Tobacco essence prepared based on compound enzyme and preparation process

Abstract

The invention belongs to the technical field of tobacco essence preparation and biological catalysis application, and particularly relates to tobacco essence prepared on the basis of compound enzyme and a preparation process. The process comprises the following steps: firstly, compounding pectinase, beta-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase in proportion, and forming microcapsules through a sodium alginate-chitosan system; the method comprises the following steps: pre-treating tobacco powder, folium eriobotryae and fructus momordicae by steam explosion to prepare an extracting solution; adding microcapsule enzymes to carry out three-stage segmented enzymolysis; then adding hydroxypropyl-beta-cyclodextrin to wrap aroma molecules, carrying out enzyme deactivation, filtering and concentrating, and adding vitamin E to stabilize the quality of the essence, so as to finally prepare the tobacco essence with rich aroma levels and good thermal stability. According to the preparation method, directional release and construction of flavor substances in the natural raw materials are realized through coordination of the compound enzyme and multi-stage reaction regulation, and the obtained essence is suitable for various application scenes such as cigarette perfuming and papermaking perfuming.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tobacco flavor preparation and biocatalysis application, and particularly relates to a tobacco flavor prepared based on a composite enzyme and a preparation process. BACKGROUND

[0002] Tobacco flavor is an important component in cigarette formula, and has a significant influence on the flavor quality, aroma concentration, taste coordination and aftertaste of tobacco products. The preparation of traditional tobacco flavor mainly relies on three methods: plant extraction, thermal reaction flavoring and chemical synthesis. Although these methods are widely used in the tobacco industry, there are still many shortcomings in flavor complexity, component coordination and natural properties, especially in the context of increasing attention to health and safety and green and sustainable development, the existing technology needs to be optimized and innovated.

[0003] Plant extraction method usually uses aromatic plants or spice raw materials to obtain essential oils or aromatic components through steam distillation, solvent extraction or supercritical extraction. This method has the advantage of "natural source", but also has the problems of low extraction efficiency, easy thermal degradation of active components, poor product flavor stability, etc. In addition, the limitation of natural plant resources also causes great pressure on raw material acquisition and cost control.

[0004] Thermal reaction flavoring method relies on heat-induced processes such as Maillard reaction and caramelization to generate complex aroma components, and is widely used in reconstituted tobacco and tobacco flavor. This method can effectively enhance the "tobacco flavor" and "baking flavor" of smoke, but the heat treatment process is easy to produce off-flavors or harmful gases, and some reaction by-products also have potential health risks. At the same time, due to the high uncertainty of thermal reaction, the stability of the obtained flavor batch is poor, and it is difficult to form a controllable and standardized flavor system.

[0005] Chemical synthesis method can improve the yield and cost-effectiveness by artificially synthesizing natural spices or artificial spices. However, there is a significant gap between chemical synthesis spices and natural spices in terms of flavor delicacy and coordination, and some artificial spices are controversial in safety, especially under the increasingly strict regulatory restrictions, the use of chemical flavors is constantly compressed. In addition, there are many synergistic and antagonistic effects in flavor matching, and simple splicing flavoring often cannot achieve the effect of high integration with natural tobacco aroma.

[0006] In recent years, with the development of biocatalytic technology, enzyme-catalyzed flavoring has gradually attracted industry attention. Single enzyme reactions such as lipase-catalyzed esterification and oxidase-catalyzed dehydrogenation have been used for the construction of spice precursors or the transformation of natural substances. However, in practical application, single enzyme reaction has the problems of narrow substrate selectivity, insufficient flavor levels, and monotonous reaction system, which is difficult to meet the needs of high complexity and multi-dimensional flavor construction.

[0007] Especially in the field of tobacco flavor, aroma not only requires the intensity of the fragrance, but also has rich "top notes", "main notes" and "after notes" changes, and the traditional single enzyme system is difficult to achieve the synergistic construction of multi-dimensional flavor. In addition, most of the current enzyme catalytic methods still use single substrate or binary substrate system, and the synergistic conversion mechanism between multiple components in natural complex substrate still lacks systematic research. This also leads to the lack of sufficient stability and flavor performance of the existing enzyme flavor, which is difficult to replace the traditional flavor system in application.

[0008] Therefore, it is urgent to develop a new preparation method with multi-pathway conversion ability, strong flavor construction diversity, high safety and industrial control, to improve the quality performance and product differentiation ability of tobacco flavor, and meet the multiple needs of new generation consumers for health, safety and natural flavor. SUMMARY

[0009] The purpose of the present application is to provide a tobacco flavor prepared based on complex enzymes and a preparation process, to solve the problems of insufficient naturalness, single flavor level, poor stability and preparation process relying on high temperature or chemical synthesis of the existing tobacco flavor, to realize the mild and efficient conversion of multiple natural raw materials, and to obtain a complex tobacco flavor with rich aroma, safe ingredients and good stability.

[0010] In order to achieve the above purpose, the present application provides the following technical solutions:

[0011] The first aspect of the present application provides a process for preparing a tobacco flavor based on complex enzymes, comprising the following steps:

[0012] (1) mixing pectinase, beta-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase to obtain complex enzyme powder, and packaging the complex enzyme powder in sodium alginate-chitosan microcapsules to obtain complex enzyme microcapsules;

[0013] (2) mixing tobacco crumbs, loquat leaves and monk fruit, then steam blasting, and then sequentially extracting with ethanol aqueous solution and supercritical CO2 extraction, and combining the extract;

[0014] (3) adding 2-4% of the mass of the complex enzyme microcapsules to the extract obtained in step (2) to perform three-stage enzymatic reaction, and obtaining a mixture;

[0015] (4) adding 0.4-0.6% of the mass of hydroxypropyl-beta-cyclodextrin to the mixture obtained in step (3), stirring, inactivating the enzyme, filtering, and collecting the filtrate;

[0016] (5) concentrating the filtrate, adding vitamin E, and obtaining a tobacco flavor.

[0017] Further, the pectinase, beta-glucosidase, lipase, lipoxygenase, glucose oxidase, and acetyltransferase in step (1) are mixed in the ratio of (1.0±0.2):(1.0±0.2):(0.5±0.1):(0.5±0.1):(0.3±0.06):(0.03±0.006) of enzyme dosage (active units U). For example, in the above ratio, 1.0±0.2 means a range of 0.8-1.2, and the same applies to the others.

[0018] Further, the method for encapsulating the complex enzyme powder in the sodium alginate-chitosan microcapsule in step (1) comprises the following steps:

[0019] (a) The complex enzyme powder is added to a 1.5-4% sodium alginate aqueous solution in the mass ratio of 1:3-5, and stirred to form a complex solution;

[0020] (b) The complex solution is added dropwise to a 1-4% CaCl2 aqueous solution in the mass ratio of 1:4-7, and stirred for 20-40 minutes to form a gelled calcium alginate enzyme ball;

[0021] (c) The calcium alginate enzyme ball is taken out, washed with deionized water, and transferred to a 0.2-0.8% chitosan acetic acid aqueous solution, and stirred for 30-50 minutes to obtain a chitosan-coated microcapsule;

[0022] (d) The chitosan-coated microcapsule is transferred to a 0.8-2% sodium tripolyphosphate solution for crosslinking for 10-20 minutes, and then washed and dried to obtain a complex enzyme microcapsule.

[0023] Pectinase and β-glucosidase in the complex enzyme microcapsule mainly act on the plant cell wall and phenolic glycoside structure, and can release a large amount of potential aroma components combined by glycosidic bonds under mild conditions, such as phenylethanoid glycoside, syringin, etc. These substances have higher volatility and olfactory perception intensity in free state, and provide a "pre-fragrance" basis for aroma construction. Lipase can catalyze the transesterification of natural esters and alcohols in the substrate, on the one hand, it can hydrolyze trace components of plant surface wax esters, and on the other hand, it can also act with the released alcohols in the system to generate ester aroma molecules. These components can help to improve the coordination and softness in tobacco flavor, and are important aroma building factors for "roundness".

[0024] In addition, the synergistic time window of the six enzymes in the reaction does not completely coincide, which is another consideration for adopting microcapsule embedding. By coating a layer of chitosan on the sodium alginate gel ball, and then cross-linking and stabilizing with sodium tripolyphosphate, the release of the enzymes can be controlled to avoid premature inactivation or mutual interference, which helps to maintain the peak of enzyme activity at different pH and temperature stages, and provides a technical basis for subsequent phased enzymolysis.

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

[0026] Further, the temperature of the steam explosion in step (2) is 118-122°C, the pressure is 1.1-1.3 MPa, and the time is 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] The application adopts a three-stage enzymatic reaction strategy to release and convert aroma precursors step by step. In the first stage, pectinase and beta-glucosidase are activated under acidic conditions to facilitate the release of glycosides and cell wall-bound substances. In the second stage, the reaction is adjusted to neutral to alkaline conditions, and L-leucine is added to guide the participation of amino acid precursors in flavor construction. In the third stage, the reaction is maintained at neutral conditions, and UDP-glucose is added to promote the formation of glycoside structures and improve aroma stability and retention. The three-stage reaction cooperatively completes the release of precursors, aroma construction, and stability modification.

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

[0036] In step (4), hydroxypropyl-beta-cyclodextrin is introduced as a stabilizer to "wrap up" the hydrophobic components in the essence through inclusion to prevent their oxidative degradation under high temperature, light, or air. At the same time, inclusion can also improve the water solubility of some hydrophobic molecules, making the essence more suitable for modern cigarette flavoring or spraying processes.

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

[0038] The addition of vitamin E has an antioxidant effect and can inhibit free radical chain reactions during the storage and use of the essence, providing obvious protection for 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 application provides a tobacco essence prepared by the above process based on a composite enzyme.

[0040] Compared with the prior art, the application has the following advantages and benefits:

[0041] The application realizes the precise release, structure regulation, and slow-release protection of natural aroma precursor substances by constructing a composite enzyme system based on multiple functional enzymes, supplemented by microcapsule embedding, 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 levels through phased enzymatic reactions, significantly enhancing the "top note performance", "main note fullness", and "afternote persistence" of the tobacco essence. At the same time, through cyclodextrin inclusion and vitamin E antioxidant protection measures, the thermal stability and use adaptability of the finished product during high-temperature flavoring are effectively improved. The overall process is green and environmentally friendly, the raw materials are widely sourced, the reaction conditions are mild, and the product is highly safe, making it suitable for industrialized application in the fields of cigarette making, spice making, and papermaking. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of, rather than all of, the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0043] The raw materials used in the embodiments are all commercially available products unless otherwise specified, and the following sources are exemplary:

[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 Yuan Ye Biotechnology Co., Ltd., with an enzyme activity of 20-40 U / mg.

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

[0047] Lipoxygenase 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 Biochem Technology, with an enzyme activity of 50 U / mg.

[0050] Cellulase was purchased from Shanghai Huashangxiangyang Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0051] Catalase was purchased from Macklin Biotechnology Co., Ltd., with an enzyme activity of 3,500 U / mg.

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

[0053] Embodiment 1

[0054] The present embodiment provides a process for preparing tobacco flavor based on composite enzyme, comprising the following steps:

[0055] (1) Preparation of composite enzyme microcapsules: Pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase were weighed according to the enzyme dosage (active unit U) ratio of 1.0:1.0:0.5:0.5:0.3:0.03, mixed, and composite enzyme powder was prepared; the composite enzyme powder was added to a 2.5% sodium alginate aqueous solution according to a mass ratio of 1:4, stirred at 600 rpm for 15 minutes at room temperature to form a composite liquid; then, the composite liquid was added dropwise into a 3% CaCl2 solution, the mass ratio of the composite liquid to the CaCl2 solution was 1:5, the dropwise rate was controlled at 1 mL / min, stirring was maintained, and the reaction was carried out for 30 minutes to form a calcium alginate gel ball; the gel ball was taken out, washed with deionized water three times, and then transferred to a 0.4% chitosan acetic acid aqueous solution (the mass ratio of acetic acid to water was 1:80), the mass ratio of the calcium alginate gel ball to the 0.4% chitosan acetic acid aqueous solution was 1:5, and the reaction was carried out at room temperature for 40 minutes to form a primary microcapsule. Then, the microcapsule was put into a 1.5% sodium tripolyphosphate aqueous solution, the mass ratio of the primary microcapsule to the 1.5% sodium tripolyphosphate aqueous solution was 1:7, and the cross-linking reaction was carried out at room temperature for 15 minutes. After that, the microcapsule was washed twice with deionized water and vacuum dried at 40°C for 12 hours to obtain the composite enzyme microcapsule.

[0056] (2) 50 g of tobacco powder, 15 g of dried loquat leaves, and 20 g of dried momordica grosvenori were weighed, mixed, and then placed in a steam explosion device for treatment at 120°C and 1.2 MPa for 90 seconds. After treatment, the mixture was cooled to room temperature and transferred to a 1000 mL three-necked flask. 400 mL of 60% ethanol aqueous solution was added, and the mixture was stirred at 60°C for 2 hours. After filtration, the filtrate was collected. The residue was added with 400 mL of 80% ethanol aqueous solution, and the mixture was extracted under the same conditions for 2 hours. After filtration, the two extracts were combined and concentrated to 150 mL. Then, the concentrated solution was transferred to a supercritical extraction device, and CO2 was used as the fluid for supercritical extraction at 35°C and 25 MPa for 2 hours. The extract was collected as the substrate solution for the subsequent reaction.

[0057] (3) 3% of the composite enzyme microcapsules were added to the substrate solution, and the three-stage enzymatic reaction was started. In the first stage, the pH was adjusted to 5.0, and the reaction was carried out at 40°C for 2 hours. In the second stage, the pH was adjusted to 6.8, 0.1% of L-leucine based on the mass of the substrate solution was added, and the reaction was carried out at 50°C for 1.5 hours. In the third stage, the pH was maintained at 6.8, 0.05% of UDP-glucose based on the mass of the substrate solution was added, and the reaction was carried out at 45°C for 1 hour.

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

[0059] (5) The crude essence solution is concentrated under reduced pressure to 20% of the original volume, and then 1.0% of vitamin E by mass of the concentrated solution is added, and stirred uniformly at room temperature, and thus a tobacco flavor essence product is obtained.

[0060] Example 2

[0061] The present embodiment provides a process for preparing a tobacco flavor essence based on composite enzymes, comprising the following steps:

[0062] (1) Preparation of composite enzyme microcapsules: pectinase, β-glucosidase, lipase, lipoxygenase, glucose oxidase and acetyltransferase are weighed according to the enzyme dosage (active unit U) ratio of 1.2:1.0:0.6:0.4:0.32:0.03, and mixed uniformly to prepare a composite enzyme powder. The composite enzyme powder is added to a 3.5% sodium alginate aqueous solution at a mass ratio of 1:3, and stirred at 550 rpm at room temperature for 18 minutes to form a composite solution. Then, the composite solution is added dropwise into a 2.5% CaCl2 solution, and the mass ratio of the composite solution to the CaCl2 solution is 1:6, the dropwise rate is controlled at 0.8 mL / min, and the stirring is maintained for 35 minutes to form calcium alginate gel balls. The gel balls are taken out, washed with deionized water three times, and then transferred to a 0.6% chitosan acetic acid aqueous solution (the mass ratio of acetic acid to water is 1:80) at room temperature, and the mass ratio of the calcium alginate gel balls to the 0.6% chitosan acetic acid aqueous solution is 1:4.8. The stirring reaction is carried out at room temperature for 35 minutes to form primary microcapsules. Then, the microcapsules are put into a 1.2% sodium tripolyphosphate aqueous solution, and the mass ratio of the primary microcapsules to the 1.2% sodium tripolyphosphate aqueous solution is 1:7.5. The crosslinking reaction is carried out at room temperature for 20 minutes, and then washed with deionized water twice, and vacuum dried at 48°C for 10 hours to obtain composite enzyme microcapsules.

[0063] (2) Tobacco powder 40 g, dried loquat leaves 12 g and dried momordica grosvenori 18 g were weighed and mixed, and then placed in a steam explosion device for treatment at 122°C and 1.3 MPa for 85 seconds. After treatment, the mixture was cooled to room temperature, and then transferred to a 1000 mL three-necked flask. 450 mL of 60% ethanol aqueous solution was added, and the mixture was stirred at 60°C for 2 hours. The mixture was filtered, and the filtrate was collected. The residue was added with 4000 mL of 80% ethanol aqueous solution, and the mixture was stirred at 60°C for 2 hours. The mixture was filtered, and the filtrate was collected. The two filtrates were combined and concentrated to 120 mL. The concentrated mixture was transferred to a supercritical extraction device, and extracted with CO2 at 37°C and 24 MPa for 2.5 hours. The extract was collected and used as a substrate for subsequent reaction.

[0064] (3) 2.5% of the complex enzyme microcapsules by mass were added to the substrate, and a three-stage enzymatic reaction was started. In the first stage, the pH was adjusted to 5.2, and the mixture was reacted at 38°C for 2.2 hours. In the second stage, the pH was adjusted to 7.0, and 0.08% of L-leucine by mass of the substrate was added, and the mixture was reacted at 52°C for 1.3 hours. In the third stage, the pH was maintained at 7.0, and 0.04% of UDP-glucose by mass of the substrate was added, and the mixture was reacted at 47°C for 0.8 hours.

[0065] (4) After the enzymatic reaction was completed, a mixed solution was obtained. 0.6% of hydroxypropyl-β-cyclodextrin by mass of the mixed solution was added, and the mixture was stirred at 48°C for 35 minutes. Then, the mixture was heated to 85°C for 10 minutes to inactivate the enzymes. After cooling to room temperature, the mixture was filtered, and the filtrate was collected. The filtrate was a crude essence solution.

[0066] (5) The crude essence solution was concentrated to 18% of the original volume under reduced pressure. Then, 1.2% of vitamin E by mass of the concentrated solution was added, and the mixture was stirred at room temperature to obtain a tobacco essence product.

[0067] Comparative Example 1

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

[0069] Comparative Example 2

[0070] The difference between the present comparative example and Example 1 is that: step (1) is that: 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 composite enzyme powder is prepared; the composite enzyme powder is added to a 2.5% by mass sodium alginate aqueous solution according to a mass ratio of 1:4, stirred at 600 rpm for 15 minutes at room temperature to form a uniform composite liquid; the composite liquid is added dropwise to a 3% by mass CaCl2 solution (the mass ratio of the composite liquid to the CaCl2 solution is 1:5), the dropwise rate is controlled at 1 mL / min, and stirring is maintained; after the dropwise addition is completed, the reaction is continued for 30 minutes to form calcium alginate gel balls; the gel balls are rinsed with deionized water three times, vacuum dried at 40°C for 12 hours, and directly used as composite enzyme microcapsules.

[0071] Comparative Example 3

[0072] The difference between the present comparative example and Example 1 is that: dry momordica grosvenori is replaced by dry licorice.

[0073] Comparative Example 4

[0074] The difference between the present comparative example and Example 1 is that: the enzyme hydrolysis reaction process of step (3) is that: 3% by mass composite enzyme microcapsules are added to the substrate liquid prepared in the previous step, and 0.1% by mass L-leucine and 0.05% by mass UDP-glucose are added at one time, and the three-stage enzyme hydrolysis reaction is started; 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 test

[0076] The tobacco flavor prepared in Examples 1-2 and Comparative Examples 1-4 is subjected to performance testing.

[0077] Total aroma component content: according to GB / T 14454.2-2008 “Spices-Aroma Evaluation Method”, the total peak area of volatile components in the flavor is determined by gas chromatography-mass spectrometry (GC-MS) (normalization method).

[0078] Ester content: according to GB / T 14455.6-2008 “Spices-Ester Value Determination”, the total ester content is determined by saponification titration (calculated as ethyl acetate).

[0079] Thermal stability: according to GB / T 16447-2004 “Tobacco and Tobacco Products-Regulating and Testing Atmospheric Environment”, the flavor is sealed and placed in a 60°C constant temperature box for 7 days, and the residual rate of ester substances is 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] Total amount of aroma components (x 10 6 AU) Ester content (mg / L) Thermal stability (ester residual rate, %) Sensory score (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.

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 method of encapsulating the compound enzyme powder in sodium alginate-chitosan microcapsules as described in step (1) includes the following steps: (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 of 0.8-2% by mass for cross-linking for 10-20 minutes, and then washed and dried to obtain the composite enzyme microcapsules.

4. The process for preparing tobacco flavoring 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).

5. The process for preparing tobacco flavorings 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.

6. 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.

7. The process for preparing tobacco flavorings based on compound enzymes according to claim 1, characterized in that, The conditions for the tertiary enzymatic hydrolysis reaction in step (3) 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.

8. 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.

9. 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.

10. 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 1-9.