A tobacco flavor prepared based on a composite enzyme and a preparation process thereof

Abstract

The present application belongs to the technical field of tobacco essence preparation and biocatalysis application, and particularly relates to a tobacco essence prepared based on composite enzymes and a preparation process. The process first proportionally compounds pectinase, beta-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase, and forms microcapsules through a sodium alginate-chitosan system; prepares an extract after steam explosion pretreatment of tobacco powder, loquat leaves and monk fruit; adds the microcapsule enzymes for three-stage segmented enzymolysis; further adds hydroxypropyl-beta-cyclodextrin to include aroma molecules, and after enzyme inactivation and filtration, concentrates and adds vitamin E to stabilize the quality of the essence, so as to finally obtain a tobacco essence with rich aroma levels and good thermal stability. Through the synergistic effect of composite enzymes and multi-stage reaction regulation, the present application realizes the directional release and construction of flavor substances in natural raw materials, and the obtained essence is suitable for various application scenarios such as cigarette flavoring and paper spraying.

Description

Technical Field

[0001] This invention belongs to the field of tobacco flavoring preparation and biocatalysis application technology, specifically relating to a tobacco flavoring prepared based on a compound enzyme and its preparation process. Background Technology

[0002] Tobacco flavorings are an important component of cigarette formulations, significantly influencing the flavor quality, aroma concentration, taste harmony, and post-smoking pleasure of tobacco products. Traditional methods for preparing tobacco flavorings primarily rely on plant extraction, thermal reaction flavoring, and chemical synthesis. While these methods are widely used in the tobacco industry, they still have many shortcomings in terms of flavor complexity, component harmony, and natural properties. Especially given the increasing emphasis on health, safety, and sustainable development, existing technologies urgently need optimization and innovation.

[0003] Plant extraction typically uses aromatic plants or fragrance raw materials, obtaining essential oils or aromatic components through methods such as steam distillation, solvent extraction, or supercritical fluid extraction. While this method offers the advantage of "natural sources," it also suffers from low extraction efficiency, susceptibility to thermal degradation of active components, and poor product flavor stability. Furthermore, the limited availability of natural plant resources puts significant pressure on this method in terms of raw material acquisition and cost control.

[0004] Thermal flavoring relies on heat-induced processes such as the Maillard reaction and caramelization to generate complex aroma components, and is widely used in reconstituted tobacco and tobacco flavorings. This method can effectively enhance the "tobacco aroma" and "roasted aroma" of tobacco smoke, but the heat treatment process is prone to producing off-flavors or unpleasant gases, and some reaction byproducts also pose potential health risks. Furthermore, due to the high uncertainty of thermal reactions, the resulting flavorings exhibit poor batch-to-batch stability, making it difficult to establish a controllable and standardized flavor system.

[0005] Chemical synthesis methods, through the artificial synthesis of natural or artificial flavorings, can improve yield and cost-effectiveness to some extent. However, there is a significant gap between chemically synthesized and natural flavorings in terms of flavor delicacy and harmony, and some artificial flavorings are subject to safety controversies. Especially under increasingly stringent regulations, the use of chemical flavorings is constantly being squeezed. Furthermore, there are many synergistic and antagonistic effects in flavor formulation, and simple blending often fails to achieve a high degree of integration with the aroma of natural tobacco.

[0006] In recent years, with the development of bio-enzyme technology, enzyme-catalyzed flavoring has gradually attracted industry attention. Single enzyme reactions, such as lipase-catalyzed esterification and oxidase-catalyzed dehydrogenation, have been used in the construction of flavor precursors or the conversion of natural products. However, in practical applications, single enzyme reactions suffer from problems such as narrow substrate selectivity, insufficient flavor complexity, and monotonous reaction systems, making it difficult to meet the needs of constructing highly complex and multi-dimensional flavors.

[0007] Especially in the field of tobacco flavorings, aroma not only requires intensity but also a rich variety of "top notes," "main notes," and "after notes." Traditional single-enzyme systems struggle to achieve the synergistic construction of multidimensional aroma profiles. Furthermore, most current enzyme catalysis methods still employ single-substrate or binary substrate systems, lacking systematic research on the synergistic transformation mechanisms between multiple components in natural complex substrates. This results in existing enzymatic flavorings lacking sufficient stability and flavor expressiveness, making them difficult to replace traditional flavoring systems in practical applications.

[0008] Therefore, there is an urgent need to develop a new preparation method with multi-pathway conversion capabilities, strong flavor construction diversity, high safety, and industrial controllability, in order to improve the quality performance and product differentiation of tobacco flavorings and meet the multiple needs of the new generation of consumers for health, safety and natural flavor. Summary of the Invention

[0009] The purpose of this invention is to provide a tobacco flavoring based on compound enzymes and its preparation process, in order to solve the problems of insufficient naturalness, single flavor layers, poor stability, and reliance on high temperature or chemical synthesis in existing tobacco flavorings. This invention achieves a gentle and efficient conversion of various natural raw materials, resulting in a compound tobacco flavoring with rich aroma, safe ingredients, and good stability.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] The first aspect of this invention provides a process for preparing tobacco flavorings based on compound enzymes, comprising the following steps:

[0012] (1) Pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase are mixed to obtain a compound enzyme powder. The compound enzyme powder is then encapsulated in sodium alginate-chitosan microcapsules to obtain compound enzyme microcapsules.

[0013] (2) Mix tobacco fragments, loquat leaves and monk fruit and steam-explode them, then extract with ethanol aqueous solution and supercritical CO2 in sequence, and combine the extracts;

[0014] (3) Add 2-4% of the mass of the compound enzyme microcapsules to the extract obtained in step (2) and carry out a three-stage enzymatic hydrolysis reaction to obtain a mixture;

[0015] (4) Add 0.4-0.6% by mass of hydroxypropyl-β-cyclodextrin to the mixture obtained in step (3), stir, inactivate the enzyme, filter, and collect the filtrate;

[0016] (5) Concentrate the filtrate and add vitamin E to obtain tobacco flavoring.

[0017] Further, the pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase, and acetyltransferase described in step (1) are mixed in the following ratio (activity units U): (1.0±0.2):(1.0±0.2):(0.5±0.1):(0.5±0.1):(0.3±0.06):(0.03±0.006). For example, in the above ratio, 1.0±0.2 represents a range of 0.8-1.2, and the others are the same.

[0018] Further, the method of encapsulating the compound enzyme powder in sodium alginate-chitosan microcapsules in step (1) includes the following steps:

[0019] (a) Add the compound enzyme powder to a sodium alginate aqueous solution with a mass fraction of 1.5-4% at a mass ratio of 1:3-5, stir, and form a compound solution;

[0020] (b) Add the above composite solution dropwise to a CaCl2 aqueous solution with a mass fraction of 1-4% at a mass ratio of 1:4-7, stir and react for 20-40 minutes to form gel-like alginate calcium spheres;

[0021] (c) Take out the above alginate calcium spheres, rinse with deionized water, transfer to a chitosan acetic acid aqueous solution with a mass fraction of 0.2-0.8%, react with stirring for 30-50 minutes to obtain chitosan-coated microcapsules;

[0022] (d) The chitosan-coated microcapsules were transferred into a sodium tripolyphosphate solution of 0.8-2% by mass for cross-linking for 10-20 minutes, and then washed and dried to obtain the composite enzyme microcapsules.

[0023] In the compound enzyme microcapsules, pectinase and β-glucosidase primarily act on plant cell walls and phenolic glycoside structures, releasing a large amount of latent aroma components bound by glycosidic bonds, such as phenylethanol glycosides and eugenol, under mild conditions. These substances have higher volatility and olfactory intensity in their free state, providing a foundation for aroma top notes. Lipases catalyze the transesterification of natural esters and alcohols in the substrate. On the one hand, they hydrolyze residual plant surface wax esters; on the other hand, they react with alcohols released in the system to generate ester aroma molecules. These components help enhance harmony and smoothness in tobacco flavorings and are important aroma-constructing factors for "roundness." Lipoxygenases catalyze the oxidative modification of certain polyphenols or terpenes containing double bonds, forming small aldehydes or ketones. These intermediate products often manifest as grassy or fruity aromas in flavor construction, enhancing the freshness and penetration of the top notes. Glucose oxidase oxidizes glucose to gluconic acid, generating trace amounts of hydrogen peroxide in the process. This process regulates the concentration of free sugars in the reaction system, preventing sugars from participating in the non-selective Maillard reaction and causing unpleasant odors. Furthermore, low concentrations of H₂O₂ can have a mild promoting effect in certain esterification reactions or aldehyde-alcohol oxidation, thus this enzyme plays a guiding role in regulating flavor reaction pathways. Acetyltransferase, added in a very small proportion to the complex system, primarily catalyzes the transfer of acetyl groups from acetyl-CoA to alcohol substrates, generating esters. Many key components contributing to the "high-quality" feel of tobacco flavorings, such as phenethyl acetate, geraniol acetate, and linalool acetate, belong to this type of structure. These substances exhibit high stability during tobacco combustion and can penetrate the smoke to create a pleasant "aftertaste." Simultaneously, acetyltransferase can synergistically work with lipases to increase ester yields and enhance the aromatization capacity of the enzymatic hydrolysis system.

[0024] Furthermore, the synergistic time windows of these six enzymes in the reaction do not completely overlap, which is another consideration for using microencapsulation. By coating a chitosan layer onto a gel sphere formed from sodium alginate and then stabilizing it with sodium tripolyphosphate crosslinking, the release of enzymes can be controlled, avoiding premature inactivation or mutual interference. This helps to maintain the staggered distribution of enzyme activity peaks at different pH and temperature stages, providing a technical basis for subsequent staged enzymatic hydrolysis.

[0025] Further, the mass ratio of tobacco shreds, loquat leaves and monk fruit in step (2) is (2.5-5):(1-2):(1-2).

[0026] Furthermore, the steam explosion in step (2) is performed at a temperature of 118-122°C, a pressure of 1.1-1.3 MPa, and a time of 85-95 seconds.

[0027] Furthermore, the temperature of the supercritical CO2 extraction in step (2) is 33-37℃, the pressure is 24-26 MPa, and the time is 1.8-3 hours.

[0028] The substrate materials selected for this invention are tobacco dust, loquat leaves, and monk fruit. Tobacco dust, as the base material, not only contains nicotine but is also rich in roasting aroma precursors, maleic coumarin aroma compounds, amino acids, polyphenols, and a certain amount of sugars, forming the core of the "authentic tobacco aroma." Loquat leaves are rich in ursolic acid, oleanolic acid, tannins, and bitter glycosides, possessing a herbal aroma, particularly suitable for adjusting the "bitter aftertaste" in tobacco aroma and balancing the harshness caused by excessive roasting. Monk fruit's main components are triterpenoid saponins, which, in a highly diluted state, exhibit a mellow fruity aroma and sweetness, providing aroma support and mitigating the harshness of tobacco aroma. The combination of these three raw materials creates a complementary aroma profile, possessing a typical "tobacco + herb + fruit" triple structure, providing extremely rich flavor precursors for the subsequent enzymatic hydrolysis process.

[0029] Traditional extraction methods often employ boiling or alcohol maceration, but these methods frequently suffer from low extraction efficiency and blockage of active ingredients in plant tissues with dense cell structures or high degrees of lignification. This invention utilizes steam explosion technology to instantly rupture cell walls and loosen tissue structures, releasing previously inaccessible intracellular components. Simultaneously, it allows some bound flavor precursors to enter a free state without significant thermal degradation, significantly improving subsequent extraction and enzymatic hydrolysis efficiency. After the explosion treatment, polar and moderately polar components, such as phenols, glycosides, and amino acids, are first extracted from the raw material using an ethanol-water solution. Subsequently, supercritical CO2 extraction is used to extract non-polar substances, such as monoterpenes, sesquiterpenes, and lipid-soluble aromatic components like alcohols, compensating for the shortcomings of the water-alcohol extraction system. Finally, the two extracts are combined to obtain a complex substrate solution rich in flavor components and with complementary polar structures, providing diverse precursors for subsequent enzymatic hydrolysis.

[0030] Furthermore, the conditions for the tertiary enzymatic hydrolysis reaction described in step (3) are as follows:

[0031] First stage: pH 4.8-5.2, 38-42℃, reaction time 1.8-2.2 hours;

[0032] Second stage: pH 6.6-7.0, 48-52℃, add 0.08-0.12% L-leucine by weight of the extract, and react for 1.3-1.7 hours;

[0033] Third stage: pH 6.6-7.0, 43-47℃, add 0.04-0.06% of UDP-glucose by weight of the extract, and react for 0.8-1.2 hours.

[0034] This invention employs a three-stage enzymatic hydrolysis strategy to release and transform aroma precursors stepwise. The first stage activates pectinase and β-glucosidase under acidic conditions, promoting the full release of glycosides and cell wall-bound substances. The second stage adjusts the pH to a slightly alkaline state and adds L-leucine to guide amino acid precursors to participate in flavor-building reactions. The third stage maintains a neutral pH and adds UDP-glucose to promote the formation of glycoside structures, improving aroma stability and longevity. These three stages synergistically complete precursor release, aroma building, and stabilization modification.

[0035] Furthermore, the stirring temperature in step (4) is 48-52℃ and the stirring time is 25-35 minutes.

[0036] In step (4), hydroxypropyl-β-cyclodextrin is introduced as a stabilizer to "encapsulate" the hydrophobic components in the flavoring through inclusion, preventing them from oxidizing and degrading under high temperature, light, or air. At the same time, inclusion can also improve the water solubility of some hydrophobic molecules, making the flavoring more suitable for modern cigarette flavoring or spraying processes.

[0037] Further, in step (5), the filtrate is concentrated to 18-22% of its original volume, and the added vitamin E is 0.8-1.2% of the concentrated filtrate.

[0038] The addition of vitamin E has an antioxidant effect and can also inhibit free radical chain reactions during the storage and use of flavorings. It has a significant protective effect on easily oxidized components such as polyphenols and alcohols in the aroma system, thereby improving the flavor retention and color stability of the finished product during use.

[0039] The second aspect of the present invention provides a tobacco flavoring prepared by the above-mentioned process for preparing tobacco flavoring based on compound enzymes.

[0040] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0041] This invention achieves precise release, structural regulation, and sustained-release protection of natural aroma precursors by constructing a complex enzyme system based on multiple functional enzymes and supplementing it with microencapsulation, multi-source plant extraction, three-stage enzymatic hydrolysis, and cyclodextrin inclusion. Compared with traditional processes, this method not only improves the conversion rate of flavor substances but also forms rich aroma structure layers through staged enzymatic reactions, significantly enhancing the "top note performance," "main aroma fullness," and "after-scent persistence" of tobacco flavorings. Simultaneously, through cyclodextrin inclusion and vitamin E antioxidant protection, it effectively improves the thermal stability and usability of the finished product during high-temperature flavoring. The overall process is green and environmentally friendly, with widely available raw materials, mild reaction conditions, and high product safety, making it suitable for industrial application in the cigarette, fragrance, and paper industries. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0043] Unless otherwise specified, all raw materials used in the embodiments are commercially available products. The following sources are for illustrative purposes only:

[0044] Pectinase was purchased from Sinopharm Chemical Reagent Co., Ltd., with an enzyme activity of 30,000 U / g.

[0045] β-glucosidase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 20-40 U / mg.

[0046] The lipase was purchased from Beijing Bailingwei Technology Co., Ltd., with an enzyme activity of 30,000 U / g.

[0047] Lipooxygenase was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., with an enzyme activity of 50,000 U / mg.

[0048] Glucose oxidase was purchased from Yunbang Biotechnology Co., Ltd., with an enzyme activity of 100 U / mg.

[0049] Acetyltransferase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with an enzyme activity of 50 U / mg.

[0050] The cellulase was purchased from Shanghai Huashang Xiangyang Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0051] Catalase was purchased from Maclean Biotechnology Co., Ltd., with an enzyme activity of 3500 U / mg.

[0052] The transaminase was purchased from Unicore (Shanghai) Life Science Co., Ltd., with an enzyme activity of 75 U / mg.

[0053] Example 1

[0054] This embodiment provides a process for preparing tobacco flavorings based on compound enzymes, including the following steps:

[0055] (1) Preparation of compound enzyme microcapsules: Pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase were weighed and mixed in an enzyme dosage (activity unit U) ratio of 1.0:1.0:0.5:0.5:0.3:0.03 to obtain compound enzyme powder; the compound enzyme powder was added to a 2.5% sodium alginate aqueous solution at a mass ratio of 1:4 and stirred at 600 rpm for 15 minutes at room temperature to form a compound solution; then, the compound solution was added dropwise to a 3% CaCl2 solution at a mass ratio of 1:5, and the dropping rate was controlled at 1. The mixture was stirred at a rate of mL / min for 30 minutes to form calcium alginate gel spheres. The gel spheres were then removed, rinsed three times with deionized water, and transferred to a 0.4% (w / w) chitosan-acetic acid aqueous solution (acetic acid to water mass ratio 1:80), with a mass ratio of calcium alginate gel spheres to 0.4% chitosan-acetic acid aqueous solution of 1:5. The mixture was stirred at room temperature for 40 minutes to form primary microcapsules. These microcapsules were then added to a 1.5% (w / w) sodium tripolyphosphate aqueous solution, with a mass ratio of primary microcapsules to 1.5% sodium tripolyphosphate aqueous solution of 1:7. The mixture was cross-linked at room temperature for 15 minutes, washed twice with deionized water, and vacuum dried at 40°C for 12 hours to obtain composite enzyme microcapsules.

[0056] (2) Weigh 50g of tobacco powder, 15g of dried loquat leaves and 20g of dried monk fruit, mix them and place them in a steam explosion device. Treat them at 120℃ and 1.2 MPa for 90 seconds. After treatment, cool them to room temperature and transfer the explosion raw material to a 1000 mL three-necked flask. Add 400 mL of 60% ethanol aqueous solution and stir and extract at 60℃ for 2 hours. Filter and collect the filtrate. Add 400 mL of 80% ethanol aqueous solution to the residue again and extract it under the same conditions for 2 hours. Filter and combine the two extracts and concentrate them to 150 mL. Then transfer the concentrate to a supercritical extraction device and perform supercritical extraction at 35℃ and 25 MPa with CO2 as the fluid for 2 hours. Collect the extract as the substrate liquid for subsequent reactions.

[0057] (3) Add 3% by mass of the compound enzyme microcapsules to the above substrate solution to start the three-stage enzymatic hydrolysis reaction. In the first stage, adjust the pH to 5.0 and react at 40°C for 2 hours. In the second stage, adjust the pH to 6.8, add 0.1% by mass of L-leucine to the substrate solution, and react at 50°C for 1.5 hours. In the third stage, maintain the pH at 6.8, add 0.05% by mass of UDP-glucose to the substrate solution, and react at 45°C for 1 hour.

[0058] (4) After the enzymatic hydrolysis is completed, a mixture is obtained. 0.5% of hydroxypropyl-β-cyclodextrin by mass of the mixture is added to the mixture. The mixture is stirred at 50°C for 30 minutes, then heated to 85°C and kept warm for 10 minutes to inactivate the enzyme. After cooling to room temperature, the mixture is filtered and the filtrate is collected. The filtrate obtained is crude flavoring liquid.

[0059] (5) Concentrate the crude flavoring liquid to 20% of its original volume under reduced pressure, then add 1.0% of its mass of vitamin E to the concentrate and stir evenly at room temperature to obtain the finished tobacco flavoring product.

[0060] Example 2

[0061] This embodiment provides a process for preparing tobacco flavorings based on compound enzymes, including the following steps:

[0062] (1) Preparation of compound enzyme microcapsules: Pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase were weighed according to the enzyme dosage (activity unit U) ratio of 1.2:1.0:0.6:0.4:0.32:0.03, mixed evenly to obtain compound enzyme powder. The compound enzyme powder was added to a 3.5% sodium alginate aqueous solution at a mass ratio of 1:3 and stirred at 550 rpm for 18 minutes at room temperature to form a compound solution. Subsequently, the compound solution was added dropwise to a 2.5% CaCl2 solution at a mass ratio of 1:6, and the dropping rate was controlled at 0.8%. The mixture was stirred at a rate of mL / min for 35 minutes to form calcium alginate gel spheres. The gel spheres were then removed, rinsed three times with deionized water, and transferred to a 0.6% (w / w) chitosan-acetic acid aqueous solution (acetic acid to water mass ratio 1:80). The mass ratio of the calcium alginate gel spheres to the 0.6% chitosan-acetic acid aqueous solution was 1:4.8. The mixture was stirred at room temperature for 35 minutes to form primary microcapsules. These microcapsules were then added to a 1.2% (w / w) sodium tripolyphosphate aqueous solution (the mass ratio of the primary microcapsules to the 1.2% sodium tripolyphosphate aqueous solution was 1:7.5). The mixture was cross-linked at room temperature for 20 minutes, washed twice with deionized water, and vacuum dried at 48°C for 10 hours to obtain composite enzyme microcapsules.

[0063] (2) Weigh 40g of tobacco powder, 12g of dried loquat leaves and 18g of dried monk fruit, mix them and place them in a steam explosion device. Treat them at 122℃ and 1.3MPa for 85 seconds. After treatment, cool them to room temperature and transfer the explosion raw material to a 1000mL three-necked flask. Add 450mL of 60% ethanol aqueous solution and stir and extract at 60℃ for 2 hours. Filter and collect the filtrate. Add 4000mL of 80% ethanol aqueous solution to the residue and extract it again under the same conditions for 2 hours. Filter and combine the two extracts and concentrate to 120mL. Then transfer the concentrate to a supercritical extraction device and perform supercritical extraction with CO2 as the fluid at 37℃ and 24MPa for 2.5 hours. Collect the extract as the substrate liquid for subsequent reactions.

[0064] (3) Add 2.5% by mass of the compound enzyme microcapsules to the above substrate solution to start the three-stage enzymatic hydrolysis reaction. In the first stage, adjust the pH to 5.2 and react at 38°C for 2.2 hours. In the second stage, adjust the pH to 7.0 and add 0.08% by mass of L-leucine to the substrate solution and react at 52°C for 1.3 hours. In the third stage, maintain the pH at 7.0 and add 0.04% by mass of UDP-glucose to the substrate solution and react at 47°C for 0.8 hours.

[0065] (4) After the enzymatic hydrolysis is completed, a mixture is obtained. 0.6% of hydroxypropyl-β-cyclodextrin by mass of the mixture is added to the mixture. The mixture is stirred at 48°C for 35 minutes, then heated to 85°C and kept warm for 10 minutes to inactivate the enzyme. After cooling to room temperature, the mixture is filtered and the filtrate is collected. The filtrate is crude flavoring liquid.

[0066] (5) Concentrate the crude flavoring liquid to 18% of its original volume under reduced pressure, then add 1.2% of its mass of vitamin E to the concentrate and stir evenly at room temperature to obtain the finished tobacco flavoring product.

[0067] Comparative Example 1

[0068] The difference between this comparative example and Example 1 is that pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase are replaced with cellulase, β-glucosidase, lipase, catalase, glucose oxidase and transaminase.

[0069] Comparative Example 2

[0070] The difference between this comparative example and Example 1 is as follows: Step (1) is as follows: pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase are weighed according to the enzyme activity unit ratio of 1.0:1.0:0.5:0.5:0.3:0.03, mixed, and a compound enzyme powder is prepared; the compound enzyme powder is added to a 2.5% sodium alginate aqueous solution at a mass ratio of 1:4, and stirred at 600 rpm for 15 minutes at room temperature to form a uniform compound solution; the compound solution is added dropwise to a 3% CaCl2 solution (compound solution to CaCl2 solution mass ratio of 1:5), the dropping rate is controlled at 1 mL / min, and stirring is maintained. After the addition is completed, the reaction continues for 30 minutes to form calcium alginate gel balls; the gel balls are rinsed three times with deionized water, and vacuum dried at 40℃ for 12 hours, and used directly as compound enzyme microcapsules.

[0071] Comparative Example 3

[0072] The difference between this comparative example and Example 1 is that dried monk fruit is replaced with dried licorice.

[0073] Comparative Example 4

[0074] The difference between this comparative example and Example 1 is as follows: The enzymatic hydrolysis process in step (3) is as follows: 3% by mass of compound enzyme microcapsules are added to the substrate solution prepared in the previous step, and 0.1% by mass of L-leucine and 0.05% by mass of UDP-glucose are added at the same time to start the three-stage enzymatic hydrolysis reaction. In the first stage, the pH is adjusted to 5.0 and the reaction is carried out at 40°C for 2 hours; in the second stage, the pH is adjusted to 6.8 and the reaction is carried out at 50°C for 1.5 hours; in the third stage, the pH is maintained at 6.8 and the reaction is carried out at 45°C for 1 hour.

[0075] Performance testing

[0076] The performance of the tobacco flavorings prepared in Examples 1-2 and Comparative Examples 1-4 was tested.

[0077] Total aroma components: According to GB / T 14454.2-2008 "Fragrance Evaluation Method", the total peak area of ​​volatile components in the fragrance was determined by gas chromatography-mass spectrometry (GC-MS) (normalization method).

[0078] Ester content: The total ester content (calculated as ethyl acetate) was determined by saponification titration according to GB / T 14455.6-2008 "Determination of Ester Value of Fragrances".

[0079] Thermal stability: According to GB / T 16447-2004 "Atmospheric environment for conditioning and testing of tobacco and tobacco products", the flavoring was sealed and placed in a constant temperature chamber at 60℃ for 7 days, and the residual rate of ester substances was determined.

[0080] Sensory evaluation: Based on GB 5606.4-2005 "Cigarettes Part 4: Sensory Technical Requirements", a blind evaluation was conducted by 5 evaluators (out of 10 points). The indicators included aroma, harmony, off-flavors, and aftertaste.

[0081] The test results are shown in Table 1.

[0082] Table 1. Test Results of Tobacco Flavorings

[0083] <![CDATA[Total amount of aroma components (×10 6 AU)]]> Ester content (mg / L) Thermal stability (ester residue, %) Sensory rating (points) Example 1 8.72 830 96.5 9.1 Example 2 8.58 795 95.8 9.0 Comparative Example 1 5.31 520 82.3 6.8 Comparative Example 2 7.95 745 86.2 7.9 Comparative Example 3 6.83 610 90.1 7.2 Comparative Example 4 8.02 780 88.7 8.5

[0084] The above results demonstrate that the compound enzyme system of this invention significantly enhances flavor profile through three-stage enzymatic hydrolysis, microencapsulation technology ensures enzyme activity persistence, improves ester synthesis efficiency, steam explosion pretreatment combined with supercritical extraction fully releases the active ingredients of the raw materials, and hydroxypropyl-β-cyclodextrin stabilization treatment ultimately yields a flavoring with rich aroma and excellent stability. Comparative Example 1, due to the inability of cellulase, catalase, and transaminase to effectively synergistically catalyze esterification and glycoside hydrolysis, had a lower total aroma and ester content, resulting in a sensory score of only 6.8. This confirms the irreplaceable role of the original compound enzyme formulation in the flavor transformation of tobacco matrix. Although Comparative Example 2 exhibited excellent total aroma, its thermal stability ester residue rate was significantly lower than that of the examples, and its sensory harmony was poor. This indicates that the single-layer encapsulation structure has limited protective and sustained-release effects on the enzyme, leading to premature enzyme inactivation during the reaction, increased side reactions, and affecting both the aroma profile and stability of the final product. In Comparative Example 3, replacing monk fruit with licorice resulted in a decrease in both ester content and total aroma components compared to the original example. Sensory characteristics included a monotonous aftertaste, excessive sweetness, and a lack of the harmony and richness provided by the original ternary substrate combination. This indicates that monk fruit's contribution to flavor balance and aroma structure in the original formula is irreplaceable, and the ternary complex substrate system is more conducive to improving flavor quality and achieving multi-dimensional flavor expression. In Comparative Example 4, although the total aroma components and ester content were close to those of the original example, the thermal stability and sensory scores were lower. This suggests that the one-time addition of L-leucine and UDP-glucose affects the synergistic generation of flavor components and the overall stability of the product, hindering the optimized expression of complex aroma systems.

[0085] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for preparing tobacco flavorings based on compound enzymes, comprising the following steps: (1) Pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase are mixed to obtain a compound enzyme powder. The compound enzyme powder is then encapsulated in sodium alginate-chitosan microcapsules to obtain compound enzyme microcapsules. (2) Mix tobacco fragments, loquat leaves and monk fruit and steam-explode them, then extract with ethanol aqueous solution and supercritical CO2 in sequence, and combine the extracts; (3) Add 2-4% of the mass of the compound enzyme microcapsules to the extract obtained in step (2) and carry out a three-stage enzymatic hydrolysis reaction to obtain a mixture; (4) Add 0.4-0.6% by mass of hydroxypropyl-β-cyclodextrin to the mixture obtained in step (3), stir, inactivate the enzyme, filter, and collect the filtrate; (5) Concentrate the filtrate and add vitamin E to obtain tobacco flavoring; Methods for encapsulating compound enzyme powder in sodium alginate-chitosan microcapsules include: (a) Add the compound enzyme powder to a sodium alginate aqueous solution with a mass fraction of 1.5-4% at a mass ratio of 1:3-5, stir, and form a compound solution; (b) Add the above composite solution dropwise to a CaCl2 aqueous solution with a mass fraction of 1-4% at a mass ratio of 1:4-7, stir and react for 20-40 minutes to form gel-like alginate calcium spheres; (c) Take out the above alginate calcium spheres, rinse with deionized water, transfer to a chitosan acetic acid aqueous solution with a mass fraction of 0.2-0.8%, react with stirring for 30-50 minutes to obtain chitosan-coated microcapsules; (d) The chitosan-coated microcapsules were transferred into a sodium tripolyphosphate solution with a mass fraction of 0.8-2% for cross-linking for 10-20 minutes, and then washed and dried to obtain composite enzyme microcapsules; The conditions for a tertiary enzymatic hydrolysis reaction are as follows: First stage: pH 4.8-5.2, 38-42℃, reaction time 1.8-2.2 hours; Second stage: pH 6.6-7.0, 48-52℃, add 0.08-0.12% L-leucine by weight of the extract, and react for 1.3-1.7 hours; Third stage: pH 6.6-7.0, 43-47℃, add 0.04-0.06% of UDP-glucose by weight of the extract, and react for 0.8-1.2 hours.

2. The process for preparing tobacco flavorings based on compound enzymes according to claim 1, characterized in that, In step (1), the pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase, and acetyltransferase are mixed in the following proportions: (1.0±0.2):(1.0±0.2):(0.5±0.1):(0.5±0.1):(0.3±0.06):(0.03±0.006).

3. The process for preparing tobacco flavorings based on compound enzymes according to claim 1, characterized in that, The mass ratio of tobacco shreds, loquat leaves and monk fruit in step (2) is (2.5-5):(1-2):(1-2).

4. The process for preparing tobacco flavoring based on compound enzymes according to claim 1, characterized in that, The temperature of the steam explosion in step (2) is 118-122℃, the pressure is 1.1-1.3 MPa, and the time is 85-95 seconds.

5. The process for preparing tobacco flavorings based on compound enzymes according to claim 1, characterized in that, The temperature of the supercritical CO2 extraction in step (2) is 33-37℃, the pressure is 24-26 MPa, and the time is 1.8-3 hours.

6. The process for preparing tobacco flavorings based on compound enzymes according to claim 1, characterized in that, The stirring temperature in step (4) is 48-52℃ and the stirring time is 25-35 minutes.

7. The process for preparing tobacco flavorings based on compound enzymes according to claim 1, characterized in that, Step (5) Concentrate the filtrate to 18-22% of its original volume, and add vitamin E at a mass of 0.8-1.2% of the concentrate.

8. A tobacco flavoring, characterized in that, It is prepared using the process for preparing tobacco flavoring based on compound enzymes as described in any one of claims 1-7.