being masked, resulting in low accuracy in the analysis of the formula of a functional flavoring base. Therefore, accurate qualitative and quantitative analysis and traceability research could not be carried out when LLE is used to extract natural spices with complex matrices.

[0004] In addition, the component analysis technology provides the necessary analytical means for the "known components" of tobacco essences and spices. At present, the method of the component analysis technology includes the use of GC-MS analysis combined with in-house developed fragrance system software to analyze the components of unknown spice samples. Moreover, the volatile components of essences and monomeric spice materials could be extracted by di chloromethane ultrasonication and analyzed by GC-MS. Both are with the help of the powerful qualitative capabilities of GC-MS combined with the corresponding chemometrics and system software to analyze the components of spice samples. Although GC-MS detection could obtain a wealth of three-dimensional data, it is not only time-consuming and laborious to obtain the implicit relationship in chemical components by manual screening and checking, but also it may have the problem of incomplete information discovery. Therefore, it is urgent to provide a method for analyzing a formula of a functional flavoring base with high efficiency and high accuracy of detection results. SUMMARY

[0005] An object of the present disclosure is to provide a method for analyzing a formula of a functional flavoring base with high efficiency and high accuracy of detection results.

[0006] In order to achieve the above object, the present disclosure provides the following technical solutions:

[0007] A method for analyzing a formula of a functional flavoring base is provided, including:

[0008] (1) separately subjecting aqueous solutions of extracts of a plurality of natural spices to polar gradient extractions, to obtain organic phases from each gradient extraction of an aqueous solution of an extract of each natural spice and an aqueous phase from last extraction; wherein the polar gradient extractions are conducted in order from small to large sequentially according to polarity parameters of organic solvents used, and the organic solvents each have a polarity parameter of 0-4;

[0009] (2) separately drying the organic phases from each gradient extraction of the aqueous solution of the extract of each natural spice and the aqueous phase obtained in step (1) and then separately subjecting resulting dried products to qualitative analysis by gas chromatography-mass spectrometry (GC-MS), to obtain a pretreatment data; wherein the pretreatment data comprises relative percentage contents of extracted components in the organic phases from each gradient extraction and the aqueous phase;

[0010] (3) subjecting the pretreatment data obtained in step (2) to stoichiometric pattern identification analysis with a supervised mode, establishing a partial least squares discriminant analysis (PLS-DA) equal regression model or an orthogonal partial least squares discrimination analysis (OPLS-DA) equal regression model, and screening out symbolic differentiated compounds as characteristic components of the natural spices according to standards of variable importance factor (VIP) >1 and analysis of variance (ANOVA) p<0.05 to obtain a characteristic component database of the natural spices; and

[0011] (4) treating an aqueous solution of a functional flavoring base to be analyzed in accordance with processes for treating the aqueous solutions of extracts of natural spices in steps (1) to (3) to obtain characteristic components of the functional flavoring base, modeling and analyzing the characteristic components of the functional flavoring base and the characteristic component database of the natural spices obtained in step (3) to obtain a PLS-DA score plot or an OPLS-DA score plot; taking squared distances between the characteristic components of the functional flavoring base and the characteristic components of the natural spices in the PLS-DA score plot or OPLS-DA score plot as a judgement basis, ranking the nature spices in order from small to large according to the squared distances, taking top eight natural spices, and making a judgement that the functional flavoring base to be analyzed contains one or more of the eight natural spices.

[0012] In some embodiments, in step (1), the polar gradient extractions are conducted with 3-5 gradients.

[0013] In some embodiments, the organic solvents include at least three selected from the group consisting of n-hexane, methyl tert-butyl ether, 1,2-dichloroethane, n-butyl acetate and n-butyl alcohol.

[0014] In some embodiments, in step (1), natural spice extracts in the aqueous solutions of extracts of natural spices each include one selected from the group consisting of chicory extract, carob bean extract, tamarind extract, alfalfa extract, angelica extract, raisin extract, fig extract, plum extract, Roman chamomile extract, wolfberry concrete, dandelion concrete, malt concrete, jujube tincture, clotrimazole, and valerin tincture.

[0015] In some embodiments, in step (1), the aqueous solutions of extracts of natural spices each have a mass concentration of 5-20%.

[0016] In some embodiments, in step (1), during the polar gradient extractions, a volume ratio of an organic solvent for each gradient extraction to an aqueous phase to be extracted is in a range of 3: 1 to 1: 2.

[0017] In some embodiments, in step (1), during the polar gradient extraction, each gradient extraction is conducted for 10-50 min.

[0018] In some embodiments, in step (2), after drying the aqueous phase, a resulting dried product is subjected to derivatization treatment and the qualitative analysis by GC-MS sequentially.

[0019] In some embodiments, a derivatization reagent for the derivatization treatment is a mixed solution of bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS), and a volume percentage of the TMCS relative to the BSTFA is in a range of 1-10%.

[0020] In some embodiments, in step (2), parameters for the qualitative analysis by GC-MS include:

[0021] chromatographic parameters comprising: a chromatographic column of SH-Rxi-5MS capillary gas chromatography column with a specification of 30 m x 0.25 mm and 0.25 pm; a high-purity helium as a carrier gas with a flow rate of 1-3 mL / min; adopting a split-flow mode, a split-flow ratio of an organic phase sample being 10:1, and a split-flow ratio of an aqueous phase sample being 15: 1; a sample inlet temperature being in a range of 200-300 °C, a sample volume of the organic phases being 1 pL, and a sample volume of the aqueous phase being 0.2 pL; and a temperature procedure being set as: keeping an initial temperature at 40 °C for 2 min, then raising to 300 °C at a rate of 6 °C / min, and holding at 300 °C for 20 min; and

[0022] mass spectrometry conditions comprising: an ion source temperature being in a range of200-230 °C; an ionization voltage being 70 eV; and adopting a full scanning mode, a scanning range of an organic phase being in a range of 33 amu (atomic mass unit) to 550 amu, and a scanning range of an aqueous phase sample being in a range of 33 amu to 1050 amu.

[0023] The present disclosure provides a method for analyzing a formula of a functional flavoring base. In the present disclosure, the aqueous solutions of extracts of natural spices are separately subjected to polar gradient extractions, and the chemical components having different partition coefficients in aqueous phase and organic phase are subjected to gradient separation to obtain chemical components having different polarity ranges. After the polar gradient extraction treatment, the influence of the complex matrix on the natural spice extracts could be effectively reduced, and the masking effect of the high-content chemical components on the trace chemical components could be reduced, which is conducive to grasping the key secondary components masked by the main components, so as to get richer information about the extracted substances, and provide a rich database for the accurate analysis of the formula of a functional flavoring base. In the present disclosure, the organic phases from each gradient extraction and the aqueous phase from last extraction obtained in the polar gradient extractions are subjected to qualitative analysis by GC-MS, respectively, and then the resulting data are subjected to stoichiometric pattern discriminatory analysis with supervised modes (PLS-DA and OPLS-DA). The PLS-DA equal regression model or the OPLS-DA equal regression model is established, and the symbolic differentiated compounds are screened out as the characteristic components of the natural spices according to the standards of variable importance factor (VIP)>1 and ANOVA p<0.05 to obtain the characteristic component database of the natural spices. Then the aqueous solution of a functional flavoring base to be analyzed is treated according to the same process to obtain the characteristic components of the functional flavoring base. The characteristic components of the functional flavoring base and the characteristic component database of the natural spices are modeled and analyzed to obtain the PLS-DA score plot or the OPLS-DA score plot. Taking squared distances between the characteristic components of the natural spices and the characteristic components of the functional flavoring base in the PLS-DA score plot or OPLS-DA score plot as a judgement basis, the natural spices are ranked in order from small to large according to the squared distances, and the corresponding top eight natural spices are taken, and a judgement that the functional flavoring base to be analyzed contains one or more of the eight natural spices is made. The regression model for the extracted characteristic components provided by the present disclosure has good cross-validation and prediction ability. By the standards of limiting VIP>1 and ANOVA p<0.05, the characteristic components of the natural spices could be screened out, which can simplify the data and is more conducive to the accurate analysis of the formula of the functional flavoring base. The results of the examples show that the traceability accuracy of the pattern recognition can reach 87.5% after the four functional flavoring bases are subj ected to the same separation, detection and analysis by the method provided by the present disclosure, and according to the company standard, the traceability of the flavoring base formula is considered to be successful if the accuracy rate of the first eight natural spices in a formula of a functional flavoring base analyzed reaches 70% or higher. Therefore, the method provided by the present disclosure has a higher accuracy for the analysis of the formula of a functional flavoring base.

[0024] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying diagrammatic drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows an experimental flow chart of the polar gradient extractions according to an embodiment of the present disclosure.

[0026] FIG. 2A to FIG. 2B are graphs showing the results of the influence of different extraction processes on the extraction effect according to an embodiment of the present disclosure.

[0027] FIG. 3 is a graph showing the results of the influence of the extraction time on the extraction efficiency of polar extraction according to an embodiment of the present disclosure.

[0028] FIG. 4 is a graph showing the results of the influence of O / W on the extraction effect according to an embodiment of the present disclosure.

[0029] FIG. 5 A to FIG. 5B are graphs showing the results of the chemical composition analysis of 15 natural spices according to an embodiment of the present disclosure.

[0030] FIG. 6A to FIG. 6D show multivariate statistical analysis diagrams of the characteristic components of the four functional flavoring bases of the present disclosure and the characteristic component database screened from 15 natural spices. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] A method for analyzing a formula of a functional flavoring base is provided, including:

[0032] (1) separately subjecting aqueous solutions of extracts of a plurality of natural spices to polar gradient extractions, to obtain organic phases from each gradient extraction of an aqueous solution of an extract of each natural spice and an aqueous phase from last extraction; wherein the polar gradient extractions are conducted in order from small to large sequentially according to polarity parameters of organic solvents used, and the organic solvents each have a polarity parameter of 0-4;

[0033] (2) separately drying the organic phases from each gradient extraction of the aqueous solution of the extract of each natural spice and the aqueous phase obtained in step (1) and then separately subjecting resulting dried products to qualitative analysis by gas chromatography-mass spectrometry (GC-MS), to obtain a pretreatment data; wherein the pretreatment data comprises relative percentage contents of extracted components in the organic phases from each gradient extraction and the aqueous phase;

[0034] (3) subjecting the pretreatment data obtained in step (2) to stoichiometric pattern identification analysis with a supervised mode, establishing a PLS-DA equal regression model or an OPLS-DA equal regression model, and screening out symbolic differentiated compounds as characteristic components of the natural spices according to standards of VIP >1 and ANOVA p<0.05 to obtain a characteristic component database of the natural spices; and

[0035] (4) treating an aqueous solution of a functional flavoring base to be analyzed in accordance with processes for treating the aqueous solutions of extracts of natural spices in steps (1) to (3) to obtain characteristic components of the functional flavoring base, modeling and analyzing the characteristic components of the functional flavoring base and the characteristic component database of the natural spices obtained in step (3) to obtain a PLS-DA score plot or an OPLS-DA score plot; taking squared distances between the characteristic components of the functional flavoring base and the characteristic components of the natural spices in the PLS-DA score plot or OPLS-DA score plot as a judgement basis, ranking the nature spices in order from small to large according to the squared distances, taking top eight natural spices, and making a judgement that the functional flavoring base to be analyzed contains one or more of the eight natural spices.

[0036] In the present disclosure, aqueous solutions of extracts of a plurality of natural spices are separately subjected to polar gradient extractions, to obtain organic phases from each gradient extraction of an aqueous solution of an extract of each natural spice and an aqueous phase from last extraction.

[0037] In some embodiments of the present disclosure, natural spice extracts in the aqueous solutions of extracts of natural spices each include one selected from the group consisting of chicory extract, carob bean extract, tamarind extract, alfalfa extract, angelica extract, raisin extract, fig extract, plum extract, Roman chamomile extract, wolfberry concrete, dandelion concrete, malt concrete, jujube tincture, clotrimazole, and valerin tincture. In the present disclosure, one of the above natural spice extracts is used for gradient extraction so that the extract in the extracted organic phase and aqueous phase of each natural spice could be known. In some embodiments of the present disclosure, the aqueous solutions of extracts of natural spices each have a mass concentration of 5-20%, and preferably 10-20%. In some embodiments of the present disclosure, a total number of the natural spice extracts is adjusted when the database is established, and the total number of the natural spice extracts is in a range of 10-15, and preferably 15. In some embodiments of the present disclosure, the above natural spice extracts are common essence and spice components in the tobacco industry and contain rich aroma components. Therefore, it is more beneficial for the accurate analysis of the formula of a functional flavoring base to take the above natural spice extracts as raw material to obtain the characteristic component database of the natural spices. There is no specific limitation on the source of the natural spice extracts, and conventional commercial products could be used. In some embodiments of the present disclosure, the natural spice extracts are provided by China Tobacco Hunan Industrial Co., Ltd. (Changsha, China).

[0038] There is no specific limitation on the operation process of the polar gradient extractions, and a conventional operation process of the polar gradient extraction could be used. In some embodiments of the present disclosure, the polar gradient extractions are conducted by a process including: (a) mixing a first organic solvent with an aqueous solution of a natural spice extract, subjecting a resulting first mixture to first gradient extraction, centrifugation and layering to obtain a first gradient extracted organic phase and an aqueous phase from the first gradient extraction; (b) mixing a second organic solvent with the aqueous phase from the first gradient extraction obtained in step (a), subjecting a resulting second mixture to second extraction, centrifugation and layering to obtain a second gradient extracted organic phase and an aqueous phase from the second gradient extraction phase, which are repeated similarly, i.e., mixing an aqueous phase obtained after each gradient extraction with an organic solvent for the next gradient extraction, and subjecting a resulting mixture to extraction, until the last gradient extraction is performed to reach a set number of times, to obtain the last extracted organic phase and the aqueous phase from the last extraction.

[0039] In the present disclosure, the polar gradient extractions are conducted in order from small to large sequentially according to polarity parameters of organic solvents used. In order from small to large according to polarity parameters of organic solvents used, the organic solvents having different polarities are used for the polar gradient extractions on the aqueous solutions of extracts of natural spices so that chemical components having different partition coefficients in the aqueous phase and the organic phase could be subjected to gradient separation to obtain chemical components having different polarity ranges. There is no specific limitation on the number of gradients of the polar gradient extraction, and it could be adjusted as required. In some embodiments of the present disclosure, the polar gradient extractions are conducted with 3-5 gradients, and preferably 5 gradients.

[0040] In some embodiments of the present disclosure, the organic solvents each have a polarity parameter of 0-4. In the present disclosure, the organic solvents having the above polarity parameters are used for the polar gradient extractions on the aqueous solutions of extracts of natural spices so that the extracts in the aqueous solutions of extracts of natural spices could be completely separated. For the polar gradient extractions, there is no specific limitation on the polarity parameter of the organic solvent used for each gradient extraction, and the polarity parameter of the organic solvent could be selected within the range of 0-4, and the polar gradient extractions are conducted according to the order of the polarity parameters of the organic solvents from small to large.

[0041] In some embodiments of the present disclosure, the organic solvents include at least three selected from the group consisting of n-hexane, methyl tert-butyl ether, 1,2-di chloroethane, n-butyl acetate and n-butyl alcohol, and preferably n-hexane, methyl tert-butyl ether, 1,2-dichloroethane, n-butyl acetate and n-butanol. In the present disclosure, it is more advantageous to separate out more extracts by selecting the above organic solvents for the polar gradient extractions.

[0042] In some embodiments of the present disclosure, during the polar gradient extractions, a volume ratio of an organic solvent for each gradient extraction to an aqueous phase to be extracted is in a range of 3: 1-1:2, and preferably 1:1. In the disclosure, during the polar gradient extractions, the volume ratio of the organic solvent for each gradient extraction to the aqueous phase to be extracted is recorded as O / W. In the disclosure, by controlling the O / W in the above range, a larger extraction capacity could be avoided, which is caused by a large quantity of organic phase involved when the O / W is too large; the too large O / W further leads to the chemical components having a small partition coefficient or low concentration being partially extracted to the organic phase with substances having a large partition coefficient and high content, and these substances could be masked due to too low content during detection. In addition, by controlling the O / W in the above range, it could also prevent severe emulsification and unclear layer interface, caused by large quantity of aqueous phase when the O / W ratio is too small, which is not conducive to the smooth progress of the extraction experiment. Therefore, in the present disclosure, the O / W is controlled in the above range to improve the extraction efficiency.

[0043] In some embodiments of the present disclosure, during the polar gradient extractions, each gradient extraction is conducted for 10-50 min, and preferably 30-40 min. In the present disclosure, by controlling the extraction time of each gradient within the above range, the extraction balance could be achieved, and the degradation or oxidation of heat sensitive or easily-oxidized components caused by too long time could be prevented, thereby improving the extraction efficiency.

[0044] In some embodiments of the present disclosure, during the polar gradient extractions, each gradient extraction is conducted at a temperature of 5-25 °C, and preferably 5-10 °C. In the present disclosure, during the polar gradient extractions, by controlling the extraction temperature of each gradient within the above range, the degradation or oxidation of heat sensitive or easily-oxidized components caused by excessively high temperature could be prevented, thereby improving the extraction efficiency.

[0045] In the present disclosure, aqueous solutions of extracts of a plurality of natural spices are subjected to polar gradient extractions, respectively, so that the influence of the complex matrix could be effectively reduced, and the masking effect of the high content chemical components on the trace chemical components could be reduced, which is conducive to grasping the key secondary components masked by the main components, so as to get richer information about the extracted substances, and provide a database for the accurate analysis of the formula of a functional flavoring base.

[0046] In the present disclosure, after obtaining the organic phases from each gradient extraction and the aqueous phase from last extraction, the organic phases from each gradient extraction and the aqueous phase are separately dried, and then resulting dried products are separately subjected to qualitative analysis by GC-MS, to obtain a pretreatment data, wherein the pretreatment data includes relative percentage contents of extracted components in the organic phases from each gradient extraction and the aqueous phase.

[0047] There is no specific limitation on the processes for drying the organic phases from each gradient extraction and the aqueous phase, respectively, and conventional drying processes could be used. In some embodiments of the present disclosure, the drying of the organic phases from each gradient extraction is conducted by a process including: adding anhydrous sodium sulfate to the organic phases from each gradient extraction, completely drying, and filtering to obtain organic phase samples. There is no specific limitation on the filtering process, and a conventional filtering process could be used. In some embodiments of the present disclosure, the filtration is conducted with a 0.22 pm organic needle filter membrane. In some embodiments of the present disclosure, the drying of the aqueous phase is conducted by freeze-drying. There are no specific limitations on the temperature and time of the freeze-drying, and it could be adjusted as required to completely dry the aqueous phase.

[0048] In some embodiments of the present disclosure, after drying the aqueous phase, a resulting solid obtained after the drying is subjected to derivatization treatment and qualitative analysis by GC-MS sequentially. There is no specific limitation on the process for the derivatization treatment, and a conventional derivatization treatment process could be used. In some embodiments of the present disclosure, the derivatization treatment is conducted by a process including: dissolving the resulting solid obtained after drying the aqueous phase in a solution of hydroxylamine hydrochloride in anhydrous pyridine, heating at 70 °C for 30 min, and cooling to room temperature to obtain a mixed solution; adding a derivatization reagent into the mixed solution, heating at 70 °C for 30 min, and cooling to room temperature. In the present disclosure, pyridine could be used as an acid scavenging solvent in the above derivatization treatment process so that by-product HC1 could be removed prior to introduction into a GC-MS system. In some embodiments of the present disclosure, the solution of hydroxylamine hydrochloride in anhydrous pyridine has a concentration of 30 mg / mL; a reagent for the derivatization treatment is a mixed solution of BSTFA and TMCS; and a volume percentage of the TMCS relative to the BSTFA is in a range of 1-10%, and preferably 1%.

[0049] In some embodiments of the present disclosure, parameters for the qualitative analysis by GC-MS include:

[0050] chromatographic parameters including: a chromatographic column of SH-Rxi-5MS capillary gas chromatography column with a specification of 30 m * 0.25 mm and 0.25 pm; a high-purity helium being used as a carrier gas, preferably with a purity of 99.999%, and at a flow rate of 1-3 mL / min, and preferably 1 mL / min; adopting a split-flow mode, a split-flow ratio of an organic phase sample being 10: 1, and a split-flow ratio of an aqueous phase sample being 15: 1; a sample inlet temperature being in a range of 200-300 °C, and preferably 300 °C, a sample volume of the organic phase being 1 pL, and a sample volume of the aqueous phase being 0.2 pL; and a temperature procedure being set as follows: keeping an initial temperature at 40 °C for 2 min, then raising to 300 °C at a rate of 6 °C / min, and holding at 300 °C for 20 min; and

[0051] mass spectrometry conditions including: an ion source temperature being in a range of 200-230 °C; an ionization voltage being 70 eV; and adopting a full scanning mode, a scanning range of an organic phase being in a range of 33 amu-550 amu, and a scanning range of an aqueous phase sample being in a range of 33 amu-1050 amu. There is no specific limitation on the solvent delay time for the qualitative analysis of GC-MS, and the solvent delay time could be selected according to different solvents. In the present disclosure, most substances with complex components could reach the best separation state by limiting the above parameters of the qualitative analysis by GC-MS, which is convenient for detecting the most substances by this process. Specifically, in the present disclosure, the maximum separation effect within the least time could be achieved by limiting the types and specifications of chromatographic columns. The use of high-purity helium as the carrier gas can avoid abnormal chromatographic peaks (such as high baselines, ghost peaks, and negative peaks), as well as the reaction of impurity gases (such as hydrogen and oxygen) with samples, and reduce the loss of chromatographic columns. In the present disclosure, different extracts could be completely separated by limiting the flow rate of the carrier gas. In the present disclosure, the influence of solvent saturation could be removed by adjusting the solvent delay time according to the selection of the solvent.

[0052] In some embodiments of the present disclosure, the qualitative analysis by GC-MS is conducted by a process including: collecting a total ion current chromatogram and a mass spectrogram of an analyte in a full scanning mode, preprocessing data by GC-MS Shimadzu reanalysis software (Shimadzu, Japan), and comparing a mass spectrometry fragmentation diagram of each chromatographic peak with a NIST 17 standard library, selecting substances with matching scores of not less than 80 as a preliminary qualitative basis, subjecting peak area of components obtained from detection of the organic phases from each gradient extraction to normalization treatment to calculate a relative percentage content, and subjecting peak area of aqueous phase derivatized samples from the last extraction to normalization treatment to calculate a relative percentage content.

[0053] In the present disclosure, after obtaining the pretreatment data, the pretreatment data are subjected to stoichiometric pattern identification analysis with a supervised mode, a PLS-DA equal regression model or an OPLS-DA equal regression model is established, and symbolic differentiated compounds are screened out as characteristic components of the natural spices according to standards of variable importance factor >1 and ANOVA p<0.05 to obtain a characteristic component database of the natural spices.

[0054] There is no specific limitation on the process for operating the supervised modes (PLS-DA and OPLS-DA), and a conventional analysis process could be used.

[0055] In some embodiments of the present disclosure, the pretreatment data are subjected to analysis with an unsupervised mode (PCA and HCA) to understand the distribution of compounds in the spice as a whole. There is no specific limitation on the process for operating the unsupervised mode, and it can be obtained by a conventional analysis process.

[0056] In some embodiments of the present disclosure, the characteristic components are the relative expression change after normalization, shown as a clustering heat map. In the present disclosure, the database could be visualized by the clustering heat map. In the present disclosure, the clustering heat map is to classify the object variables according to the similarity degree and distance so that the similarity of elements in the same class is stronger than that of other classes, and the homogeneity of elements in the same class and the heterogeneity of elements between different classes can be maximized.

[0057] In the present disclosure, after obtaining the characteristic component database of natural spices, an aqueous solution of a functional flavoring base to be analyzed is treated in accordance with processes for treating the aqueous solutions of extracts of natural spices to obtain characteristic components of the functional flavoring base, the characteristic components of the functional flavoring base and the characteristic component database of the natural spices are modeled and analyzed to obtain a PLS-DA score plot or an OPLS-DA score plot; squared distances between the characteristic components of the natural spices and the characteristic components of the functional flavoring base in the PLS-DA score plot or OPLS-DA score plot are taken as a judgement basis, natural spices are ranked in order from small to large according to the squared distances, and the corresponding top eight natural spices are taken, and a judgement that the functional flavoring base to be analyzed contains one or more of the eight natural spices is made.

[0058] In the present disclosure, the process for treating the aqueous solution of the functional flavoring base to be analyzed is conducted the same as the above process for treating the aqueous solutions of extracts of natural spices, and the details are not repeated here.

[0059] There is no specific limitation on the type of the functional flavoring base to be analyzed in the aqueous solution of functional flavoring base to be analyzed, and it could be selected according to the flavoring base to be analyzed. In the present disclosure, in order to judge the accuracy of the traceability method, the functional flavoring base having a known formula is taken as a research object. In some embodiments of the present disclosure, the functional flavoring base is flavoring base A, flavoring base B, flavoring base C, and flavoring base D provided by China Tobacco Hunan Industrial Co., Ltd. (Changsha, China). In some embodiments of the present disclosure, the aqueous solution of functional flavoring base has a mass concentration of 75-90%.

[0060] In some embodiments of the present disclosure, the experimental flow chart of the polar gradient extractions and qualitative analysis is shown in FIG 1. In the present disclosure, the aqueous solutions of extracts of natural spices are separately subjected to extraction by polar gradient extractions, and then the organic phases from each gradient extraction and the aqueous phase from last extraction are subjected to drying and qualitative analysis by GC-MS sequentially, respectively. In the present disclosure, the aqueous solutions of extracts of natural spices are subjected to extraction by polar gradient extractions, respectively, so as to get richer information about extracted substances, and provide a database for the accurate analysis of the formula of a functional flavoring base. In the present disclosure, the organic phases from each gradient extraction and the aqueous phase from last extraction are subjected to qualitative analysis by GC-MS and stoichiometric pattern identification analysis with a supervised mode sequentially; a PLS-DA equal regression model or an OPLS-DA equal regression model is established, and symbolic differentiated compounds are screened out as the characteristic components of the natural spices according to the standards of VIP>1 and ANOVA p<0.05 to obtain the characteristic component database of the natural spices. Therefore, the method provided by the present disclosure has a higher accuracy for the analysis of the formula of a functional flavoring base.

[0061] The technical solutions provided by the present disclosure will be clearly and completely described below with reference to the examples in the present disclosure. Obviously, the described examples are merely a part of, but not all of, the examples of the present disclosure. Based on the examples of the present disclosure, all other examples obtained by a person of ordinary skill in the art without making creative labor shall fall within the scope of the present disclosure.

[0062] Example 1

[0063] A formula of a functional flavoring base was analyzed by a method, which was performed as follows:

[0064] (1) Aqueous solutions of extracts of 15 natural spices were subjected to polar gradient extractions, respectively, to obtain organic phases from each gradient extraction of an aqueous solution of an extract of each natural spice and an aqueous phase from last extraction, wherein the extracts of 15 natural spices were as follows: chicory extract, carob bean extract, tamarind extract, alfalfa extract, angelica extract, raisin extract, fig extract, plum extract, Roman chamomile extract, wolfberry concrete, dandelion concrete, malt concrete, jujube tincture, clotrimazole, and valerin tincture.

[0065] The polar gradient extractions were conducted as follows: natural spices were dissolved with deionized water to prepare aqueous solutions of extracts of natural spices having a mass concentration of 10%, and the experiment was designed as a five-gradient liquid-liquid extraction according to magnitudes of polar gradients of organic solvents. The organic solvents in the gradient design were in order of n-hexane, methyl tert-butyl ether, 1,2-dichloroethane, n-butyl acetate, and n-butyl alcohol.

[0066] n-Hexane and an aqueous solution of an extract of each natural spice O / W(18:18, v:v) were taken, completely oscillated for 30 min in a constant temperature oscillator with a water bath at 5 °C, and subjected to a first gradient extraction and then centrifugation for 3-5 min at a high-speed rotating centrifuge at a speed of 3000 r / min for layering, to obtain an organic layer and an aqueous phase from the first gradient extraction; the aqueous phase from the first gradient extraction was taken into the next extraction step, methyl tert-butyl ether was used as a second organic solvent, the O / W ratio was kept at 1: 1, and the operations were repeated to obtain a second gradient extracted organic phase and an aqueous phase from the second gradient extraction; the aqueous phase from the second gradient extraction was taken into the next extraction step, 1,2-dichloroethane was used as a third organic solvent, the O / W ratio was kept at 1: 1, and the operations were repeated to obtain a third gradient extracted organic phase and an aqueous phase from the third gradient extraction; the aqueous phase from the third gradient extraction was taken into the next extraction step, n-butyl acetate was used as a fourth organic solvent, the O / W ratio was kept at 1: 1, and the operations were repeated to obtain a fourth gradient extracted organic phase and an aqueous phase from the fourth gradient extraction; and the aqueous phase from the fourth gradient extraction was taken into the next extraction step, n-butyl alcohol was used as a fifth organic solvent, the O / W ratio was kept at 1: 1, and the operations were repeated to obtain a fifth gradient extracted organic phase and an aqueous phase from the fifth gradient extraction.

[0067] (2) The organic phases from each gradient extraction of aqueous solutions of extracts of various natural spices obtained in step (1) were completely dried by adding appropriate amount of anhydrous sodium sulfate thereto, and a resulting mixture was filtered through a 0.22 pm organic needle filter membrane. A resulting liquid was then subjected to qualitative analysis by GC-MS. The aqueous phase obtained in step (1) was subjected to freeze-drying and derivatization treatment sequentially, which were conducted as follows: 0.1 mL of a raffinate aqueous phase from the last extraction of the polar gradient extractions was transferred to a 1.5 mL derivatization vial, and subjected to vacuum freeze-drying; 0.2 mL of a solution of hydroxylamine hydrochloride in anhydrous pyridine (30 mg / mL) was added thereto, heated at 70 °C for 30 min, and then cooled to room temperature; 0.6 mL of BSTFA (containing 1% TMCS) was quickly added thereto, heated at 70 °C for 30 min, and cooled to room temperature. Samples were taken, filtered through a 0.22 pm organic needle filter membrane and then analyzed by GC-MS.

[0068] Parameters of the qualitative analysis by GC-MS were as follows: SH-Rxi-5MS (30 m x 0.25 mm, 0.25 pm) capillary gas chromatography column was used for separation. Chromatographic conditions were as follows: a high-purity helium (purity: 99.999%) was used as a carrier gas with a flow rate of 1 mL / min; a split-flow mode was adopted, a split-flow ratio of an extracted organic phase was 10:1, and a split-flow ratio of a raffinate aqueous phase derivatization sample was 15: 1; a sample inlet temperature was 300 °C, a sample volume of the extracted organic phase was 1 pL, and a volume of the raffinate aqueous phase derivatization sample was 0.2 pL; and a temperature procedure was set as follows: the temperature was set at an initial temperature of 40 °C for 2 min, raised to 300 °C at a rate of 6 °C / min, and held at 300 °C for 20 min. Mass spectrometry conditions were as follows: an ion source temperature was 230 °C; an ionization voltage was 70 eV; and a scanning mode was a full scanning mode (Scan), a scanning range of an extracted organic phase was in a range of 33 amu-550 amu, and a scanning range of an aqueous phase derivatization was in a range of 33 amu-1050 amu. A solvent delay time was 4.5 min for n-hexane phase, methyl tert-butyl ether phase and 1,2-dichloroethane phase, respectively, 12 min for n-butyl acetate phase, 13.5 min for n-butanol phase, and 7.5 min for aqueous phase derivatization samples.

[0069] The qualitative analysis was conducted as follows: a total ion current chromatogram and a mass spectrogram of an analyte were collected in a full scanning mode, data were preprocessed by GC-MS Shimadzu reanalysis software (Shimadzu, Japan), and a mass spectrometry fragmentation diagram of each chromatographic peak was compared with a NIST 17 standard library, substances with matching scores of not less than 80 were selected as a preliminary qualitative basis, peak area of the five extracted organic phases was subjected to normalization treatment to calculate a relative percentage content, and peak area of the raffinate aqueous phase derivatization samples was subjected to normalization treatment to calculate a relative percentage content to obtain pretreatment data.

[0070] (3) The pretreatment data obtained in step (2) was subjected to stoichiometric pattern identification analysis with supervised modes (PLS-DA and OPLS-DA), a PLS-DA equal regression model and an OPLS-DA equal regression model were established, and symbolic differentiated compounds were screened out as characteristic components of the natural spices according to standards of variable importance factor (VIP)>1 and ANOVA p<0.05 to obtain a characteristic component database of the natural spices.

[0071] Specifically, the analysis was performed as follows: the PLS-DA equal regression model and the OPLS-DA equal regression model were established to find characteristic substances. The values of R2 and Q2 were used to evaluate the quality and reliability of these models. Generally speaking, R2Y-Q2 <0.3, and their values closer to 1 indicated that the established regression models had better cross-validation prediction ability. SPSS.IBM statistical software (IBM, Armonk, NY, USA) was used to analyze the significant differences of compounds (VIP >1) in 15 natural spices by Duncan test for one-way ANOVA. Under the condition that ANOVA p<0.05, the difference has statistical significance. The symbolic differentiated compounds were screened out as characteristic components of the various natural spices according to standards of VIP>1 and ANOVA p<0.05 to obtain the characteristic component database of the natural spices. In order to realize the visualization of the database, the characteristic components with statistical significance were subjected to hierarchical clustering analysis of heat map using a Lianchuan biological cloud platform, and the different components in different groups of natural spice samples were subjected to visualized analysis.

[0072] (4) An aqueous solution of a functional flavoring base to be analyzed having a mass concentration of 75% was treated according to the process for treating aqueous solutions of extracts of natural spices in steps (1) to (3) to obtain characteristic components of the functional flavoring base, wherein the functional flavoring base to be analyzed was flavoring base A provided by China Tobacco Hunan Industrial Co., Ltd. (Changsha, China), which was composed of wolfberry, plum, raisin and fig in a volume ratio of 10: 10: 10: 10. The functional flavoring base was modeled and analyzed with the characteristic component database of the natural spices to obtain an OPLS-DA score plot. Taking squared distances between the characteristic components of the natural spices and the characteristic components of the functional flavoring base in the OPLS-DA score plot as a judgement basis, natural spices were ranked in order from small to large according to the squared distances, and the corresponding top eight natural spices were taken, and made is a judgement that the functional flavoring base to be analyzed contains one or more of the eight natural spices.

[0073] Example!

[0074] This example differed from Example 1 in that: in step (4), the aqueous solution of functional flavoring base to be analyzed was flavoring base B provided by China Tobacco Hunan Industrial Co., Ltd. (Changsha, China), which was composed of dandelion, tamarind, chicory, carob bean, raisin and malt in a volume ratio of 10: 5: 10: 5: 15: 15.

[0075] Example 3

[0076] This example differed from Example 1 in that: in step (4), the aqueous solution of functional flavoring base to be analyzed was flavoring base C provided by China Tobacco Hunan Industrial Co., Ltd. (Changsha, China), which was composed of Roman chamomile, clotrimazole, jujube tincture, angelica, alfalfa, plum, fig, and valerin tincture in a volume ratio of 15: 5: 5: 8: 12: 8: 8: 5.

[0077] Example 4

[0078] This example differed from Example 1 in that: in step (4), the aqueous solution of functional flavoring base to be analyzed was flavoring base D provided by China Tobacco Hunan Industrial Co., Ltd. (Changsha, China), which was composed of Roman chamomile, chicory, wolfberry, fig, angelica, carob bean, alfalfa, clotrimazole, malt and dandelion in a volume ratio of 12: 8: 5: 5: 12: 10: 10:5:8: 5.

[0079] Example 5

[0080] This example differed from Example 1 in that: in step (4), the concentration of the aqueous solution of functional flavoring base to be analyzed was 90%, and the remaining steps were the same as in Example 1.

[0081] Example 6

[0082] This example differed from Example 2 in that: in step (4), the concentration of the aqueous solution of functional flavoring base to be analyzed was 90%, and the remaining steps were the same as in Example 2.

[0083] Example 7

[0084] This example differed from Example 3 in that: in step (4), the concentration of the aqueous solution of functional flavoring base to be analyzed was 90%, and the remaining steps were the same as in Example 3.

[0085] Example 8

[0086] This example differed from Example 4 in that: in step (4), the concentration of the aqueous solution of functional flavoring base to be analyzed was 90%, and the remaining steps were the same as in Example 4.

[0087] Example 9

[0088] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, each gradient extraction was conducted for 10 min, and the remaining steps were the same as in Example 1.

[0089] Example 10

[0090] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, each gradient extraction was conducted for 20 min, and the remaining steps were the same as in Example 1.

[0091] Example 11

[0092] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, each gradient extraction was conducted for 40 min, and the remaining steps were the same as in Example 1.

[0093] Example 12

[0094] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, each gradient extraction was conducted for 50 min, and the remaining steps were the same as in Example 1.

[0095] Example 13

[0096] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, O / W of each gradient extraction was 3: 1, and the remaining steps were the same as in Example 1.

[0097] Example 14

[0098] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, O / W of each gradient extraction was 2: 1, and the remaining steps were the same as in Example 1.

[0099] Example 15

[0100] The subject of the extraction was chicory extract. This example differed from Example 1 in that: during the polar gradient extractions, O / W of each gradient extraction was 1: 2, and the remaining steps were the same as in Example 1.

[0101] Comparative Example 1

[0102] A chicory extract was used as a subject and subjected to extraction by single-stage liquidliquid extraction (SLLE) with n-hexane, methyl tert-butyl ether, 1,2-dichloroethane, n-butyl acetate, and n-butyl alcohol.

[0103] The extraction was conducted by a process as follows: n-hexane and an aqueous solution of chicory extract O / W (18: 18, v: v) was taken, completely oscillated for 30 min in a constant temperature oscillator with a water bath at 5 °C, and subjected to extraction to obtain organic phases and aqueous phases. According to the same process, methyl tert-butyl ether, 1,2-dichloroethane, n-butyl acetate, and n-butyl alcohol were used for SLLE on the chicory extract to obtain organic phases and aqueous phases. The obtained organic phases and aqueous phases were dried according to the process of Example 1. Then resulting dried products were subjected to qualitative analysis by GC-MS.

[0104] Test Example

[0105] (1) FIG. 2A to FIG. 2B are graphs showing the results of the influence of different extraction processes on the extraction effect according to an embodiment of the present disclosure. The total ion current chromatograms of the organic phases obtained after the polar gradient extractions of the aqueous solution of the chicory extract in Example 1 is shown in FIG. 2A. The effects of the extraction agents and the extraction gradient settings regarding the extractions on the aqueous solution of the chicory extract in Example 1 and Comparative Example 1 are shown in FIG. 2B. 97 substances were detected in the organic phases by the process of Example 1, 70 substances were detected in the organic phases by the process of Comparative Example 1, and the unique chemical components detected by the process provided by Example 1 were more than those detected by the SLLE process of Comparative Example 1. As can also be seen from FIG. 2B, except that the number of the chemical components detected by polar gradient extraction in n-hexane phase is slightly lower than that of chemical components detected by SLLE and that the number of the unique chemical components detected by them is equal, the number of the chemical components obtained by polar gradient extractions in other organic solvents and the number of the unique chemical components detected in this solvent is greater than or equal to that of SLLE process. The number of the unique chemical substances detected in the solvents can indicate the necessity of the presence of each selected solvent. Compared with SLLE, polar gradient extractions could result in more substances being extracted, which may be because that the rich chemical components and the great content differences thereof in natural spices, and that different substances have different distribution behaviors in different organic solvents. When the content of substances with large partition coefficient is reduced by gradient extractions and is lower than the extraction capacity of organic solvents, substances with small partition coefficient could be extracted into organic solvents for enrichment. Therefore, gradient extraction on natural spices with complex systems with multiple polar solvents would have better effects than traditional single-stage extraction, which could provide a rich data basis for the analysis of functional flavoring base.

[0106] (2) FIG. 3 is a graph showing the results of the influence of the extraction time on the extraction efficiency of polar extractions according to an embodiment of the present disclosure. That is, the influence of the extraction time on the number of substances of the chicory in the extracted organic phases and the raffinate aqueous phase derivatization as well as the peak area after the polar gradient extractions on the aqueous solution of chicory extract in Examples 1 and 9-12. As can be seen from FIG. 3, under the condition that the extraction time is 10-50 min, the extraction effects are good, but under the condition that the extraction time is 30 min, the number of substances in the organic phases is slightly lower than those at other time points, while the number of substances in the aqueous phase derivatization as well as the peak area of the organic phases and the aqueous phase derivatization samples are the maximum, possibly because the extraction equilibrium is not reached under the condition that the extraction time is less than 30 min, but some heat-sensitive or easily-oxidized components are degraded or oxidized under the condition that the extraction time is more than 30 min. Therefore, 30 min is chosen as the best liquid-liquid extraction time for natural spices.

[0107] (3) FIG. 4 is a graph showing the results of the influence of O / W on the extraction effect under the condition that the aqueous solution of chicory extract is subjected to polar gradient extractions in Examples 1 and 13-15, i.e., the influence of O / W on the number of substances of the chicory in the extracted organic phases and the raffinate aqueous phase derivatization, as well as the peak area. As can be seen from FIG. 4, during the polar gradient extractions, with the O / W ratio from 3: 1 (v / v) to 1: 2 (v / v), the number of substances in the extracted organic phases of the chicory shows a process of first decreasing and then increasing, and the peak area of the extracted organic phases shows an effect of first increasing and then slightly decreasing due to the addition of more aqueous phase. Under the condition that the O / W ratio is 1: 2 (v / v), a reason for the decrease of the peak area of the organic phases may be due to the serious emulsification during the experiment. With the increase of ratio, the peak area of aqueous phase derivatization sample of the chicory shows a gradual increase trend. Under the condition that the O / W ratio is 1: 1 (v:v), and the number of substances in the aqueous phase derivatization sample is the largest; and under the condition that the O / W ratio is 1: 2 (v / v), the number of substances decreases slightly. A possible reason for this is that under the condition that the O / W ratio is 1: 2, the organic solvents reach the extraction capacity so that many substances are remained in the aqueous phase from the last extraction, which leads to an increase in the concentration of substances in the derivatization samples and masks some trace chemical components. Considering the experimental results and the simple and efficient experimental process comprehensively, O / W=l: 1 (v / v) is selected as the best extraction phase ratio condition for natural spices.

[0108] (4) The detection results of step (2) of Example 1 show that 143 substances are detected in the chicory extract in the polar gradient extraction system, among which 96 substances are in the organic phase samples and 47 substances are in the aqueous phase derivatization samples. 30 substances are detected in the n-hexane phase, 55 substances are detected in the methyl tert-butyl ether phase, 33 substances are detected in the 1,2-di chloroethane phase, 28 substances are detected in the n-butyl acetate phase, 16 substances are detected in the n-butanol phase, and there is an overlapping phenomenon of substances in each extraction phase. The components detected in the polar gradient extraction of the chicory are classified according to functional groups as hydrocarbons, ethers, esters, carbonyl groups (including aldehydes and ketones), amines, alcohols, phenols, acids, and others. The results of the number and content of substances detected in the different extracted organic phases and raffinate aqueous phase derivatization samples show that most of the substances are esters, alcohols, acids, and saccharides, and saccharides and alcohols account for 65.55% of the relative content of the aqueous phase derivatization samples. The polar gradient extraction system provided by the present disclosure could reduce the masking effect of high-content substances on trace substances so that more chemical component information in the spices could be obtained for subsequent characteristic component search, and the traceability research of the functional flavoring base could be conducted through the established characteristic component database.

[0109] (5) Chemical composition analysis of the 15 natural spices in Example 1

[0110] FIG. 5A and FIG. 5B are graphs showing the distribution of the relative contents of different kinds of chemical components and the distribution of the total number of substances detected by the 15 natural spices in the organic phases and the aqueous phase derivatization. Combining the two figures, it can be seen that hydrocarbons, carbonyls, and phenol compounds are almost all detected in the extracted organic phases, whereas alcohols and saccharides are mostly detected in the aqueous phase derivatization samples. The information about the chemical components obtained from the dandelion and wolfberry in the organic phases is the most, whereas the information about the chemical components obtained from the aqueous phase derivatization has little difference, except for slightly less information for tamarind. The possible reason for this is that in the raffinate aqueous phase itself left are high-polarity and high-boiling-point chemicals, mainly amino acids, organic acids, and sugars. The aim of derivatization is to change specific functional groups to obtain the target products, so the substances obtained by the same derivatization solution have little difference. In the chicory, the carob bean, the tamarind, and the dandelion, amines are hardly detected; in the raisin, the fig, and the malt, the contents of acid substances are smaller; the dandelion contains more acid substances; and in the carob bean, the sugar content is higher.

[0111] (6) A PCA model was established based on the qualitative and quantitative results of the 15 natural spices in Example 1. The 2D scatter plots of PCI and PC2 scores explain 69.73% of the total square deviation (R2X = 0.642, Q2 = 0.0553) together. Using the qualitative and quantitative analysis data of 45 samples of the 15 natural spices obtained in Example 1, a data matrix is constructed by using squared Euclidean distance. The 45 samples of natural spices (i.e., 15 natural spices, three paralleling experiments being repeated for each spice) were subjected to HCA with the square sum of deviations. The HCA results show that the 45 samples could be divided into 15 categories within a certain distance, which is consistent with the PCA results.

[0112] (7) Extraction and identification of the characteristic components of the 15 natural spices in Example 1

[0113] The regression model parameters of the 15 natural spices are shown in Table 1, and all of them have good fitting parameters to show the accuracy of the model (R2Y-Q2<0.3, R2>0.73 and Q2>0.73). Variable Importance Factor (VIP) could quantify the contribution of each variable to classification. It is generally believed that the greater the VIP value, the greater the contribution rate of the corresponding characteristic variable. In the regression model provided by the present disclosure established by the 15 natural spices, 203 substances with VIP>1 and AVOVA p<0.05 are screened out to establish the characteristic component database of the 15 natural spices, and the specific characteristic component classification information is shown in Table 2. The existence of these characteristic components endows the natural spices with special smell and efficacy, which makes differences among the natural spices. The identification of these different compounds is beneficial to the subsequent traceability research of the formula of the functional flavoring base according to the present disclosure.

[0114] Table 1 Regression model parameters established for the 15 natural spices in Example 1 Natural spice Scaling type Model R2X R2X Q2 Chicory Par OPLS-DA 0.336 0.987 0.832 Carob bean Par OPLS-DA 0.443 0.997 0.91 Tamarind Par OPLS-DA 0.398 0.996 0.933 Alfalfa Par OPLS-DA 0.284 0.975 0.876 Angelica Par OPLS-DA 0.263 0.984 0.883 Raisin UV PLS-DA 0.15 0.983 0.736 Fig UV PLS-DA 0.197 0.987 0.734 Plum Par OPLS-DA 0.397 0.995 0.871 Roman chamomile Par OPLS-DA 0.286 0.996 0.953 Wolfberry UV PLS-DA 0.107 0.982 0.845 Dandelion Par OPLS-DA 0.258 0.995 0.977 Malt UV PLS-DA 0.165 0.995 0.912 Jujube tincture Par OPLS-DA 0.383 0.997 0.896 Clotrimazole Par OPLS-DA 0.277 0.995 0.958 Valerin tincture Par OPLS-DA 0.216 0.991 0.952

[0115] Table 2 Classification of the characteristic component data of the 15 natural spices in Example 1 Natural spice Hydrocarbons Ethers Ethers Carbonyl group Amines Alcohols Phenols Acids Saccharides Others Chicory 1 1 4 2 - 2 - 1 1 - Carob bean 1 - 1 1 - 3 - 4 - 1 Tamarind 1 - 5 3 - - - 3 - - Alfalfa 1 - 3 1 1 2 1 5 3 - Angelica - - 6 4 - 4 1 2 - 2 Raisin 3 - - - 1 1 - - - - Fig 8 - 4 - - - 1 1 1 1 Plum 1 - 2 - - 1 - 1 1 - Roman chamomile - 3 5 3 2 3 2 2 Wolfberry 3 - 1 1 - - - 2 1 2 Dandelion 1 - - 3 - 4 2 8 - - Malt 9 - 5 1 2 2 - 1 1 2 Jujube tincture - 3 1 - - 2 2 - Clotrimazole - - 3 1 - 1 - 2 - 1 Valerin tincture - - 5 1 2 5 - 3 1 1

[0116] (8) Analysis results of the four functional flavoring components in Examples 1 to 8

[0117] From the qualitative and quantitative analysis by GC-MS, it can be known that the functional flavoring base A having mass concentrations of 75% and 90% match 133 substances and 166 substances, respectively. The functional flavoring base B having mass concentrations of 75% and 90% match 172 substances and 141 substances, respectively. The numbers the substances matched with the functional flavoring base C having mass concentrations of 75% and 90% are 186 and 215, respectively. The numbers the substances matched with the functional flavoring base D having mass concentrations of 75% and 90% are 203 and 143, respectively. As the flavoring base per se is made up of different spices in different proportions, the mass concentration to achieve the best extraction state is inconsistent. The stoichiometry pattern identification method could capture a large amount of trace component information, thereby eliminating the interference caused by concentration change, and successfully tracing the source of flavoring base formula.

[0118] (9) FIG. 6A to FIG. 6D show the results of the multivariate statistical analysis between the characteristic components of the four functional flavoring bases in Examples 1 to 4 and the characteristic component databases (VIP>1 and ANOVA p<0.05) screened out from the 15 natural spices. Based on the company standard, if the accuracy rate of the first eight natural spices in a formula of a functional flavoring base analyzed reaches 70% or higher, the traceability of the flavoring base formula is considered to be successful. The OPLS-DA (UV) score plot established between the flavoring base A and the characteristic component data is shown in FIG. 6A, i.e., R2X=O.87, R2Y=0.976, and Q2=0.923. It shows that the regression model has good fitting degree and credibility. In the OPLS-DA score plot of the flavoring base A, the data are analyzed around the flavoring base A, and taking the squared distance between the natural spice and the flavoring base A as the judgment basis, the closer distance indicates the higher the possibility of containing this natural spice in the flavoring base A. The specific existence possibility relationship is as follows: chicory >tamarind> carob bean >jujube tincture >wolfberry >angelica >plum >fig >raisin >clotrimazole >Roman chamomile >malt >dandelion >valerin tincture >alfalfa. Compared with the correct formula, the accuracy rate of the prediction result of the flavoring base A is 75%. The OPLS-DA (UV) score plot of the flavoring base B and the characteristic components is shown in FIG. 6B, i.e., R2X=0.869, R2Y=0.973, and Q2=0.919. In the same way, it is concluded that the existence possibility relationship between the flavoring base B and the characteristic components is as follows: dandelion >tamarind> chicory >wolfberry >jujube tincture >carob bean >raisin >plum >clotrimazole >fig >alfalfa >angelica >Roman chamomile >malt >valerin tincture. Compared with the correct formula, the accuracy rate of the prediction result of the flavoring base B is 83.3%. The OPLS-DA (Par) score plot of the flavoring base C and the characteristic components is shown in FIG. 6C, i.e., R2X=0.904, R2Y=0.958, and Q2=0.884. According to the squared distance relationship, it is concluded that the order of the existence possibility of the characteristic components in the flavoring base C is as follows: Roman chamomile >clotrimazole >chicory >jujube tincture >wolfberry >angelica >alfalfa >plum: carob bean >fig >tamarind> raisin >malt >valerin tincture >dandelion. Compared with the correct formula, the accuracy rate of the prediction result of the flavoring base C is 75%. The OPLS-DA (Par) score plot of the flavoring base D and the characteristic components is shown in FIG. 6D, i.e., R2X=0.921, R2Y=0.959, and Q2=0.9. The order of the existence possibility of spices is as follows: Roman chamomile >chicory >wolfberry >fig >jujube tincture >angelica >carob bean >alfalfa >clotrimazole >tamarind >raisin >plum >malt >dandelion >valerin tincture. Compared with the correct formula, the accuracy rate of the prediction result of the flavoring base D is 87.5%. The traceability research of the four functional flavoring bases all reach the company recognition standards, which proves that the present disclosure achieves excellent results in the identification of the formula of the functional flavoring bases. As can be seen from the above experimental results, the method provided by the present disclosure could accurately analyze a formula of a functional flavoring base.

[0119] The above are merely preferred embodiments of the present disclosure. It should be noted that several improvements and modifications may further be made by a person of ordinary skill in the art without departing from the principle of the present disclosure, and such improvements and modifications should also be deemed as falling within the scope of the present disclosure.

Claims

1. A method for analyzing a formula of a functional flavoring base, comprising:(1) separately subjecting aqueous solutions of extracts of a plurality of natural spices to polar gradient extractions, to obtain organic phases from each gradient extraction of an aqueous solution of an extract of each natural spice and an aqueous phase from last extraction; wherein the polar gradient extractions are conducted in order from small to large sequentially according to polarity parameters of organic solvents used, and the organic solvents each have a polarity parameter of 0-4;(2) separately drying the organic phases from each gradient extraction of the aqueous solution of the extract of each natural spice and the aqueous phase obtained in step (1) and then separately subjecting resulting dried products to qualitative analysis by gas chromatography-mass spectrometry (GC-MS), to obtain a pretreatment data; wherein the pretreatment data comprises relative percentage contents of extracted components in the organic phases from each gradient extraction and the aqueous phase;(3) subjecting the pretreatment data obtained in step (2) to stoichiometric pattern identification analysis with a supervised mode, establishing a partial least squares discriminant analysis (PLS-DA) equal regression model or an orthogonal partial least squares discrimination analysis (OPLS-DA) equal regression model, and screening out symbolic differentiated compounds as characteristic components of the natural spices according to standards of variable importance factor (VIP) >1 and analysis of variance (ANOVA) p<0.05 to obtain a characteristic component database of the natural spices; and(4) treating an aqueous solution of a functional flavoring base to be analyzed in accordance with processes for treating the aqueous solutions of extracts of natural spices in steps (1) to (3) to obtain characteristic components of the functional flavoring base, modeling and analyzing the characteristic components of the functional flavoring base and the characteristic component database of the natural spices obtained in step (3) to obtain a PLS-DA score plot or an OPLS-DA score plot; taking squared distances between the characteristic components of the functional flavoring base and the characteristic components of the natural spices in the PLS-DA score plot or OPLS-DA score plot as a judgement basis, ranking the nature spices in order from small to large according to the squared distances, taking top eight natural spices, and making a judgement that the functional flavoring base to be analyzed contains one or more of the eight natural spices.

2. The method of claim 1, wherein in step (1), the polar gradient extractions are conducted with 3-5 gradients.

3. The method of claim 1 or 2, wherein the organic solvents comprise at least three selected from the group consisting of n-hexane, methyl tert-butyl ether, 1,2-dichloroethane, n-butyl acetate, and n-butyl alcohol.

4. The method of any preceding claim, wherein in step (1), natural spice extracts in the aqueous solutions of extracts of natural spices each comprise one selected from the group consisting of chicory extract, carob bean extract, tamarind extract, alfalfa extract, angelica extract, raisin extract, fig extract, plum extract, Roman chamomile extract, wolfberry concrete, dandelion concrete, malt concrete, jujube tincture, clotrimazole, and valerin tincture.

5. The method of any preceding claim, wherein in step (1), the aqueous solutions of extracts of natural spices each have a mass concentration of 5-20%.

6. The method of any preceding claim, wherein in step (1), during the polar gradient extractions, a volume ratio of an organic solvent for each gradient extraction to an aqueous phase to be extracted is in a range of 3: 1 to 1: 2.

7. The method of any preceding claim, wherein in step (1), during the polar gradient extractions, each gradient extraction is conducted for 10-50 min.

8. The method of any preceding claim, wherein in step (2), after drying the aqueous phase, a resulting dried product is subjected to derivatization treatment and the qualitative analysis by GC-MS sequentially.

9. The method of claim 8, wherein a derivatization reagent for the derivatization treatment is a mixed solution of bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS), and a volume percentage of the TMCS relative to the BSTFA is in a range of 1-10%.

10. The method of any preceding claim, wherein in step (2), parameters for the qualitative analysis by GC-MS comprise:chromatographic parameters comprising: a chromatographic column of SH-Rxi-5MS capillary gas chromatography column with a specification of 30 m x 0.25 mm and 0.25 pm; a high-purity helium as a carrier gas with a flow rate of 1-3 mL / min; adopting a split-flow mode, a split-flow ratio of an organic phase sample being 10: 1, and a split-flow ratio of an aqueous phase sample being 15: 1; a sample inlet temperature being in a range of 200-300 °C, a sample volume of the organic phasesbeing 1 pL, and a sample volume of the aqueous phase being 0.2 pL; and a temperature procedure being set as: keeping an initial temperature at 40 °C for 2 min, then raising to 300 °C at a rate of 6 °C / min, and holding at 300 °C for 20 min; andmass spectrometry conditions comprising: an ion source temperature being in a range of 200-230 °C; an ionization voltage being 70 eV; and adopting a full scanning mode, a scanning range of an organic phase being in a range of 33 amu to 550 amu, and a scanning range of an aqueous phase sample being in a range of 33 amu to 1050 amu.

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