Cocoa flavor tobacco flavor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cigarette perfuming, in particular to a cocoa flavor tobacco flavor and a preparation method and application thereof. The preparation method comprises the following steps: diluting a cocoa extract with an ethanol aqueous solution, carrying out multi-stage membrane separation according to the principle that the pore diameters of membranes are sequentially reduced, and carrying out reverse osmosis treatment on a permeate of the last stage of membrane separation to obtain a reverse osmosis trapped fluid; collecting trapped fluid of each stage to obtain a plurality of first separation groups; performing two-dimensional column chromatography separation on the reverse osmosis trapped fluid to obtain a plurality of second separation groups; the yield of each separation group is calculated, and the separation groups with the yield larger than or equal to 2% are reserved; adding the retained separated group into blank cigarettes for sensory evaluation; screening out a characteristic flavor group; performing thermal cracking-gas chromatography-mass spectrometry analysis on the characteristic flavor group, and constructing characteristic flavor data information; and compounding the characteristic flavor group with the characteristic flavor substance to obtain the cocoa flavor tobacco flavor. According to the method, endogenous flavor enhancement can be realized, and uncontrollable factors and ineffective components of the natural perfume are reduced to the greatest extent while the style characteristics of the natural perfume are highlighted.

Description

Technical Field

[0001] This invention relates to the field of cigarette flavoring technology, and in particular to a cocoa-flavored tobacco flavoring, its preparation method, and its application. Background Technology

[0002] Flavorings are the foundation of cigarette flavoring and a core element in cigarette product development. Natural flavorings have complex compositions. On the one hand, their rich composition can endow and enhance the aroma and style of cigarettes. On the other hand, the presence of some negative components or suboptimal proportions of characteristic components can often adversely affect the sensory quality of cigarettes or lead to inaccurate and inefficient flavoring and product design.

[0003] Cocoa extracts, used in cigarette flavoring, enrich the smoky aroma, increase smoke concentration, impart cocoa notes, and enhance roasted and sweet aromas. However, cocoa extracts obtained through conventional solvent extraction and concentration have complex internal compositions, which may lead to undesirable effects such as astringency, irritation, and residue when applied to cigarette products, or introduce unwanted flavor characteristics such as burnt or bitter tastes. Refined processing technologies such as multi-stage solvent extraction, supercritical fluid extraction, and membrane separation have been explored for the preparation of specialized cocoa extracts, highlighting the beneficial functions of cocoa and reducing undesirable effects. However, the functions of cocoa extracts are quite complex; functions such as increasing smoke concentration, sweetening, imparting cocoa and roasted aromas originate from different material bases. Single refined processing technologies often result in the loss of other functions while acquiring some flavoring properties. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this invention provides a cocoa-flavored tobacco flavoring, its preparation method, and its application.

[0005] This invention provides a method for preparing cocoa-flavored tobacco flavoring, comprising:

[0006] Step 1) After diluting the cocoa extract with an ethanol-water solution, multi-stage membrane separation is performed according to the principle of decreasing membrane pore size. The permeate from the last stage of membrane separation is subjected to reverse osmosis treatment to obtain reverse osmosis retentate. Each stage retentate is collected to obtain several first separation groups.

[0007] Step 2) The reverse osmosis retentate is separated by two-dimensional column chromatography. The first-dimensional column is packed with dextran gel and the second-dimensional column is packed with reversed-phase silica gel to obtain several second separation groups.

[0008] Step 3) Calculate the yield of each segregating group and retain the segregating groups with a yield ≥ 2%; the yield is calculated according to Formula I:

[0009] y=a / A×100% Formula I

[0010] In Formula I, y is the yield, a is the dry matter mass of a certain separation group, and A is the dry matter mass of the cocoa extract; the dry matter mass is the mass after removing organic solvents and water.

[0011] Step 4) Add the separated groups retained in Step 3) to blank cigarettes with the same dry weight for sensory evaluation; use smoke characteristics, comfort characteristics, taste style and aroma style as evaluation indicators to screen out the distinctive flavor groups.

[0012] Step 5) Perform pyrolysis-gas chromatography-mass spectrometry analysis on the characteristic flavor groups to construct characteristic flavor data information, which includes: the names of characteristic flavor substances after pyrolysis, and the corresponding aroma characteristics.

[0013] Step 6) Combine the aforementioned flavor groups with characteristic flavor substances to obtain cocoa-flavored tobacco flavoring.

[0014] Further, in step 1), the multi-stage membrane separation specifically involves:

[0015] Multi-stage membrane separation is performed sequentially through a 50nm ceramic membrane, a 50kDa spiral-wound organic membrane, a 5nm ceramic membrane, and a 1kDa spiral-wound organic membrane; or,

[0016] Separation is achieved sequentially through a 200nm ceramic membrane, a 50nm ceramic membrane, and a 2nm ceramic membrane.

[0017] Furthermore, in step 2), the ultraviolet detection wavelengths of the first-dimensional chromatographic column and the second-dimensional chromatographic column are 245 nm and 326 nm, respectively.

[0018] Further, step 2) specifically includes:

[0019] The stationary phase of the first-dimensional chromatographic column is dextran gel packing, and the mobile phase is distilled water or an aqueous ethanol solution with a volume concentration not exceeding 10%. According to the ultraviolet detection wavelength signal, the eluent at different time periods is introduced into different trapping columns, and the stationary phase of the trapping column is reversed-phase silica gel packing.

[0020] The effluent from each collection column was collected separately to obtain several second separation groups B1;

[0021] After collection is complete, gradient elution is performed on the collection column using an ethanol-water solution as the mobile phase.

[0022] The stationary phase of the second-dimensional chromatographic column is reversed-phase silica gel packing, and the eluent of the trapping column is used as the mobile phase. The eluent is collected according to the ultraviolet detection wavelength signal, and the eluents with similar absorption peaks and the continuous eluents with low yields are combined to obtain several second separation groups B2.

[0023] Step 2) yields several second segregation groups, including several second segregation groups B1 and several second segregation groups B2.

[0024] Furthermore, in the gradient elution, the initial volume concentration of the ethanol aqueous solution is 10%, and the final volume concentration is 90-95%.

[0025] Further, in step 4), the smoke characteristics include: aroma, richness, smoothness and roundness, impurities, smoke concentration and strength; the comfort characteristics include: oral irritation / tongue burning, oral residue / dryness, astringency, throat irritation, throat dryness and nasal irritation; the flavor profile includes: sweetness, sourness, bitterness and coolness; the aroma profile includes: flue-cured tobacco aroma, fresh aroma, fruity aroma, spicy aroma, woody aroma, bean aroma, cocoa aroma, milky aroma, paste aroma, roasted aroma and sweet aroma.

[0026] Furthermore, in step 5), the characteristic flavor data information also includes the content of characteristic flavor substances after pyrolysis in each characteristic flavor group.

[0027] Furthermore, in step 5), the analytical conditions for the pyrolysis-gas chromatography-mass spectrometry analysis are as follows:

[0028] Pyrolysis conditions:

[0029] Pyrolysis temperature: 900℃; pyrolysis time: 0.2 min; connection temperature: 280℃; sample addition amount: 1 μL;

[0030] GC-MS conditions:

[0031] Chromatographic column: DB-5MS capillary column, 30m × 0.25mm × 0.25μm;

[0032] Carrier gas: He; Flow rate: 1.0 mL / min; Split ratio: 50:1;

[0033] Temperature program: 40℃, hold for 3 minutes; increase to 240℃ at a rate of 10℃ / min; increase to 280℃ at a rate of 20℃ / min, hold for 15 minutes;

[0034] GC-MS transfer line temperature: 280℃; ionization mode: EI; ion source temperature: 230℃; quadrupole temperature: 150℃; ionization energy: 70eV; solvent delay time: 4.5min; mass scan range: 29-450a.mu.

[0035] The present invention also provides a cocoa-flavored tobacco flavoring, which is prepared according to any one of the methods described above.

[0036] The present invention also provides the application of the above-mentioned method for preparing cocoa-flavored tobacco flavoring in cigarette flavoring.

[0037] The method for preparing cocoa-flavored tobacco flavoring provided by this invention can include the following beneficial effects:

[0038] 1. This invention utilizes multi-stage membrane separation and two-dimensional column chromatography to screen and obtain a variety of distinctive cocoa flavor groups with different sensory characteristics. This highlights the diverse functions of cocoa extracts for tobacco use, enriches the product categories of cocoa extracts, and increases the flexibility and precision of using cigarette flavorings. The combined application of multi-stage membrane separation and two-dimensional column chromatography technologies offers complementary advantages, and the separation process involves almost no thermal, chemical, or biological changes, preserving the natural characteristics of the flavorings. Both dextran gel and reversed-phase silica gel can be reused multiple times, and efficient production of two-dimensional column-separated flavor groups can be achieved using production-grade automated two-dimensional medium-pressure column chromatography equipment.

[0039] 2. Natural flavorings in tobacco products can only fully exert their flavor-regulating effects through combustion. This method uses pyrolysis-gas chromatography-mass spectrometry (GC-MS) to further analyze the characteristic flavor compounds that have a substantial impact on cigarette smoke after being added. Finally, the characteristic flavor compounds and characteristic flavor groups are compounded to achieve endogenous flavor enhancement. This method highlights the style characteristics of natural flavorings while minimizing uncontrollable factors and ineffective components, thereby increasing the quality stability and controllability of refined natural flavoring products. It has practical application value.

[0040] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0041] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same parts.

[0042] Figure 1 These are ultraviolet chromatograms of two-dimensional column chromatography separation in Example 1 of this invention; in the figure, a is an ultraviolet chromatogram of one-dimensional column chromatography separation; b is an ultraviolet chromatogram of second-dimensional column chromatography separation of component 1; c is an ultraviolet chromatogram of second-dimensional column chromatography separation of component 2; d is an ultraviolet chromatogram of second-dimensional column chromatography separation of component 3; and e is an ultraviolet chromatogram of second-dimensional column chromatography separation of component 4.

[0043] Figure 2 The results show the sensory evaluation of the compound product and the original cocoa-extracted cigarette in Example 1.

[0044] Figure 3The results show the sensory evaluation of the compound product and the original cocoa-extracted cigarette in Example 2. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0047] It should be understood that although the terms "first," "second," "third," etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this invention, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0048] To address the aforementioned issues, the inventors of this application propose to fully highlight the various functional characteristics of natural flavorings while removing impurities, thereby obtaining a cocoa flavor group with multiple functional focuses, improving the functional focus and product diversification of homologous natural flavorings, and providing a richer and more effective supply of flavoring raw materials for precision flavoring and digital product design.

[0049] The inventors of this application considered that the characteristics of natural flavorings for tobacco products determine that they consist of two parts: indirect flavor-contributing components (contributing flavor after combustion) and direct flavor-contributing components (transferred from the original form to the smoke). Membrane separation technology can segment the components contained in flavorings within the range of 0.0001–100 μm, separating indirect flavor-contributing components (flavor precursors), such as polysaccharides, pigments, and long-chain acids, and separating and enriching direct flavor-contributing components. Furthermore, pressure-driven membrane separation processes are purely physical separations, involving almost no thermal, chemical, or biological changes; simultaneously, membrane separation equipment is constructed in modular form, adaptable to different production capacities, and suitable for large-scale production. Preparative column chromatography technology features mild separation conditions and high resolution, enabling the separation of direct flavor-contributing components in different functional regions of natural flavorings, and is an important supplement to the fine processing technology of natural flavorings. The combined use of membrane separation and preparative column chromatography technologies offers complementary advantages and can be flexibly applied to the refined production of natural flavorings for tobacco products.

[0050] Based on the above inventive concept, embodiments of the present invention provide a method for preparing cocoa-flavored tobacco flavoring, which includes the following steps:

[0051] Step 1) After diluting the cocoa extract with an ethanol-water solution, multi-stage membrane separation is performed according to the principle of decreasing membrane pore size. The permeate from the last stage of membrane separation is subjected to reverse osmosis treatment to obtain reverse osmosis retentate. Each stage retentate is collected to obtain several first separation groups.

[0052] Step 2) The reverse osmosis retentate is separated by two-dimensional column chromatography. The first-dimensional column is packed with dextran gel and the second-dimensional column is packed with reversed-phase silica gel to obtain several second separation groups.

[0053] Step 3) Calculate the yield of each segregating group and retain the groups with a yield ≥ 2%; the yield is calculated according to Formula I:

[0054] y=a / A×100% Formula I

[0055] In Formula I, y is the yield, a is the dry matter mass of a certain group, and A is the dry matter mass of the cocoa extract; the dry matter mass is the mass after removing organic solvents and water.

[0056] Step 4) Add the groups retained in Step 3) to blank cigarettes with the same dry weight for sensory evaluation; use smoke characteristics, comfort characteristics, taste style and aroma style as evaluation indicators to screen out distinctive flavor groups;

[0057] Step 5) Perform pyrolysis-gas chromatography-mass spectrometry analysis on the characteristic flavor groups to construct characteristic flavor data information, which includes: the names of characteristic flavor substances after pyrolysis, and the corresponding aroma characteristics.

[0058] Step 6) Combine the aforementioned flavor groups with characteristic flavor substances to obtain cocoa-flavored tobacco flavoring.

[0059] Step 1) above is the step of separating the cocoa extract in stages. First, the cocoa extract needs to be diluted with an ethanol-water solution. For the concentration of the ethanol solution, the solubility of the cocoa extract in ethanol can be evaluated using the YC / T 145.4-1998 method. Based on the results, an appropriate volume fraction of ethanol-water solution is selected to dilute the cocoa extract. After dilution, it is preferable to also include a step of centrifugation or filtration to remove precipitates, so as to facilitate subsequent separation operations.

[0060] In this step, the separation membranes used can be microfiltration, ultrafiltration, or nanofiltration membranes for multi-stage separation. Different pore sizes of inorganic or organic membranes can be used, following the principle of decreasing pore size in each stage. The permeate from each stage is used as the separation target for the next stage. Finally, reverse osmosis is used to concentrate the permeate from the last stage, and the reverse osmosis retentate is collected. The retentates obtained from each stage of separation (including the reverse osmosis retentate) are collected as the first separation group.

[0061] As the preferred method for this step, multi-stage membrane separation specifically involves:

[0062] Multi-stage membrane separation is performed sequentially through a 50nm ceramic membrane, a 50kDa spiral-wound organic membrane, a 5nm ceramic membrane, and a 1kDa spiral-wound organic membrane; or,

[0063] Separation is achieved sequentially through a 200nm ceramic membrane, a 50nm ceramic membrane, and a 2nm ceramic membrane.

[0064] Step 2) involves further separating the reverse osmosis retentate using two-dimensional column chromatography, thereby refining it into more distinct groups. Steps 1) and 2) allow for the screening of various cocoa flavor groups with distinct sensory characteristics. Two-dimensional column chromatography (2D-LC) is a highly efficient separation method that combines two different chromatographic techniques. Orthogonal separation significantly improves the resolution of complex mixtures. In this embodiment, the first-dimensional column uses dextran gel packing, and the second-dimensional column uses reversed-phase silica gel packing, preferably C18 bonded silica gel. Preferably, the UV detection wavelengths for the first and second-dimensional columns are 245 nm and 326 nm, respectively.

[0065] Furthermore, step 2) can be specifically as follows:

[0066] The stationary phase of the first-dimensional chromatographic column is dextran gel packing, and the mobile phase is distilled water or an aqueous ethanol solution with a volume concentration not exceeding 10%. According to the ultraviolet detection wavelength signal, the eluent at different time periods is introduced into different trapping columns, and the stationary phase of the trapping column is reversed-phase silica gel packing.

[0067] The effluent from each collection column was collected separately to obtain several second separation groups B1;

[0068] After collection is completed, gradient elution is performed on the collection column using an ethanol-water solution as the mobile phase; furthermore, in the gradient elution, the initial volume concentration of the ethanol-water solution is 10%, and the final volume concentration is 90-95%.

[0069] The stationary phase of the second-dimensional chromatographic column is reversed-phase silica gel packing, and the eluent of the trapping column is used as the mobile phase. The eluent is collected according to the ultraviolet detection wavelength signal, and several second separation groups B2 are obtained by combining elutions with similar absorption peaks and continuous elutions with low yield.

[0070] The second segregation groups obtained in this step include several second segregation groups B1 and several second segregation groups B2.

[0071] Step 3) is the group selection step based on the yield. The yield is calculated according to Formula I above. Specifically, the content of the corresponding solvent in the sample can be determined by gas chromatography according to YC / T 242-2020 "Determination of Ethanol, 1,2-Propanediol and Glycerol Content in Tobacco Flavorings", and the water content of the sample can be determined by Karl Fischer method. The dry matter content is obtained based on the solvent content and water content, and then the mass of dry matter is obtained. Using the aforementioned method, the dry matter mass of a certain group and the dry matter mass of cocoa extract can be obtained. Then, the yield of each group is obtained according to Formula I, and groups with a yield ≥2% are retained, that is, groups with a yield less than 2% are removed.

[0072] Step 4) involves selecting distinctive flavor groups through sensory evaluation. This is done by adding these groups to unflavored blank cigarettes for sensory evaluation, identifying those groups that demonstrate good sensory effects and focus on different flavor characteristics. Evaluation indicators include smoke characteristics, comfort characteristics, flavor profile, and aroma profile. Preferably, smoke characteristics include: aroma, richness, smoothness and roundness, impurities, smoke concentration, and strength; comfort characteristics include: oral irritation / tongue burning, oral residue / dryness, astringency, throat irritation, throat dryness, and nasal irritation; flavor profile includes: sweetness, sourness, bitterness, and coolness; aroma profile includes: flue-cured tobacco aroma, fresh aroma, fruity aroma, spicy aroma, woody aroma, bean aroma, cocoa aroma, milky aroma, paste aroma, roasted aroma, and sweet aroma. Specifically, the sensory evaluation is conducted according to YC / T 497—2014 "Sensory Evaluation Method for Chinese-style Cigarette Styles".

[0073] Furthermore, the preferred method for selecting distinctive flavor groups is as follows:

[0074] Step S1: If a group's indicators meet any of the following conditions, it is directly identified as a distinctive flavor group:

[0075] The score for cocoa aroma increased by more than 3.0 points compared to the score for blank cigarettes;

[0076] The score for roasted aroma increased by more than 1.0 point compared to the score for blank cigarettes;

[0077] Step S2: If a group's indicators meet any of the following conditions, it is removed from the list:

[0078] The score for throat stimulation increased by more than 5% compared to the score for blank cigarettes;

[0079] The tongue burning score increased by more than 5% compared to the score of a blank cigarette.

[0080] Step S3: Among the separated groups retained after step S2, if a group's index meets any of the following conditions, it is considered a group to be evaluated:

[0081] The score for cocoa aroma increased by more than 2.5 points compared to the score for blank cigarettes;

[0082] The aroma score increased by more than 5% compared to the score of blank cigarettes;

[0083] The richness score increased by more than 5% compared to the score of blank cigarettes;

[0084] Step S4: Calculate the comprehensive score of the groups to be evaluated according to the following formula, and select the top 2-3 groups as the distinctive flavor groups;

[0085] Overall score = 0.3 × cocoa aroma + 0.3 × (aroma + richness) + 0.1 × roasting aroma - 0.2 × off-flavors

[0086] Step 5) involves performing pyrolysis-gas chromatography-mass spectrometry (GC-MS) analysis on the selected characteristic flavor groups to obtain characteristic flavor data. Natural flavorings used in tobacco products must be burned to fully exert their flavor-regulating effects. GC-MS involves heating the sample under strict control, rapidly decomposing it into volatile small molecules, and then directly sending the decomposition products into the GC-MS instrument for analysis. This method can simulate the cigarette combustion environment to a certain extent and is suitable for analyzing flavor substances that have a substantial impact on cigarette smoke after being added to cigarettes. To facilitate subsequent blending processes, the aforementioned characteristic flavor data also includes the content of the decomposed characteristic flavor substances in each characteristic flavor group.

[0087] In this step, the preferred analytical conditions for pyrolysis-GC-MS analysis are as follows:

[0088] Pyrolysis conditions:

[0089] Pyrolysis temperature: 900℃; pyrolysis time: 0.2 min; connection temperature: 280℃; sample addition amount: 1 μL;

[0090] GC-MS conditions:

[0091] Chromatographic column: DB-5MS capillary column, 30m × 0.25mm × 0.25μm;

[0092] Carrier gas: He; Flow rate: 1.0 mL / min; Split ratio: 50:1;

[0093] Temperature program: 40℃, hold for 3 minutes; increase to 240℃ at a rate of 10℃ / min; increase to 280℃ at a rate of 20℃ / min, hold for 15 minutes;

[0094] GC-MS transfer line temperature: 280℃; ionization mode: EI; ion source temperature: 230℃; quadrupole temperature: 150℃; ionization energy: 70eV; solvent delay time: 4.5min; mass scan range: 29-450a.mu.

[0095] Step 6) is the process of compounding the distinctive flavor groups with characteristic flavor substances. Those skilled in the art can, based on the tobacco leaf formulation, auxiliary material design parameters, style requirements, and characteristic flavor data, compound the separated distinctive flavor groups and characteristic flavor substances screened by pyrolysis-gas chromatography-mass spectrometry analysis in appropriate proportions to obtain cocoa-flavored tobacco flavorings.

[0096] The method for preparing cocoa-flavored tobacco flavoring provided by this invention has the following advantages:

[0097] 1. This invention utilizes multi-stage membrane separation and two-dimensional column chromatography to screen and obtain a variety of distinctive cocoa flavor groups with different sensory characteristics. This highlights the diverse functions of cocoa extracts for tobacco use, enriches the product categories of cocoa extracts, and increases the flexibility and precision of using cigarette flavorings. The combined application of multi-stage membrane separation and two-dimensional column chromatography technologies offers complementary advantages, and the separation process involves almost no thermal, chemical, or biological changes, preserving the natural characteristics of the flavorings. Both dextran gel and reversed-phase silica gel can be reused multiple times, and efficient production of two-dimensional column-separated flavor groups can be achieved using production-grade automated two-dimensional medium-pressure column chromatography equipment.

[0098] 2. Natural flavorings in tobacco products can only fully exert their flavor-regulating effects through combustion. This method uses pyrolysis-gas chromatography-mass spectrometry (GC-MS) to further analyze the characteristic flavor compounds that have a substantial impact on cigarette smoke after being added. Finally, the characteristic flavor compounds and characteristic flavor groups are compounded to achieve endogenous flavor enhancement. This method highlights the style characteristics of natural flavorings while minimizing uncontrollable factors and ineffective components, thereby increasing the quality stability and controllability of refined natural flavoring products. It has practical application value.

[0099] Another embodiment of the present invention provides a cocoa-flavored tobacco flavoring, which is prepared according to the method described in the above embodiments. The specific implementation is the same as that in the foregoing embodiments, and will not be repeated here.

[0100] Another embodiment of the present invention provides the application of the above-mentioned method for preparing cocoa-flavored tobacco flavoring in cigarette flavoring.

[0101] The technical solution of the present invention will be further described below with reference to specific embodiments:

[0102] Example 1

[0103] 1. A commercially available cocoa extract was tested, and its optimal solubility in 60% ethanol was determined according to method YC / T 145.4-1998. The cocoa extract was diluted with 60% ethanol to a 5% (w / w) solution, centrifuged to remove insoluble matter, and the supernatant was used for membrane separation. Separation was performed sequentially using a 50nm ceramic membrane, a 50kDa spiral-wound organic membrane, a 5nm ceramic membrane, and a 1kDa spiral-wound organic membrane. The permeate from each membrane stage was used as the separation target for the next stage. The process parameters and rejection ratios (the ratio of rejection liquid to feed mass in each process stage) for each membrane stage are shown in Table 1. The rejection liquid obtained from each stage was collected. The 1kDa membrane permeate was concentrated using a reverse osmosis (RO) membrane, and the RO membrane rejection liquid was collected. This step yielded five first separation groups, denoted as the 50nm rejection group, the 50kDa rejection group, the 5nm rejection group, the 1kDa rejection group, and the RO rejection group.

[0104] Table 1. Process parameters for each stage of membrane separation process.

[0105]

[0106] 2. The RO membrane retentate was separated using a preparative-grade fully automated two-dimensional column chromatography platform (Zhengzhou Tobacco Research Institute, system pressure resistance 5 MPa, one-dimensional pump flow rate 0-200 mL / min, two-dimensional pump flow rate 0-50 mL / min). This equipment has one one-dimensional separation column, followed by four collection columns, and then two two-dimensional separation columns. The system has a maximum pressure resistance of 5 MPa. UV detectors are connected after the one-dimensional column and the two two-dimensional columns, and an automatic fraction collector is also connected after the two-dimensional column.

[0107] One-dimensional column chromatography was performed using a 50mm × 400mm column, with dextran gel as the packing material, distilled water as the eluent, a flow rate of 8 mL / min, and an elution time of 200 min. The UV detection wavelengths were 245 nm and 326 nm. Based on the UV absorption signal (… Figure 1 a) The eluents from the one-dimensional column chromatography separation at time points of 0–46 min, 46–67 min, 67–99 min, and 99–200 min were introduced into four trapping columns, each 26 mm × 200 mm in size, packed with C18 bonded silica gel. During the trapping process, the eluent from the trapping columns was collected to obtain four second separation groups B1, denoted as T1–T4. After trapping, the trapping columns were eluted with a gradient of ethanol from 10% to 90% (ethanol volume percentage increased uniformly by 0.8% per minute until reaching 90%, then maintained at this concentration until the two-dimensional separation was complete). The eluents were then introduced into second-dimensional column chromatography. The second-dimensional column was 20 mm × 460 mm in size, packed with C18 bonded silica gel, with a two-dimensional separation time of 100 min and UV detection wavelengths of 245 nm and 326 nm, respectively. Based on the UV absorption signal of the second-dimensional column chromatography (…),… Figure 1 (b~e) The elution fractions corresponding to similar absorption peaks are merged. At the same time, considering the actual production application, the continuous fractions with lower yields are also merged, resulting in a total of 12 second separation groups B2, which are denoted as A1, A2, A3, B1, B2, C1, C2, C3, C4, D1, D2, D3.

[0108] This step yielded a total of 16 second segregating groups: A1, A2, A3, B1, B2, C1, C2, C3, C4, D1, D2, D3, T1, T2, T3, T4.

[0109] 3. For the segregating groups obtained in steps 1 and 2, calculate the yield of each segregating group according to Equation I:

[0110] y=a / A×100% Formula I

[0111] In Formula I, y is the yield, a is the dry matter mass of a certain separation group, and A is the dry matter mass of the cocoa extract; the dry matter mass is the mass after removing organic solvents and water.

[0112] The contents of the corresponding solvents in the samples were determined by gas chromatography according to YC / T 242-2020 "Determination of Ethanol, 1,2-Propanediol and Glycerol Content in Tobacco Flavorings". The water content of the samples was determined by the Karl Fischer method. The dry matter content of the sample was taken as the mass percentage after deducting solvents and water. The dry matter mass was calculated based on the dry matter content and the total sample mass. The yields of each separation group are shown in Table 2. Separation groups with a yield ≥2% were retained.

[0113] Table 2. Yields of each segregating group

[0114]

[0115]

[0116] Note: ① A1, A2, A3, B1, B2, C1, C2, C3, C4, D1, D2, D3 represent the groups of substances obtained by two-dimensional column separation from the four collection columns; T1 to T4 correspond to the effluent groups of the four collection columns; ② "-" indicates that the yield is very low and the accurate value cannot be calculated.

[0117] 4. Prepare solutions of the same concentration from the separated groups retained in step 3) and number them. Using an automatic flavoring injector, add each group solution to the reference cigarette at a rate of 50 μg / g dry matter of tobacco shreds. Seal and store for at least 48 hours at a temperature of (22±1)℃ and a relative humidity of 60%±2%. Sensory evaluation of the sample cigarettes after flavoring injection of the cocoa extract separated groups is conducted according to YC / T497—2014 "Sensory Evaluation Method for Chinese-style Cigarettes". The sensory evaluation results are shown in Table 3.

[0118] Table 3 Sensory evaluation results of the separated groups

[0119]

[0120] Continued from Table 3: Sensory evaluation results of the segregated groups

[0121]

[0122] Note: The scores for each of the evaluation indicators for cigarettes, such as "coolness", "sun-dried tobacco aroma", "fresh aroma", "floral aroma" and "herbal aroma", are all 0.

[0123] Table 3 shows that the sensory effects of the groups obtained from cocoa extract separation by membrane separation and column chromatography differed significantly, with clear distinctions between superior and inferior groups. The group separated by 50nm membrane separation retained the cocoa and roasted aromas of the original sample. Group A1, separated by two-dimensional column chromatography, had a prominent roasted aroma. Group A2 significantly enhanced the cocoa and roasted aromas, increased the smoke concentration, and to some extent improved the sweetness of the reference cigarette. In addition, the 50nm membrane separation retentate, groups A1, and A2 all improved the aroma, richness, and smoothness of the reference cigarette, resulting in good overall sensory performance. The remaining groups all exhibited varying degrees of increased irritation, residual sensation, and off-flavors (e.g., B1, T2), and decreased the aroma and richness of the reference cigarette (e.g., 5nm retentate, 1kDa retentate), among other negative sensory characteristics.

[0124] Follow these steps to select distinctive flavor groups:

[0125] Step S1: If a group's indicators meet any of the following conditions, it is directly identified as a distinctive flavor group:

[0126] The score for cocoa aroma increased by more than 3.0 points compared to the score for blank cigarettes;

[0127] The score for roasted aroma increased by more than 1.0 point compared to the score for blank cigarettes;

[0128] Step S2: If a group's indicators meet any of the following conditions, it is removed from the list:

[0129] The score for throat stimulation increased by more than 5% compared to the score for blank cigarettes;

[0130] The tongue burning score increased by more than 5% compared to the score of a blank cigarette.

[0131] Step S3: Among the separated groups retained after step S2, if a group's index meets any of the following conditions, it is considered a group to be evaluated:

[0132] The score for cocoa aroma increased by more than 2.5 points compared to the score for blank cigarettes;

[0133] The aroma score increased by more than 5% compared to the score of blank cigarettes;

[0134] The richness score increased by more than 5% compared to the score of blank cigarettes;

[0135] Step S4: Calculate the comprehensive score of the groups to be evaluated according to the following formula, and select the top 2-3 groups as the distinctive flavor groups;

[0136] Overall score = 0.3 × cocoa aroma + 0.3 × (aroma + richness) + 0.1 × roast aroma - 0.2 × off-flavors. Based on the above method, the selected characteristic flavor groups are: 50nm retention group, A1 and A2.

[0137] 5. The three distinctive flavor groups were analyzed by pyrolysis-gas chromatography-mass spectrometry. Using the NIST20 spectral library, qualitative analysis was performed on samples with a matching degree higher than 80%, and semi-quantitative analysis was conducted using peak area normalization. Each sample was measured twice, and the average value was taken.

[0138] Pyrolysis conditions:

[0139] EGA / PY-3030D pyrolysis instrument (FRONTIER LAB, Japan); pyrolysis temperature: 900℃; pyrolysis time: 0.2min; connection temperature: 280℃; sample addition amount: 1μL.

[0140] GC-MS conditions:

[0141] 7890A-5975C Gas Chromatography-Mass Spectrometry (Agilent Technologies, USA); Column: DB-5MS capillary column (30m × 0.25mm × 0.25μm); Carrier gas: He; Flow rate: 1.0mL / min; Split ratio: 50:1; Temperature program: 40℃, hold for 3 minutes; ramp to 240℃ at 10℃ / min; ramp to 280℃ at 20℃ / min, hold for 15 minutes; GC-MS transfer line temperature: 280℃; Ionization mode: EI; Ion source temperature: 230℃; Quadrupole temperature: 150℃; Ionization energy: 70eV; Solvent delay time: 4.5min; Mass scan range: 29-450 a.mu.

[0142] By extracting the TIC peaks of compounds and analyzing the co-elution components from the raw Py-GC / MS data of three characteristic flavor groups, a total of 78 pyrolysis products were identified. Characteristic flavor data information was constructed, as detailed in Table 4, including: the names of characteristic flavor substances after pyrolysis, their corresponding aroma characteristics, and their contents.

[0143] As shown in Table 4, the pyrolysis products are mainly heterocyclic and aromatic compounds; their aroma types are rich, including nutty aroma (such as 2-methylpyrazine, 2-acetylfuran), sweet aroma (such as 2-methylpyridine, styrene), cocoa aroma (such as 2,6-dimethylpyrazine, 2-methylpyrazine), fruity aroma (such as 1-hydroxy-2-butanone, 2,3-butanediol), and milky aroma (γ-butyrolactone), etc.

[0144] Table 4. 900℃ pyrolysis components and aroma characteristics of the three distinctive flavor groups

[0145]

[0146]

[0147] Note: ① Refer to information from http: / / perflavory.com / and https: / / www.chemcalbook.com / ; " / " indicates no aroma characteristics reported; ② "-" indicates not detected.

[0148] 6. Although the selected characteristic flavor groups have prominent styles, they also have shortcomings such as uneven sensory effects and insufficient richness. In combination with the requirements of tobacco leaf blends and flavorings, the characteristic flavor groups are compounded with the characteristic flavor substances in Table 4 to enhance the endogenous style. The composition of the compounded cocoa flavorings for tobacco is shown in Table 5.

[0149] The sensory evaluation results of the compound product and the original cocoa extract in cigarettes are shown in the figure. Figure 2 The compounded products have a significant effect on enhancing the aroma, richness, smoothness and roundness of cigarettes and masking off-flavors. At the same time, they are outstanding in increasing sweetness, sweet aroma, roasted aroma, milky aroma and cocoa aroma. The overall effect is significantly better than that of the original flavoring.

[0150] Table 5. Blending Schemes for Cocoa Characteristic Flavor Groups and Significant Flavor Compounds

[0151]

[0152]

[0153] In practical production applications, by flexibly changing the compounding method, more diverse compound products can be obtained to meet the personalized flavoring needs of different cigarettes.

[0154] Example 2

[0155] 1. A commercially available cocoa extract was tested, and its optimal solubility in 80% ethanol was determined according to method YC / T 145.4-1998. The cocoa extract was diluted with 80% ethanol to a mass fraction of 8%, and insoluble matter was removed by centrifugation. The supernatant was then subjected to membrane separation. 200nm, 50nm, and 2nm ceramic membranes were used sequentially for separation. The permeate from each membrane stage was used as the target for the next stage. The process parameters and rejection ratios (the ratio of retentate to feed mass in each process stage) for each membrane stage are shown in Table 6. The retentate from each stage was collected. The permeate from the 2nm membrane was concentrated using a reverse osmosis (RO) membrane, and the RO membrane retentate was collected. This step yielded four first separation groups, denoted as the 200nm rejection group, the 50nm rejection group, the 2nm rejection group, and the RO rejection group.

[0156] Table 6 Process parameters for each stage of membrane separation process

[0157]

[0158] 2. A production-grade fully automated two-dimensional column chromatography platform was used for further separation of the RO membrane-restricted groups of cocoa extract. The device uses a 30×60cm one-dimensional column, followed by four 20×20cm trap columns, and then four 20×40cm two-dimensional separation columns. The system has a maximum pressure rating of 2MPa and a maximum flow rate of 2L / min. UV detectors are connected after both the one-dimensional and four two-dimensional columns, and an automatic fraction collector is also connected after the two-dimensional columns.

[0159] The one-dimensional column chromatography column had a size of 30 × 60 cm, packed with dextran gel, with a loading volume of 3000 mL, using 10% ethanol as the eluent, a flow rate of 300 mL / min, and UV detection wavelengths of 245 nm and 326 nm. Based on the UV absorption signal, the eluents from the one-dimensional column chromatography separations at time points of 0–115 min, 115–140 min, and 140–300 min were introduced into the first three trapping columns, packed with reversed-phase silica gel. During the trapping process, the eluent from the trapping columns was collected, yielding three second separation groups B1, denoted as T1–T3. After collection, a gradient elution column was used with ethanol at a volume fraction ranging from 10% to 95% (ethanol volume fraction increased uniformly by 0.8% per minute until the ethanol volume fraction reached 95%, which was then maintained until the two-dimensional separation was completed). The eluents were then introduced into the second-dimensional column for chromatographic separation. The second-dimensional column was packed with reversed-phase silica gel, with dimensions of 20×40 cm. The two-dimensional separation time was 200 min, and the UV detection wavelengths were 245 nm and 326 nm, respectively. Based on the UV absorption signal of the second-dimensional column chromatography, eluents corresponding to similar absorption peaks were combined. Considering practical production applications, continuous fractions with lower yields were also combined, resulting in six second-separation groups B2, denoted as A1, A2, B1, B2, C1, and C2.

[0160] This step yielded a total of 9 second segregation groups: A1, A2, B1, B2, C1, C2, T1, T2, and T3.

[0161] 3. The operation is the same as step 3 in Example 1.

[0162] 4. The operation is the same as step 4 in Example 1. The sensory evaluation results are listed in Table 7. The selected color and flavor groups are: 50nm retention group, RO membrane retention group and A1.

[0163] Table 7 Sensory evaluation results of the separated groups

[0164]

[0165]

[0166] Continued in Table 7: Sensory evaluation results of each segregating group

[0167]

[0168] Note: The scores for each of the evaluation indicators for cigarettes, such as "coolness", "sun-dried tobacco aroma", "fresh aroma", "floral aroma" and "herbal aroma", are all 0.

[0169] 5. The operation was the same as step 5 in Example 1. The pyrolysis products and aroma characteristics of the three characteristic flavor groups are shown in Table 8. A total of 78 pyrolysis products were detected.

[0170] Table 8. 900℃ pyrolysis components and aroma characteristics of the three distinctive flavor groups

[0171]

[0172]

[0173]

[0174] 6. Taking into account the advantages and disadvantages of the sensory characteristics of the distinctive flavor groups, compounds suitable for use as synthetic flavorings in tobacco products were selected from the pyrolysis characteristic flavor substances, focusing on substances with aromas such as cocoa, caramel sweetness, roasting, and milkiness. These compounds were then compounded according to the scheme in Table 9. A refined cocoa-flavored tobacco flavoring product was obtained. The sensory evaluation results of the compounded product and the original flavoring in cigarettes are shown in Table 9. Figure 3 The compounded products have a significant effect on enhancing the aroma, richness, and concentration of cigarette smoke, as well as masking off-flavors and reducing irritation. They also have outstanding effects on increasing sweetness, sweet aroma, roasted aroma, milky aroma, and cocoa aroma. Overall, the effects are significantly better than those of the original flavorings.

[0175] Table 9. Blending Schemes for Cocoa Characteristic Flavor Groups and Significant Flavor Compounds

[0176]

[0177] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A process for the preparation of a cocoa-flavoured tobacco flavouring, characterised in that, The application relates to a method for preparing a cocoa flavor tobacco flavoring agent. The method comprises the following steps: 1) diluting cocoa extract with an ethanol aqueous solution, and performing multi-stage membrane separation according to the principle of gradually reducing the membrane pore size, performing reverse osmosis treatment on the permeate of the last-stage membrane separation to obtain reverse osmosis retention liquid; collecting the retention liquid of each stage to obtain a plurality of first separation groups; 2) performing two-dimensional column chromatography separation on the reverse osmosis retention liquid, using dextran gel as the filler of the first-dimensional chromatographic column and using reversed-phase silica gel as the filler of the second-dimensional chromatographic column to obtain a plurality of second separation groups; 3) calculating the yield of each separation group, and retaining the separation groups with a yield greater than or equal to 2%; the yield is calculated according to formula I: y=a / A*100% formula I wherein y is the yield, a is the mass of dry matter in a certain separation group, and A is the mass of dry matter of the cocoa extract; the mass of dry matter is the mass after removing the organic solvent and water; 4) adding the separation groups retained in step 3) into blank cigarettes with the same dry matter mass to perform sensory evaluation; taking smoke characteristics, comfort characteristics, taste style and aroma style as the evaluation indexes, and screening out characteristic flavor groups; 5) performing thermal cracking-gas chromatography-mass spectrometry analysis on the characteristic flavor groups to construct characteristic flavor data information, wherein the characteristic flavor data information comprises the names of characteristic flavor substances after cracking and corresponding aroma characteristics; 6) compounding the characteristic flavor groups with the characteristic flavor substances to obtain a cocoa flavor tobacco flavoring agent. In step 1), the multi-stage membrane separation is specifically as follows: performing multi-stage membrane separation by sequentially passing through a 50 nm ceramic membrane, a 50 kDa organic membrane, a 5 nm ceramic membrane and a 1 kDa organic membrane; or 2. The production method according to claim 1, characterized by, performing separation by sequentially passing through a 200 nm ceramic membrane, a 50 nm ceramic membrane and a 2 nm ceramic membrane. In step 2), the ultraviolet detection wavelengths of the first-dimensional chromatographic column and the second-dimensional chromatographic column are 245 nm and 326 nm. In step 2), the first-dimensional chromatographic column has a dextran gel filler and a mobile phase of distilled water or an ethanol aqueous solution with a volume concentration of not more than 10%; the effluent of different time periods is introduced into different trapping columns according to the ultraviolet detection wavelength signals; the trapping columns have a reversed-phase silica gel filler; 3. The preparation method according to claim 1, characterized in that, the effluent of each trapping column is collected to obtain a plurality of second separation groups B1; 4. The production method according to claim 3, characterized by, after the trapping is completed, gradient elution is performed on the trapping columns by taking an ethanol aqueous solution as the mobile phase; the second-dimensional chromatographic column has a reversed-phase silica gel filler and takes the eluent of the trapping column as the mobile phase; the effluent is collected according to the ultraviolet detection wavelength signals, and the elution fractions with similar absorption peaks and the continuous elution fractions with low yield are combined to obtain a plurality of second separation groups B2; the plurality of second separation groups obtained in step 2) comprise the plurality of second separation groups B1 and the plurality of second separation groups B2. In the gradient elution, the initial volume concentration of the ethanol aqueous solution is 10%, and the final volume concentration is 90-95%. ​ ​ 5. The preparation method according to claim 4, characterized in that, ​ 6. The method of claim 1, wherein, In the step 4), the smoke characteristics include aroma, richness, softness, harshness, smoke density and strength; the comfort characteristics include mouth irritation / tongue burning, mouth residue / dryness, astringency, throat irritation, throat dryness and nasal irritation; the taste style includes sweetness, sourness, bitterness and coolness; and the aroma style includes roasted tobacco aroma, fresh aroma, fruity aroma, spicy aroma, woody aroma, bean aroma, cocoa aroma, milk aroma, cream aroma, baking aroma and sweet aroma.

7. The preparation method according to claim 1, characterized in that, In the step 5), the characteristic flavor data information further includes the content of the characteristic flavor substances after cracking in each characteristic flavor group.

8. The method of claim 1, wherein, In the step 5), the analysis conditions of thermal cracking-GC-MS are as follows: Cracking conditions: Cracking temperature: 900℃; cracking time: 0.2min; connecting port temperature: 280℃; sample addition amount: 1μL; GC-MS conditions: Chromatographic column: DB-5MS capillary column, 30m×0.25mm×0.25μm; Carrier gas: He; Flow rate: 1.0mL / min; split ratio: 50:1; Programmed temperature rising: 40℃, holding for 3min; rising at a rate of 10℃ / min to 240℃; rising at a rate of 20℃ / min to 280℃, holding for 15min; GC-MS transfer line temperature: 280℃, ionization mode: EI; ion source temperature: 230℃; quadrupole temperature: 150℃; ionization energy: 70eV; solvent delay time: 4.5min; mass scan range 29-450a.m.u.

9. A cocoa-flavoured tobacco flavouring characterised in that, which is prepared according to the method of any one of claims 1-8.

10. Use of the preparation method of the cocoa flavor tobacco flavor of claim 9 in cigarette flavoring.

Images