Cocoa extract containing homotheobromine, preparation method and application of cocoa extract, tobacco flavor, cigarette paper coating liquid and cigarette

Abstract

The invention provides a cocoa extract containing homotheobromine, a preparation method and application of the cocoa extract, a tobacco flavor, a cigarette paper coating liquid and a cigarette, and belongs to the technical field of tobacco flavors and fragrances. The preparation method of the cocoa extract with high theobromine content comprises the following steps: extracting or dissolving a cocoa raw material containing theobromine with water, removing insoluble substances, and collecting clear liquid; carrying out membrane separation on the clear liquid, and collecting permeate; adsorbing the permeate through macroporous adsorption resin, removing impurities, eluting, and collecting the eluent to prepare the cocoa extract containing homotheobromine; the macroporous adsorption resin is prepared from one or more of AB-8, X-5, HPD-100 and HJ-04. The theobromine in the cocoa extract is enriched by adopting membrane separation and macroporous adsorption resin, and meanwhile, other natural flavor substances are reserved. The cocoa extract with high theobromine content is singly used or compounded to be used in tobacco flavors, and the comprehensive sensory effect of cigarettes can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of tobacco flavoring technology, and in particular to a cocoa extract containing high theobromine, its preparation method and application, tobacco flavoring, cigarette paper coating liquid, and cigarettes. Background Technology

[0002] Cocoa extract is one of the most widely used natural flavorings in the tobacco industry. Adding it to cigarettes imparts a cocoa aroma, enhances roasted, sweet, and bean-like notes, and also helps to enhance the tobacco flavor while suppressing harshness and removing impurities. Theobromine (3,7-dimethylxanthine) is an important alkaloid in cocoa, related to some of the sensory flavors of foods and beverages such as chocolate and tea. It is also closely related to the sensory quality of cocoa extract, a flavoring used in tobacco. Pure theobromine has no aroma and its addition to cigarettes has no significant impact on the sensory quality of cigarettes.

[0003] Theobromine content in cocoa extract is relatively low, with theobromine content in cocoa powder around 2% and the dry matter mass fraction in commercially available cocoa extract around 0.5-3%. Currently, research on theobromine in tobacco flavorings focuses primarily on its detection methods, with less attention paid to its extraction and separation processes. The patent application number 200980104250.7 uses cation exchange resin to prepare a high-theobromine composition; however, ion exchange resins typically involve the use of acid and alkaline solutions, which alters the original pH value of the natural flavoring, changing its composition and affecting its natural flavor. Furthermore, the cation exchange resin elution in this patent involves high-temperature (40-100℃) deionized water as the eluent, which results in the loss of heat-sensitive and volatile substances in the natural flavoring. High-temperature elution also has high energy consumption and production costs, placing high demands on production equipment. Therefore, developing an industrial preparation method for cocoa extracts with high theobromine content suitable for the field of natural flavorings, which can enrich theobromine while retaining other natural flavor substances in natural flavorings and improve the flavoring effect of cocoa extracts in cigarettes, is of great significance for the intensive processing of theobromine-based tobacco flavorings. Summary of the Invention

[0004] Based on this, the main objective of the present invention is to provide a cocoa extract containing high theobromine, its preparation method and application, tobacco flavoring, cigarette paper coating liquid, and cigarettes. The preparation method of the cocoa extract containing high theobromine enriches theobromine while retaining as many other flavor substances in the natural flavoring as possible.

[0005] The first aspect of this application provides a method for preparing a cocoa extract containing high levels of theobromine, comprising:

[0006] The cocoa raw material containing theobromine is extracted or dissolved in water to remove insoluble matter and collect the clear liquid.

[0007] The supernatant is separated by membrane separation, and the permeate is collected;

[0008] The permeate is adsorbed by a macroporous adsorption resin, impurities are removed, and the eluent is collected to prepare a cocoa extract with high theobromine content.

[0009] The macroporous adsorption resin includes one or more of AB-8, X-5, HPD-100 and HJ-04.

[0010] In some embodiments, the macroporous adsorption resin includes one or more of AB-8, X-5, and HJ-04.

[0011] In some embodiments, the membrane separation method includes one or more of microfiltration, ultrafiltration, and nanofiltration.

[0012] In some embodiments, the theobromine-containing cocoa raw material includes: natural cocoa plant material; processed cocoa bean products; extracts of natural cocoa plant material; or, products prepared by one or more of the following treatment methods: microbial fermentation, enzymatic hydrolysis, and Maillard reaction.

[0013] In some embodiments, the preparation of a cocoa extract containing high theobromine specifically includes: packing a macroporous adsorption resin into a chromatography column, loading the permeate onto the chromatography column for adsorption, removing impurities with a first eluent, eluting with a second eluent, collecting the eluent, and preparing a cocoa extract containing high theobromine; the first eluent includes water; the second eluent includes an aqueous ethanol solution.

[0014] In some embodiments, the preparation of a cocoa extract containing high theobromine specifically includes: packing a macroporous adsorption resin into a chromatography column, loading the permeate onto the chromatography column for adsorption, removing impurities with a first eluent, eluting with a second eluent, collecting the eluent, and preparing a cocoa extract containing high theobromine; the first eluent includes water; the second eluent includes an aqueous ethanol solution.

[0015] The sample loading flow rate is 1-4 BV / h;

[0016] The amount of the first eluent is 1-3 BV;

[0017] The volume percentage of the ethanol-water solution is 35-95%;

[0018] The elution volume of the second eluent is 2-5 BV;

[0019] The elution flow rate of the second eluent is 1-4 BV / h.

[0020] In a second aspect of this application, a cocoa extract containing high theobromine is provided, prepared by the method for preparing the cocoa extract containing high theobromine described in the first aspect.

[0021] The third aspect of this application provides the use of a cocoa extract containing high theobromine prepared by the method described in the first aspect, or the cocoa extract containing high theobromine described in the second aspect, in tobacco flavorings.

[0022] A fourth aspect of this application provides a tobacco flavoring, comprising: a cocoa extract containing high theobromine prepared by the method described in the first aspect or the cocoa extract containing high theobromine described in the second aspect; other cocoa extracts or tobacco flavorings.

[0023] In some embodiments, the dry matter mass fraction of theobromine in the tobacco flavoring is 4.89-18.55%.

[0024] The fifth aspect of this application provides a cigarette paper coating liquid, comprising a high-theobromine cocoa extract prepared by the method described in the first aspect, a high-theobromine cocoa extract described in the second aspect, or a tobacco flavoring described in the fourth aspect.

[0025] The sixth aspect of this application provides a cigarette, the raw materials of which include a cocoa extract containing high theobromine prepared by the method of preparing a cocoa extract containing high theobromine as described in the first aspect, a cocoa extract containing high theobromine as described in the second aspect, a tobacco flavoring as described in the fourth aspect, or a cigarette paper coating liquid as described in the fifth aspect.

[0026] Compared with traditional technologies, this application has at least the following beneficial effects:

[0027] This application utilizes membrane separation and macroporous adsorption resin column chromatography to enrich theobromine in cocoa raw materials containing theobromine. Membrane separation removes some large molecules, while macroporous adsorption resin column chromatography further focuses small and medium-sized polar molecules. The resulting cocoa extract with high theobromine content retains other natural flavor compounds well while increasing the relative theobromine content. The preparation process is simple and easy to operate, making it suitable for industrial production applications. Attached Figure Description

[0028] Figure 1 Dynamic adsorption-leakage curves of theobromine for five macroporous adsorption resins are shown.

[0029] Figure 2 The image shows the HPLC-DAD chromatogram of the cocoa powder aqueous extract containing theobromine in Example 1. Detailed Implementation

[0030] The present application will be further described in detail below with reference to the embodiments and examples. These embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the invention, and the equivalent forms obtained also fall within the protection scope of the present invention. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Research on theobromine in tobacco flavorings has been limited, particularly regarding its extraction and separation processes. Patent application number 200980104250.7 uses cation exchange resin to prepare a high-theobromine composition. However, ion exchange resins typically involve the use of acidic or alkaline solutions, which alters the original pH value of the natural flavoring, changing its composition and affecting its natural flavor. Furthermore, the patent's cation exchange resin elution process uses high-temperature (40-100℃) deionized water, leading to the loss of heat-sensitive and volatile substances in the natural flavoring. This method is unsuitable for the refined processing of natural flavorings. This application utilizes membrane separation and macroporous adsorption resin column separation to enrich theobromine in cocoa extracts. The preparation process is carried out at relatively low temperatures and does not involve chemical changes, thus better preserving other natural flavor substances in the cocoa extract while achieving a high theobromine content. Using the resulting high-theobromine cocoa extract as a tobacco flavoring can improve the overall sensory experience of cigarettes.

[0033] The first aspect of this application provides a method for preparing a cocoa extract containing high levels of theobromine, comprising:

[0034] The cocoa raw material containing theobromine is extracted or dissolved in water to remove insoluble matter and collect the clear liquid.

[0035] The supernatant is separated by membrane separation, and the permeate is collected;

[0036] The permeate is adsorbed by a macroporous adsorption resin, impurities are removed, and the eluent is collected to prepare a cocoa extract containing high theobromine; the macroporous adsorption resin includes one or more of AB-8, X-5, HPD-100 and HJ-04.

[0037] This application employs membrane separation technology and one or more of macroporous adsorption resins AB-8, X-5, HPD-100, and HJ-04 to enrich theobromine in cocoa extracts containing theobromine, thereby significantly increasing the theobromine content.

[0038] In some embodiments, the theobromine-containing cocoa raw material includes: natural cocoa plant material; processed cocoa bean products; extracts of natural cocoa plant material; or, products prepared by one or more of the following treatment methods: microbial fermentation, enzymatic hydrolysis, and Maillard reaction.

[0039] In some embodiments, the theobromine-containing cocoa raw material comprises the natural plant body of the cocoa tree; the natural plant body of the cocoa tree comprises one or more of the fruit, seeds, branches and leaves of the cocoa tree, and the fruit of the cocoa tree comprises cocoa beans and / or cocoa shells.

[0040] In some embodiments, the theobromine-containing cocoa raw material includes cocoa powder or commercially available cocoa extract. The commercially available cocoa extract includes commercially available cocoa concentrate.

[0041] In some embodiments, the temperature for water extraction or dissolution ranges from room temperature to 45°C, and can be 25°C, 26°C, 27°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, or 45°C. A temperature slightly above room temperature can promote the dissolution of theobromine.

[0042] In some implementations, the dissolution process using water can also employ high shear to promote the dissolution of theobromine.

[0043] In some embodiments, the membrane separation method includes one or more of microfiltration, ultrafiltration and nanofiltration, and membrane separation can be performed using membrane cores made of materials such as ceramic membranes and organic membranes.

[0044] In some embodiments, the membrane separation method includes two or more of microfiltration, ultrafiltration, and nanofiltration. Membrane separation is performed sequentially according to the membrane pore size, from largest to smallest. The permeate from the previous stage of membrane separation serves as the feed solution for the next stage. During membrane separation, a small amount of water can be added to the feed end as a dialysate; this small amount of water can be 3-10% of the feed solution's mass to promote sufficient permeation of theobromine through the membrane material. The membrane separation is used to remove large molecular impurities from theobromine aqueous extract.

[0045] In some embodiments, membrane separation can be carried out at room temperature or under appropriate temperature conditions; appropriate temperature conditions are below 45°C, and can be 36°C, 38°C, 40°C, 42°C, 44°C, or 45°C. Membrane separation under appropriate temperature conditions can avoid fouling of the membrane material by a small amount of residual cocoa butter and is conducive to the permeation of theobromine.

[0046] In some embodiments, the macroporous adsorption resin includes one or more of AB-8, X-5, HPD-100, and HJ-04; AB-8 is a weakly polar macroporous adsorption resin, and X-5, HPD-100, and HJ-04 are non-polar macroporous adsorption resins.

[0047] In some embodiments, the macroporous adsorption resin includes one or more of AB-8, X-5, and HJ-04.

[0048] In some embodiments, the macroporous adsorption resin includes one or more of AB-8 and HJ-04.

[0049] In some embodiments, the macroporous adsorption resin includes AB-8.

[0050] In some embodiments, the preparation of a cocoa extract containing high theobromine specifically includes: packing a macroporous adsorption resin into a chromatography column, loading the permeate onto the chromatography column for adsorption, removing impurities with a first eluent, eluting with a second eluent, collecting the eluent, and preparing a cocoa extract containing high theobromine.

[0051] In some embodiments, the preparation of a cocoa extract containing high theobromine specifically includes: packing a macroporous adsorption resin into a chromatography column, loading the permeate onto the chromatography column for adsorption, removing impurities with a first eluent, eluting with a second eluent, collecting the eluent, and preparing a cocoa extract containing high theobromine; the first eluent includes water; the second eluent includes an aqueous ethanol solution. Loading the permeate onto the chromatography column for adsorption for a period of time before using the first eluent for impurity removal enhances the adsorption equilibrium effect. Using the first eluent (water) removes some highly polar substances from the permeate, resulting in a significant increase in theobromine concentration in the eluent after subsequent elution with the second eluent, which is beneficial for the enrichment of theobromine while retaining other natural flavor compounds in the natural flavor cocoa extract.

[0052] In some embodiments, the preparation of cocoa extract containing high theobromine specifically includes: packing macroporous adsorption resin into a chromatography column, loading the permeate onto the chromatography column for adsorption, removing impurities with a first eluent, eluting with a second eluent, collecting the eluent, and preparing cocoa extract containing high theobromine.

[0053] The first eluent comprises water; the second eluent comprises an aqueous ethanol solution;

[0054] The loading flow rate is 1-4 BV / h, which can be 1 BV / h, 1.5 BV / h, 2 BV / h, 2.5 BV / h, 3 BV / h, 3.5 BV / h or 4 BV / h;

[0055] The amount of the first eluent is 1-3 BV, which can be 1 BV, 1.5 BV, 2 BV, 2.5 BV or 3 BV;

[0056] The volume percentage of the ethanol-water solution is 35-95%, and can be 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0057] The elution volume of the second eluent is 2-5 BV, which can be 2 BV, 2.5 BV, 3 BV, 3.5 BV, 4 BV, 4.5 BV or 5 BV;

[0058] The elution flow rate of the second eluent is 1-4 BV / h, which can be 1 BV / h, 1.5 BV / h, 2 BV / h, 2.5 BV / h, 3 BV / h, 3.5 BV / h or 4 BV / h.

[0059] In some embodiments, a macroporous adsorption resin is packed into a chromatography column, and the permeate is loaded onto the column for adsorption. The total theobromine content in the permeate is less than the theobromine adsorption saturation of the macroporous adsorption resin in the chromatography column. This achieves effective adsorption of theobromine in the permeate and avoids theobromine loss. Depending on the theobromine concentration in the permeate, the loading volume can be 4 BV, 5 BV, 6 BV, 7 BV, 8 BV, 9 BV, 10 BV, 11 BV, or 12 BV.

[0060] In some embodiments, macroporous adsorption resin adsorption, impurity removal, and elution are all carried out at room temperature.

[0061] In some embodiments, after collecting the eluent, a concentration or drying step is performed to prepare a cocoa extract containing high levels of theobromine; the concentration step includes reverse osmosis membrane separation or vacuum distillation; the drying step includes freeze drying or spray drying.

[0062] This application uses membrane separation and macroporous adsorption resin column chromatography to enrich theobromine in cocoa extract. The resulting high-theobromine cocoa extract has a significantly increased theobromine content, while other natural flavor substances are well preserved. Using the high-theobromine cocoa extract as a flavoring agent for cigarettes can improve the overall sensory effect of cigarettes.

[0063] In a second aspect of this application, a cocoa extract containing high theobromine is provided, prepared by the method for preparing the cocoa extract containing high theobromine described in the first aspect.

[0064] In some embodiments, the theobromine dry matter fraction in the high-theobromine cocoa extract is 10-22%, which can be 10%, 12%, 15%, 18%, 20%, 20.83%, 21.35%, 21.96%, or 22%. Compared with commercially available cocoa extracts, the theobromine dry matter fraction in the high-theobromine cocoa extract of this application is significantly increased, while other natural flavor compounds are better preserved.

[0065] The theobromine content in the cocoa extract prepared in this application is significantly increased, while other natural flavor substances are well preserved. Using the cocoa extract with high theobromine content as a flavoring agent for cigarettes can improve the overall sensory effect of cigarettes.

[0066] The third aspect of this application provides the use of a cocoa extract containing high theobromine prepared by the method described in the first aspect, or the cocoa extract containing high theobromine described in the second aspect, in tobacco flavorings.

[0067] Theobromine is enriched in cocoa raw materials containing theobromine to prepare cocoa extracts with high theobromine content. Using the prepared cocoa extracts with high theobromine content as tobacco flavoring can improve the overall sensory effect of cigarettes, enhance the aroma, richness of smoke, sensory comfort, and cocoa flavor without increasing bitterness.

[0068] A fourth aspect of this application provides a tobacco flavoring, comprising: a cocoa extract containing high theobromine prepared by the method described in the first aspect or the cocoa extract containing high theobromine described in the second aspect; other cocoa extracts or tobacco flavorings.

[0069] Style-enhancing products made by blending cocoa extracts with high theobromine content with other cocoa extracts or tobacco flavorings, such as blended theobromine extracts, can improve the overall sensory experience of cigarettes when used as tobacco flavorings.

[0070] In some embodiments, the other cocoa extract includes commercially available cocoa extracts, or the clear liquid described in the first aspect.

[0071] In some embodiments, the dry matter mass fraction of theobromine in the tobacco flavoring is 4.89-18.55%, which can be 4.89%, 9.38%, 11.67%, 11.72% or 18.55%.

[0072] In this embodiment of the application, the dry matter mass fraction of theobromine is: the mass fraction of theobromine in the dry matter after deducting the solvent.

[0073] Specifically, the mass fractions of ethanol, 1,2-propanediol, and glycerol in the sample were determined using gas chromatography according to YC / T 242-2020 "Determination of Ethanol, 1,2-Propanediol, and Glycerol Content in Tobacco Flavorings". The mass fraction of water was determined using the Karl Fischer method. The mass fractions of solvents (ethanol, 1,2-propanediol, glycerol, and water) were calculated. Then, the components after deducting solvents were used as dry matter, and the mass fraction of theobromine in the dry matter was calculated.

[0074] In some embodiments, the cocoa extract containing high levels of theobromine may be used alone as a tobacco flavoring, or blended with other cocoa extracts, or blended with other tobacco flavorings to form a flavoring.

[0075] This application uses cocoa raw materials containing theobromine to prepare cocoa extract with high theobromine content. By compounding the cocoa extract with high theobromine content with other cocoa extracts as a flavoring agent for cigarettes, it can significantly improve the overall sensory effect of cigarettes, enhance the aroma and richness of the smoke, increase the cocoa flavor, and without increasing bitterness.

[0076] The fifth aspect of this application provides a cigarette paper coating liquid, comprising a high-theobromine cocoa extract prepared by the method described in the first aspect, a high-theobromine cocoa extract described in the second aspect, or a tobacco flavoring described in the fourth aspect.

[0077] In some embodiments, the cocoa extract containing high theobromine and tobacco flavorings are used in the cigarette paper coating liquid, which can be used to flavor cigarette paper.

[0078] This application utilizes a cocoa extract containing high theobromine content, prepared in the form of a high-theobromine cocoa extract, to significantly improve the overall sensory effects of cigarettes, enhancing their aroma, strength, cocoa flavor, and flue-cured tobacco aroma, without increasing bitterness, irritation, residue, dryness, or astringency. Furthermore, combining the unenriched cocoa extract or its clear liquid with the enriched high-theobromine cocoa extract in the cigarette paper coating liquid further enhances the overall sensory effects of cigarettes, improving their aroma, smoke concentration, strength, cocoa flavor, sweetness, and flue-cured tobacco aroma, while reducing bitterness.

[0079] The sixth aspect of this application provides a cigarette, the raw materials of which include a cocoa extract containing high theobromine prepared by the method of preparing a cocoa extract containing high theobromine as described in the first aspect, a cocoa extract containing high theobromine as described in the second aspect, a tobacco flavoring as described in the fourth aspect, or a cigarette paper coating liquid as described in the fifth aspect.

[0080] In some embodiments, the cocoa extract containing high theobromine is used in cigarettes, which can be applied to the cigarette production process of adding ingredients and / or flavoring, by directly spraying the cocoa extract containing high theobromine onto the tobacco.

[0081] This application demonstrates that by using the prepared cocoa extract with high theobromine content in cigarettes, the overall sensory effects of cigarettes can be significantly improved, aroma enhanced, and bitterness reduced. Furthermore, mixing the high theobromine cocoa extract with unenriched cocoa extract or clear liquid and applying it to cigarettes can significantly improve the aroma, increase the richness, concentration, strength, and cocoa flavor of the cigarettes, without increasing oral bitterness or reducing the cigarette's fresh aroma.

[0082] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0083] Basic Example 1

[0084] Screening macroporous adsorption resins

[0085] Preparation of cocoa powder aqueous extract: 1 kg of cocoa powder was placed in a 10 L three-necked flask, and 5 L of distilled water was added. The mixture was mechanically stirred at 80 °C for 2 h in a heating mantle. The extract was centrifuged while hot to remove residue, then refrigerated at 4 °C and filtered through a 400-mesh filter to remove cocoa butter. The filtrate was stored in a sealed container at -4 °C for later use. HPLC analysis showed that the theobromine content in the cocoa powder aqueous extract was 1.3983 mg / g.

[0086] Macroporous adsorption resins: AB-8, HPD-100, D101, DA201, X-5, CAD-40, HJ-04 and DM-301 macroporous adsorption resins (all purchased from Tianjin Haoju Resin Technology Co., Ltd.); XDA-8 macroporous adsorption resin (purchased from Beijing Ruida Henghui Technology Development Co., Ltd.).

[0087] 1. Static adsorption performance of macroporous adsorption resin

[0088] To facilitate rapid initial selection of macroporous adsorption resins, a saturated theobromine aqueous solution was used as the experimental sample in the static adsorption experiment. Excess theobromine (99%, Innochem) was added to distilled water, and the mixture was magnetically stirred at room temperature for 1 hour. The filtrate was then filtered, and the concentration of theobromine was determined to be 0.453 mg / mL using an Agilent 1290 high-performance liquid chromatograph.

[0089] Static adsorption and desorption experiments were conducted on each macroporous adsorption resin according to the following steps: The treated macroporous adsorption resin was filtered to remove moisture, 5g was accurately weighed into an Erlenmeyer flask, 25 mL of saturated theobromine aqueous solution was added, the flask was sealed, and the mixture was kept at 303 K and shaken for 24 h before filtration. The theobromine concentration of the filtrate was determined by HPLC. The theobromine adsorption rate of the resin was calculated according to formula (1). The filtered macroporous adsorption resin was transferred to an Erlenmeyer flask, 25 mL of 95% (v / v) ethanol aqueous solution was added, the flask was sealed, and the mixture was kept at 303 K and shaken for 24 h. The theobromine concentration in the desorbed solution was measured, and the theobromine desorption rate was calculated according to formula (2). Two sets of experiments were performed for each resin.

[0090] Static adsorption rate (%) = (C0 - C e ) / C0 × 100% Equation (1);

[0091] Static desorption rate (%) = C / (C0 - C) e Equation (2) is calculated as follows: (1) × 100%

[0092] In the formula, C0 is the theobromine concentration of the initial sample solution, in mg / mL; C e C represents the theobromine concentration in the solution at adsorption equilibrium, in mg / mL; C represents the theobromine concentration in the desorption solution, in mg / mL.

[0093] The static adsorption rates and desorption rates of theobromine for nine macroporous adsorption resins with different polarities are shown in Table 1.

[0094] Table 1 shows that the adsorption rates of the various macroporous adsorption resins differed significantly. Among them, AB-8, HPD-100, D101, X-5, and HJ-04 exhibited static adsorption rates exceeding 89%, indicating better adsorption performance. These five macroporous adsorption resins are primarily nonpolar or weakly polar, making them more suitable for adsorbing weakly polar substances from polar solvents (water). However, the static desorption rates of all tested macroporous adsorption resins were very low. This may be due to the low solubility of theobromine in ethanol. During static adsorption experiments, once the concentration of theobromine in the desorption solution reached saturation, further desorption of theobromine adsorbed in the macroporous adsorption resins was impossible. Therefore, dynamic adsorption and desorption experiments were conducted using AB-8, HPD-100, D101, X-5, and HJ-04 to further screen suitable macroporous adsorption resins.

[0095] Table 1. Static adsorption and desorption rates of theobromine for various types of macroporous adsorption resins.

[0096]

[0097] 2. Determination of the dynamic saturation adsorption capacity of macroporous adsorption resin

[0098] Five macroporous adsorption resins—AB-8, HPD-100, D101, X-5, and HJ-04—selected from the static adsorption experiments were tested as follows: The treated macroporous adsorption resins were wet-packed into a chromatography column with an inner diameter of 26 mm, a resin packing height of 95 mm, and a column volume (BV) of approximately 50 mL. At room temperature, the aqueous extract of cocoa powder was continuously loaded at a flow rate of 2 BV / h. The eluent was collected, with each BV representing one fraction. The theobromine content of each fraction was measured, and dynamic adsorption-leakage curves were plotted.

[0099] Generally, adsorption is considered saturated when the theobromine content in the sample reaches 10% of the loading solution. The dynamic adsorption-leakage curves for theobromine of five macroporous adsorption resins—AB-8, HPD-100, D101, X-5, and HJ-04—are shown below. Figure 1 As shown, the results indicate that D101 leaked at 5 BV (the theobromine content in this fraction was 11.33% of the sample); HPD-100 was close to adsorption saturation at 5 BV (9.60%), and leaked significantly at 6 BV (19.89%); HJ-04 and X-5 leaked at 6 BV (13.53% and 14.60%, respectively); and AB-8 only leaked at 7 BV (12.54%). Clearly, AB-8 macroporous adsorption resin had the best adsorption effect, followed by HJ-04 and X-5, all significantly better than D101.

[0100] Next, the adsorption and desorption rates of the three macroporous adsorption resins, AB-8, HJ-04, and X-5, were determined using a sample loading amount of 5 BV.

[0101] 3. Determination of dynamic adsorption rate and desorption rate of macroporous adsorption resin

[0102] The three macroporous adsorption resins selected, AB-8, HJ-04, and X-5, were packed into columns and loaded with samples according to the method described in "2. Determination of Dynamic Saturated Adsorption Capacity of Macroporous Adsorption Resins". The sample loading volume was 5 BV, and the column was equilibrated for 0.5 h after loading. The column was then eluted sequentially with 2 BV of distilled water and 5 BV of 95% ethanol aqueous solution at a flow rate of 2 BV / h. All eluents were collected, with each BV fraction being a separate fraction. The theobromine content of each fraction was determined, and the dynamic adsorption rate and desorption rate were calculated according to formulas (3) and (4).

[0103] Equation (3);

[0104] Equation (4)

[0105] In the formula, V0 is the volume of the sample solution, in mL; C iV represents the theobromine concentration of the i-th fraction, in mg / mL. i The volume of the i-th fraction is expressed in mL.

[0106] Table 2 shows the adsorption rates, water washing loss rates, and alcohol elution desorption rates of theobromine for X-5, HJ-04, and AB-8 macroporous adsorption resins. The results in Table 2 indicate that AB-8 resin exhibits the highest adsorption rate for theobromine, the lowest theobromine loss rate during water washing, and the highest alcohol elution desorption rate, demonstrating the best enrichment effect for theobromine. Therefore, AB-8 was selected for the separation and purification of theobromine.

[0107] Table 2. Dynamic adsorption and desorption rates of theobromine for three macroporous adsorption resins.

[0108]

[0109] Example 1

[0110] Lightly alkalized low-fat cocoa powder (purchased from Zhengzhou Zhongyi Biotechnology Co., Ltd., product number AP600, originating from Indonesia) was added to 10 times its weight of distilled water and extracted at room temperature using an ultrasonic circulating extraction machine. The ultrasonic frequency was 40 kHz, the power was 750 W, the total extraction time was 50 min, the ultrasonic time and interval were 2 s / 1 s, the stirring speed was 1500 rpm, and the liquid was filtered through a 300-mesh filter. The filtrate was allowed to stand at -20℃ for 2 h, and then filtered through a 400-mesh filter cloth while cold to remove insoluble matter, obtaining a cocoa powder aqueous extract containing theobromine. The theobromine-containing cocoa powder aqueous extract was detected by HPLC-DAD (see [link to HPLC-DAD method]). Figure 2 The detection wavelength of the HPLC-DAD method was 271 nm, and the theobromine content was found to be 1.6328 mg / g, with a dry matter mass fraction of 1.48%.

[0111] The cocoa powder aqueous extract containing theobromine was heated to 35°C and separated by tangential flow filtration using a 50nm ceramic membrane to obtain the permeate. When the amount of liquid in the tank was very small, distilled water (5% of the original mass) was added in small amounts several times for further dialysis, and all the permeate from the 50nm membrane was collected.

[0112] AB-8 macroporous adsorption resin was soaked, packed into a column, and pretreated according to standard procedures. At room temperature, the permeate from the 50 nm membrane was loaded at a flow rate of 2 BV / h, with a loading volume of 6 BV. After loading, the mixture was allowed to stand for half an hour, followed by rinsing with distilled water to remove impurities, with an elution volume of 2 BV. Elution was then performed with a 35% (v / v) ethanol aqueous solution at a flow rate of 4 BV / h, with an elution volume of 4 BV. The eluent was collected to obtain a cocoa extract containing high theobromine content. The dry matter mass fraction of theobromine in the high theobromine cocoa extract was determined to be 21.96%.

[0113] Example 2

[0114] Commercially available cocoa extract (purchased from Henan Runqu Trading Co., Ltd., product number RQ-17999, originating from Germany) was used as the theobromine-containing cocoa extract. The theobromine content was measured to be 2.5561 mg / g, 1,2-propanediol content to be 67%, glycerol content to be 21.69%, and water content to be 3.79%. It contained no ethanol, and the dry matter mass fraction of theobromine was calculated to be 3.40%. Two times the mass of purified water was added to this theobromine-containing cocoa extract, and the mixture was thoroughly stirred and dissolved in a high-shear mixing vessel. The mixture was then centrifuged using a high-speed tubular centrifuge (16000 rpm), and the supernatant was collected.

[0115] The centrifuged supernatant was subjected to 200nm ceramic membrane separation to obtain 200nm permeate. This 200nm permeate was then used as the feed solution for 10nm membrane separation, and the 10nm permeate was finally collected. During each stage of membrane separation, when the feed solution in the tank was low, small amounts of purified water (10% of the feed solution mass) were added multiple times for further dialysis to promote the complete permeation of theobromine.

[0116] HPD-100 macroporous adsorption resin was soaked, packed into a column, and pretreated according to standard procedures. At room temperature, the 10nm permeate was loaded at a flow rate of 3 BV / h, with a loading volume of 12 BV. After loading, the sample was allowed to stand for 1 hour, followed by rinsing with distilled water to remove impurities, with an elution volume of 1 BV. Elution was then performed using a 50% (v / v) aqueous solution of ethanol as the eluent at a flow rate of 1 BV / h, with an elution volume of 5 BV. The eluent was collected to obtain the cocoa extract containing high theobromine content. The dry matter mass fraction of theobromine in the high theobromine cocoa extract was determined to be 21.35%.

[0117] Example 3

[0118] Commercially available cocoa extract (purchased from Zhengzhou Zhongyi Biotechnology Co., Ltd., product number 80028, sourced from Deweilong Flavors (China) Co., Ltd.) was used as a theobromine-containing cocoa extract. The theobromine content was measured to be 24.8745 mg / g, the moisture content was 0.69%, and it contained no ethanol, 1,2-propanediol, or glycerol. The dry matter mass fraction of theobromine was calculated to be 2.50%. Nine times the mass of purified water was added to this theobromine-containing cocoa extract, and the mixture was thoroughly stirred and dissolved in a high-shear mixing vessel. The mixture was then centrifuged using a high-speed tubular centrifuge (16000 rpm), and the supernatant was collected.

[0119] The centrifuged supernatant was subjected to 50nm ceramic membrane separation to obtain 50nm permeate. This 50nm permeate was used as the feed solution for 30kDa organic membrane separation, and the 30kDa permeate was finally collected. During each stage of membrane separation, when the feed solution in the tank was low, small amounts of purified water (3% of the feed solution mass) were added multiple times for further dialysis to promote the complete permeation of theobromine.

[0120] X-5 macroporous adsorption resin was soaked, packed into a column, and pretreated according to standard procedures. At room temperature, 30 kDa of the permeate was loaded at a flow rate of 1 BV / h, with a loading volume of 4 BV. After loading, the mixture was allowed to stand for 1 hour, followed by rinsing with distilled water to remove impurities, with an elution volume of 3 BV. Elution was then performed with a 95% ethanol aqueous solution at a flow rate of 2 BV / h, with an elution volume of 4 BV. The eluent was collected to obtain the cocoa extract containing high theobromine content. The dry matter mass fraction of theobromine in the high theobromine cocoa extract was determined to be 20.83%.

[0121] Experimental Example 1

[0122] The theobromine-containing cocoa powder aqueous extract and the high-theobromine-containing cocoa extract prepared in Example 1 were mixed sequentially at dry matter mass ratios of 5:1, 1:1, and 1:5 to obtain three cocoa extract compound products (labeled sequentially as compound ①, ②, and ③). The theobromine dry matter mass fraction in compound ① was 4.89%, in compound ② it was 11.72%, and in compound ③ it was 18.55%.

[0123] Five samples were prepared by adding the cocoa powder aqueous extract containing theobromine, cocoa extract containing high theobromine, compound ①, compound ②, and compound ③, respectively, to a blank experimental cigarette (made by Shanghai Tobacco Group) without flavoring or additives using a cigarette flavoring injection machine at a dry matter content of 20 μg / g tobacco shreds.

[0124] The sample cigarettes and blank cigarettes were sealed and stored for 7 days at a temperature of (22±1)℃ and a relative humidity of 60%±2%. The sample cigarettes and blank cigarettes were sensory evaluated by a 15-person evaluation panel in accordance with YC / T 497—2014 "Sensory Evaluation Method for Chinese Style Cigarettes".

[0125] In the sensory evaluation, all indicators are scored in units of 0.5; an evaluation team is established in accordance with the requirements of GB 5606.4.

[0126] Calculate the average score for each indicator using the following formula:

[0127] ;

[0128] In the formula: This represents the average score of a single indicator. This represents the sum of scores for a single indicator; n represents the number of participants in the evaluation.

[0129] The evaluation scores for YC / T 497—2014 "Sensory Evaluation Method for Chinese Style Cigarettes" are shown in Tables 3-6.

[0130] Table 3. Evaluation Scores of Smoke Characteristic Indicators in YC / T 497—2014 "Sensory Evaluation Method for Chinese-Style Cigarette Style"

[0131]

[0132] Table 4. Evaluation Scores of Comfort Characteristic Indicators in YC / T 497—2014 "Sensory Evaluation Method for Chinese-Style Cigarettes"

[0133]

[0134] Table 5. Flavor Style Index Scores in YC / T 497—2014 "Sensory Evaluation Method for Chinese-Style Cigarettes"

[0135]

[0136] Table 6. Aroma and Smoking Indicator Scores in YC / T 497—2014 "Sensory Evaluation Method for Chinese-Style Cigarette Style"

[0137]

[0138] The sensory evaluation results are shown in Table 7. The results show that cocoa extract with high theobromine content is better than cocoa powder water extract with theobromine content in increasing smoke concentration, strength, masking off-flavors, and reducing irritation. At the same time, it does not increase oral bitterness, does not reduce the aroma of cigarettes, and the cocoa aroma is more prominent.

[0139] Compared to using cocoa powder water extract containing theobromine and cocoa extract containing high levels of theobromine alone, the three compound products are more effective in enhancing cigarette aroma, increasing smoke richness, smoke concentration, strength, and cocoa aroma, without increasing bitterness in the mouth or reducing the cigarette's freshness. The overall effect of the compound products is significantly better. Among them, the sensory effect of compound product No. 2 is better than that of No. 1 and No. 3. It can be seen that compared with the dry matter mass fraction of theobromine in the compound products of 4.89% or 18.55%, the dry matter mass fraction of theobromine in the compound product is 11.72%, and its cigarettes have better aroma, richness, smoke concentration, strength, and cocoa aroma.

[0140] Table 7. Sensory evaluation results of sample cigarettes containing theobromine in cocoa powder aqueous extract, high-theobromine cocoa extract, and compound products in Example 1.

[0141]

[0142] Note: The scores for each of the following indicators in evaluating cigarettes are 0: "coolness", "sun-dried tobacco aroma", "fresh aroma", "fruity aroma", "spicy aroma", "floral aroma", "bean aroma", "paste aroma" and "herbal aroma".

[0143] Experimental Example 2

[0144] The cocoa extract with high theobromine content prepared in Example 2 was mixed with the commercially available cocoa extract from Example 2 at a dry matter mass ratio of 1:2 to obtain a cocoa extract blend product with enhanced style. The dry matter mass fraction of theobromine in the cocoa extract blend product was 9.38%.

[0145] Using the unflavored and unprocessed "Purple Qi Comes from the East" as a blank cigarette, the cocoa extract compound product, the commercially available cocoa extract from Example 2, and the cocoa extract containing high theobromine were added to the blank cigarette at a dry matter content of 50 μg / g of tobacco using a cigarette flavoring injection machine to prepare sample cigarettes. Separately, a blank cigarette was used to prepare a reference cigarette by adding a mixed solvent of 1,2-propanediol and glycerol in a mass ratio of 3:1 to 590 μg / g of tobacco (the same total mass ratio of 1,2-propanediol and glycerol as in the sample cigarette prepared from the commercially available cocoa extract in Example 2) using a cigarette flavoring injection machine.

[0146] The sample cigarettes and reference cigarettes were sealed and stored at (22±1)℃ and 60%±2% relative humidity for 7 days to equilibrate. The sample cigarettes and reference cigarettes were then subjected to sensory evaluation by an evaluation panel (11 people) according to the method in Experiment Example 1.

[0147] The results are shown in Table 8: Cocoa extracts with high theobromine content are more effective than commercially available cocoa extracts before separation in enhancing the strength, aroma, and cocoa flavor of cigarettes, without increasing irritation, dryness, or bitterness, or reducing the aroma of flue-cured tobacco, resulting in a better overall effect.

[0148] A cocoa extract with a dry matter content of 9.38% was obtained by blending cocoa extract containing high theobromine with commercially available cocoa extract at an appropriate mass ratio (1:2). This cocoa extract compound product has outstanding effects in improving the comfort of cigarettes, including reducing irritation in the mouth, throat, and nasal cavity, reducing oral residue and astringency, reducing throat dryness, and increasing sweetness. At the same time, the cocoa extract compound product can also increase the aroma, richness, and smoothness of cigarettes, and enhance the characteristic aromas such as cocoa and roasted aromas. The overall effect of the compound application is significantly improved.

[0149] Table 8 Sensory evaluation results of commercially available cocoa extracts, cocoa extracts containing high theobromine, and cocoa extract blends.

[0150]

[0151] 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.

[0152] Experimental Example 3

[0153] The cocoa extract containing high theobromine prepared in Example 3 was concentrated to the point of being ethanol-free, and then dissolved in water to prepare an aqueous solution of the cocoa extract containing high theobromine.

[0154] The cocoa extract from Example 3 was dissolved in water to prepare an aqueous solution of the cocoa extract.

[0155] An aqueous solution of cocoa extract containing high theobromine and an aqueous solution of cocoa extract were mixed at a dry matter ratio of 1:1 to obtain a cocoa extract blend product with enhanced flavor. The dry matter mass fraction of theobromine in the cocoa extract blend product was 11.67%.

[0156] The compound product, the aqueous solution of cocoa extract, and the aqueous solution of cocoa extract containing high theobromine were added to the coating solution of cigarette paper at a dosage of 100 μg / g dry matter. The paper was then used to make cigarette paper according to existing processes and used to roll tobacco shreds. Filter rods were then attached to the paper to make sample cigarettes. Following the preparation method of the sample cigarettes, a coating solution without added cocoa extract was used as a reference. The same method, the same tobacco shreds, and the same filter rods were used to make reference cigarettes.

[0157] The sample cigarettes and reference cigarettes were sealed and stored at (22±1)℃ and 60%±2% relative humidity for 7 days to equilibrate. The sample cigarettes and reference cigarettes were then subjected to sensory evaluation by an evaluation panel (11 people) according to the method in Experiment Example 1.

[0158] The results are shown in Table 9: When used as a coating liquid for cigarette paper in cigarettes, cocoa extract with high theobromine content has a better effect on enhancing the aroma, concentration, strength, cocoa aroma, and flue-cured tobacco aroma of cigarettes than commercially available cocoa extract (cocoa paste) before separation. It does not increase irritation, residue, dryness, or astringency, does not increase bitterness, and does not reduce the aroma and fragrance of sun-cured tobacco. Overall, it has better coordination and comfort.

[0159] A cocoa extract compound with a dry matter content of 11.67% was obtained by mixing cocoa extract with high theobromine content and commercially available cocoa extract (cocoa concentrate) in a suitable mass ratio (1:1). When added to cigarette paper coating liquid for use in cigarettes, this compound product showed superior effects in increasing cigarette aroma, smoke concentration, strength, and enhancing cocoa, sweet, and flue-cured tobacco aromas. It also demonstrated better reduction of irritation, astringency, and masking of off-flavors. This indicates that the application effect of a compound cocoa extract with high theobromine content and commercially available cocoa extract (cocoa concentrate) is superior.

[0160] Table 9. Comparison of the effects of commercially available cocoa extracts, aqueous solutions of cocoa extracts containing high theobromine, and their compound products in the flavoring of cigarette paper.

[0161]

[0162] Note: The scores for each of the evaluation indicators for cigarettes, including "coolness", "fruity aroma", "bean aroma", "green aroma", "floral aroma", "paste aroma", "milky aroma" and "herbal aroma", were all 0.

[0163] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0164] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a cocoa extract containing high levels of theobromine, characterized in that, include: The cocoa raw material containing theobromine is extracted or dissolved in water to remove insoluble matter and collect the clear liquid. The supernatant is separated by membrane separation, and the permeate is collected; The permeate is adsorbed by a macroporous adsorption resin, impurities are removed, and the eluent is collected to prepare a cocoa extract with high theobromine content. The macroporous adsorption resin includes one or more of AB-8, X-5, HPD-100 and HJ-04.

2. The method for preparing cocoa extract containing high theobromine according to claim 1, characterized in that, The macroporous adsorption resin includes one or more of AB-8, X-5 and HJ-04; Membrane separation methods include one or more of microfiltration, ultrafiltration, and nanofiltration; The theobromine-containing cocoa raw materials include: natural cocoa plant material; processed cocoa bean products; extracts of natural cocoa plant material; or products prepared by one or more of the following methods: microbial fermentation, enzymatic hydrolysis, and Maillard reaction.

3. The method for preparing cocoa extract containing high theobromine according to claim 1, characterized in that, The preparation of cocoa extracts with high theobromine content specifically includes: A macroporous adsorption resin was packed into a chromatography column, and the permeate was loaded onto the chromatography column for adsorption. The first eluent was used to remove impurities, and the second eluent was used for elution. The eluent was collected to prepare a cocoa extract containing high theobromine. The first eluent comprises water; the second eluent comprises an aqueous ethanol solution.

4. The method for preparing cocoa extract containing high theobromine according to claim 3, characterized in that, The sample loading flow rate is 1-4 BV / h; The amount of the first eluent is 1-3 BV; The volume percentage of the ethanol-water solution is 35-95%; The elution volume of the second eluent is 2-5 BV; The elution flow rate of the second eluent is 1-4 BV / h.

5. A cocoa extract containing high theobromine prepared by the method for preparing a cocoa extract containing high theobromine according to any one of claims 1-4.

6. The use of the cocoa extract containing high theobromine prepared by the method of any one of claims 1-4, or the cocoa extract containing high theobromine as described in claim 5, in tobacco flavorings.

7. A tobacco flavoring, characterized in that, include: The cocoa extract containing high theobromine prepared by the method of any one of claims 1-4, or the cocoa extract containing high theobromine as described in claim 5; other cocoa extracts or tobacco flavorings.

8. The tobacco flavoring according to claim 7, characterized in that, The dry matter mass fraction of theobromine in the tobacco flavoring is 4.89-18.55%.

9. A cigarette paper coating liquid, characterized in that, This includes the cocoa extract containing high theobromine prepared by the method for preparing the cocoa extract containing high theobromine according to any one of claims 1-4, the cocoa extract containing high theobromine according to claim 5, or the tobacco flavoring according to any one of claims 7-8.

10. A cigarette, characterized in that, The raw materials include the cocoa extract containing high theobromine prepared by the method of preparing the cocoa extract containing high theobromine according to any one of claims 1-4, the cocoa extract containing high theobromine according to claim 5, the tobacco flavoring according to any one of claims 7-8, or the cigarette paper coating liquid according to claim 9.

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