Enzyme composition, method for extracting aroma component from tobacco waste and obtained extract, tobacco composition and tobacco product

Abstract

The invention belongs to the field of tobacco treatment, and particularly relates to an enzyme composition which comprises a first enzyme obtained by fermenting bacillus subtilis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24244; and one or two of the following items: a second enzyme obtained by fermenting bacillus safensis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24246; the bacillus subtilis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24134 is fermented to obtain the third enzyme. The invention also relates to a method for extracting the aroma component from the tobacco waste, an extract containing the aroma component, a tobacco composition and a tobacco product. According to the enzyme composition, the extraction rate of aroma components of the tobacco waste can be remarkably increased; and / or the extract is added into the cigarette, so that the tobacco fragrance and the fresh and sweet fragrance of the cigarette can be increased during smoking, the soft feeling and the roundness of smoke are increased, the aftertaste comfort degree and the fragrance harmony are improved, the offensive odor and the irritation are reduced, and the overall sensory quality is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of tobacco processing, and specifically relates to an enzyme composition, and also relates to a method for extracting aroma components from tobacco waste, an extract containing the aroma components, a tobacco composition and a tobacco product. Background Art

[0002] Tobacco waste refers to tobacco raw materials that are generated during the preparation, re-roasting and cigarette production processes and cannot be directly used in cigarette production. It is mainly used as raw materials for the preparation of reconstituted tobacco leaves, flavors, pesticides, etc. The excess is buried or incinerated, resulting in low utilization efficiency of tobacco waste and easy environmental pollution.

[0003] Tobacco waste, as a tobacco raw material homologous to tobacco leaves, contains a large amount of tobacco aroma components. These aroma components can not only enrich the tobacco aroma and improve the smoking quality of cigarettes, but also have good coordination with the tobacco aroma. However, the existing technology has a low extraction rate of aroma components in tobacco waste, which reduces the flavoring effect of aroma components and is not conducive to the high-value utilization of tobacco waste.

[0004] Currently, there is an urgent need for a method to effectively increase the extraction rate of aroma components in tobacco waste. Summary of the invention

[0005] One of the purposes of the present invention is to provide an enzyme composition, which is used to prepare tobacco waste extract, which can significantly improve the extraction rate of aroma components (especially main aroma components); and / or, the prepared tobacco waste extract is added to cigarettes, which can increase the tobacco flavor and sweet aroma of cigarettes during smoking, increase the softness and roundness of smoke, improve the comfort of aftertaste, improve the coordination of aroma, reduce impurities and irritation, and significantly improve the overall sensory quality. On this basis, another purpose of the present invention is to provide a method for extracting aroma components from tobacco waste, an extract containing aroma components, a tobacco composition and a tobacco product.

[0006] To achieve the above object, the present invention provides an enzyme composition in a first aspect, comprising a first enzyme obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 24244; and

[0007] One or both of the following:

[0008] A second enzyme obtained by fermentation of Bacillus saffron with a preservation number of CGMCC No. 24246;

[0009] The third enzyme is obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No.24134.

[0010] The above Bacillus subtilis with a preservation number of CGMCC No. 24244, Bacillus safov with a preservation number of CGMCC No. 24246, and Bacillus subtilis with a preservation number of CGMCC No. 24134 are all deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC), and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101. Among them, the preservation date of Bacillus subtilis with a preservation number of CGMCC No. 24134 is December 20, 2021, and it was screened from the YLC1Y-2019 tobacco leaf in Yongding, Longyan, Fujian. Chinese patent applications have been submitted for Bacillus subtilis with a preservation number of CGMCC No. 24244 and Bacillus sabdariffa with a preservation number of CGMCC No. 24246, and relevant information about the strains has been recorded in public texts.

[0011] In any embodiment, the ratio of the enzyme activity of the first enzyme to one or both of the second enzyme and the third enzyme is 0.00005:1-0.0005:1, for example, 0.00008:1, 0.0001:1, 0.0002:1, 0.0003:1, 0.0004:1, 0.0005:1.

[0012] In any embodiment, the ratio of the enzyme activity of the first enzyme to the second enzyme is 0.0001:1-0.0005:1, for example, 0.0001:1, 0.0002:1, 0.0003:1, 0.0004:1, 0.0005:1.

[0013] In any embodiment, the ratio of the enzyme activity of the first enzyme to the third enzyme is 0.0001:1-0.0005:1, for example, 0.0001:1, 0.0002:1, 0.0003:1, 0.0004:1, 0.0005:1.

[0014] In any embodiment, the ratio of the enzyme activities of the first enzyme, the second enzyme and the third enzyme is 0.0001:1:1-0.0005:1:1, for example, 0.0001:1:1, 0.0002:1:1, 0.0003:1:1, 0.0004:1:1, 0.0005:1:1.

[0015] In any embodiment, the first enzyme, the second enzyme or the third enzyme is obtained by the following steps:

[0016] Inoculating the strain into the first culture medium and culturing it to obtain a bacterial solution; wherein the strain is Bacillus subtilis with a preservation number of CGMCC No. 24244, Bacillus sabdariffa with a preservation number of CGMCC No. 24246, or Bacillus subtilis with a preservation number of CGMCC No. 24134;

[0017] inoculating the bacterial liquid into a second culture medium and fermenting at 29-40° C. (e.g., 30° C., 33° C., 35° C., 37° C., 38° C., 40° C.) for 10-60 hours (e.g., 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours) to obtain a fermentation product;

[0018] The fermentation product is cleaned, concentrated, and optionally dried.

[0019] In any embodiment, in the step of preparing the fermentation product, the volume ratio of the bacterial liquid to the second culture medium is 0.5%-7%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%.

[0020] In any embodiment, in the step of preparing the fermentation product, fermentation is carried out under a stirring condition of 100-220 r / min, for example, 100 r / min, 130 r / min, 150 r / min, 170 r / min, 190 r / min, 200 r / min, 210 r / min, 220 r / min.

[0021] In any embodiment, impurities are removed by filtering through a 100KD-150KD (eg, 100KD, 150KD) ultrafiltration membrane; optionally, before filtering through the ultrafiltration membrane, filtering can be performed using a 150-250nm (eg, 150nm, 180nm, 200nm, 250nm) ceramic membrane.

[0022] In any embodiment, the concentration is performed by a 3KD-30KD (eg, 3KD, 3.5KD, 6KD, 10KD, 20KD, 30KD) ultrafiltration membrane.

[0023] In any embodiment, in the step of preparing the bacterial solution, the culture temperature is 29-40°C (e.g., 30°C, 33°C, 35°C, 37°C, 38°C, 40°C), and the culture time is 10-36 hours (e.g., 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 33 hours, 35 hours, 36 hours).

[0024] In any embodiment, in the step of preparing the bacterial solution, the culture is carried out under a stirring condition of 150-220 r / min, for example, 150 r / min, 170 r / min, 190 r / min, 200 r / min, 210 r / min, 220 r / min.

[0025] In any embodiment, the first culture medium is a common culture medium used in the art for strain rejuvenation or proliferation, for example, TSB liquid or solid culture medium, LB liquid or solid culture medium, etc.

[0026] In any embodiment, the second culture medium is a common culture medium for fermentation and enzyme production in the art. For example, the second culture medium can be a mixture of LB culture medium and sodium carboxymethyl cellulose, or a culture medium including a nitrogen source, a carbon source, an inorganic salt, etc.; wherein the nitrogen source, carbon source, and inorganic salt can be common types in the art, and the ratio between them is also well known to those skilled in the art.

[0027] The second aspect of the present invention provides a method for extracting aroma components from tobacco waste, wherein the aroma components include one or more compounds of alcohols, aldehydes, ketones, esters, and olefins; optionally, the aroma components include one or more of megastigmatrienone, damascenone, solanone, β-damascenone, geranylacetone, methyl palmitate, benzaldehyde, linalool, and benzyl alcohol;

[0028] The method comprises the following steps:

[0029] mixing tobacco waste with water to obtain a mixture;

[0030] The mixture is enzymatically hydrolyzed by using the enzyme composition or the first enzyme of the first aspect of the present invention to inactivate the enzyme and obtain an enzymatic hydrolysis product.

[0031] In the present application, the enzymatic hydrolysis product refers to the mixed material obtained after enzymatic hydrolysis and enzyme inactivation.

[0032] In any embodiment, the enzymatic hydrolysis temperature is 35-65°C (e.g., 35°C, 38°C, 40°C, 45°C, 47°C, 49°C, 50°C, 52°C, 55°C, 57°C, 59°C, 60°C, 62°C, 64°C, 65°C), and the enzymatic hydrolysis time is 1-7 hours (e.g., 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours).

[0033] In any embodiment, it further comprises: distilling the enzymatic hydrolysis product, collecting the distillate, and concentrating the distillate.

[0034] In any embodiment, the distillation temperature is 800-120°C, such as 100°C.

[0035] In any embodiment, the distillation time is 1-4 hours, such as 1 hour, 2 hours, 3 hours, 4 hours.

[0036] In any embodiment, the ratio of the enzyme composition or the first enzyme to the tobacco waste is 0.001-0.01U / g or 3-10U / g, for example, 0.001U / g, 0.003U / g, 0.005U / g, 0.007U / g, 0.01U / g, 3U / g, 4U / g, 5U / g, 6U / g, 7U / g, 8U / g, 9U / g, 10U / g.

[0037] In any embodiment, the enzymatic hydrolysis is carried out under stirring conditions of 20-60 r / min (e.g., 20 r / min, 30 r / min, 40 r / min, 50 r / min, 60 r / min).

[0038] In any embodiment, the concentration is performed using a reverse osmosis membrane.

[0039] In any embodiment, the sodium chloride rejection rate of the reverse osmosis membrane is 95%-99.7%, such as 95%, 96%, 97%, 98%, 99%, 99.7%.

[0040] In the present application, the sodium chloride rejection rate of the reverse osmosis membrane is tested by a conventional method in the art, and the test conditions of the method are well known to those skilled in the art. For example, a sodium chloride solution with a concentration of 2000 ppm is used to test the sodium chloride rejection rate of the reverse osmosis membrane under the conditions of a working pressure of 40 bar, a temperature of 5-45° C., and a pH value of 2.5-10.

[0041] In any embodiment, in the step of preparing the mixture, the mass ratio of the tobacco waste to water is 1:15-1:40, for example, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40.

[0042] In any embodiment, the enzyme inactivation is performed by heat treatment at 95° C. or higher (eg, 100° C.) for 8 minutes or more (eg, 10 minutes).

[0043] In any embodiment, before the mixing, the tobacco waste is crushed and sieved.

[0044] In any embodiment, the tobacco waste is selected from one or more of waste tobacco leaves, waste tobacco shreds, waste cigarette butts, and waste cigarette butt rods.

[0045] The third aspect of the present invention provides an extract containing aroma components, which is an extract obtained by the method of the second aspect of the present invention; the aroma components include one or more compounds of alcohols, aldehydes, ketones, esters, and olefins; optionally, the aroma components include one or more of megastigmatrienone, damascenone, solanone, β-damascenone, geranylacetone, methyl palmitate, benzaldehyde, linalool, and benzyl alcohol.

[0046] In any embodiment, the extract comprises one or more of the following:

[0047] The yield of alcohol compounds in the extract is ≥1.75 μg / g, preferably ≥2 μg / g, for example, 1.75 μg / g, 1.8 μg / g, 1.9 μg / g, 2 μg / g, 2.1 μg / g, 2.2 μg / g, 2.3 μg / g, 2.4 μg / g, 2.5 μg / g;

[0048] The yield of aldehyde compounds in the extract is ≥2 μg / g, preferably ≥2.4 μg / g, for example, 2 μg / g, 2.1 μg / g, 2.2 μg / g, 2.3 μg / g, 2.4 μg / g, 2.5 μg / g, 2.6 μg / g, 2.7 μg / g, 2.8 μg / g, 2.9 μg / g, 3.0 μg / g, 3.1 μg / g;

[0049] The ketone compound yield of the extract is ≥26.5 μg / g, preferably ≥30 μg / g, for example, 26.5 μg / g, 26.6 μg / g, 26.8 μg / g, 27 μg / g, 28 μg / g, 29 μg / g, 30 μg / g, 31 μg / g, 32 μg / g, 33 μg / g, 34 μg / g, 35 μg / g, 36 μg / g, 37 μg / g, 38 μg / g, 39 μg / g, 40 μg / g, 41 μg / g, 42 μg / g;

[0050] The yield of ester compounds in the extract is ≥3.3 μg / g, preferably ≥3.6 μg / g, for example, 3.3 μg / g, 3.4 μg / g, 3.5 μg / g, 3.6 μg / g, 3.7 μg / g, 3.8 μg / g, 3.9 μg / g, 4 μg / g, 4.1 μg / g, 4.3 μg / g, 4.5 μg / g, 4.7 μg / g, 4.8 μg / g, 5 μg / g, 5.1 μg / g, 5.2 μg / g, 5.3 μg / g, 5.5 μg / g, 5.7 μg / g, 5.8 μg / g, 5.9 μg / g, 6 μg / g;

[0051] The yield of olefin compounds in the extract is ≥4.3μg / g, preferably ≥5μg / g, for example, 4.3μg / g, 4.5μg / g, 4.6μg / g, 4.8μg / g, 4.9μg / g, 5μg / g, 5.1μg / g, 5.3μg / g, 5.5μg / g, 5.7μg / g, 5.9μg / g, 6μg / g, 6.2μg / g, 6.3μg / g, 6.5μg / g, 6.7μg / g, 6.8μg / g, 7μg / g, 7.1μg / g, 7.3μg / g, 7.5μg / g, 7.7μg / g, 7.8μg / g, 7.9μg / g, 8μg / g;

[0052] The total yield of aroma components of the extract is ≥44 μg / g, preferably ≥50 μg / g, for example, 44 μg / g, 45 μg / g, 46 μg / g, 48 μg / g, 50 μg / g, 52 μg / g, 55 μg / g, 56 μg / g, 57 μg / g, 59 μg / g, 60 μg / g, 62 μg / g, 63 μg / g, 65 μg / g, 67 μg / g, 68 μg / g, 69 μg / g, 70 μg / g;

[0053] The yield of megastigmatrienone in the extract is ≥20 μg / g, preferably ≥25 μg / g, for example, 20 μg / g, 21 μg / g, 23 μg / g, 25 μg / g, 26 μg / g, 28 μg / g, 29 μg / g, 30 μg / g, 32 μg / g;

[0054] The damascone yield of the extract is ≥4 μg / g, preferably ≥4.5 μg / g, for example, 4 μg / g, 4.1 μg / g, 4.3 μg / g, 4.5 μg / g, 4.7 μg / g, 4.9 μg / g, 5 μg / g, 5.1 μg / g, 5.2 μg / g, 5.3 μg / g, 5.5 μg / g, 5.7 μg / g, 5.8 μg / g, 6 μg / g;

[0055] The solanone yield of the extract is ≥3.4 μg / g, preferably ≥5 μg / g, for example, 3.4 μg / g, 3.5 μg / g, 3.7 μg / g, 3.8 μg / g, 3.9 μg / g, 4 μg / g, 4.1 μg / g, 4.2 μg / g, 4.4 μg / g, 4.5 μg / g, 4.7 μg / g, 4.8 μg / g, 5 μg / g, 5.1 μg / g, 5.3 μg / g, 5.5 μg / g, 5.7 μg / g, 5.8 μg / g, 5.9 μg / g, 6 μg / g, 6.1 μg / g, 6.3 μg / g, 6.5 μg / g, 6.7 μg / g, 7 μg / g;

[0056] The β-damascenone yield of the extract is ≥1.9 μg / g, preferably ≥2.0 μg / g, for example, 1.9 μg / g, 2 μg / g, 2.1 μg / g, 2.2 μg / g, 2.3 μg / g, 2.4 μg / g, 2.5 μg / g, 2.7 μg / g, 2.9 μg / g, 3 μg / g;

[0057] The geranylacetone yield of the extract is ≥1.2 μg / g, preferably ≥1.6 μg / g, for example, 1.2 μg / g, 1.3 μg / g, 1.4 μg / g, 1.5 μg / g, 1.6 μg / g, 1.7 μg / g, 1.8 μg / g, 1.9 μg / g, 2 μg / g, 2.1 μg / g, 2.3 μg / g, 2.5 μg / g;

[0058] The palmitic acid methyl ester yield of the extract is ≥0.95 μg / g, preferably ≥1.1 μg / g, for example, 0.95 μg / g, 0.97 μg / g, 0.98 μg / g, 1 μg / g, 1.1 μg / g, 1.3 μg / g, 1.5 μg / g, 1.7 μg / g, 1.8 μg / g, 2 μg / g;

[0059] The benzaldehyde yield of the extract is ≥0.55 μg / g, preferably ≥0.6 μg / g, for example, 0.55 μg / g, 0.58 μg / g, 0.6 μg / g, 0.61 μg / g, 0.63 μg / g, 0.65 μg / g, 0.67 μg / g, 0.68 μg / g, 0.7 μg / g, 0.71 μg / g, 0.73 μg / g, 0.75 μg / g, 0.77 μg / g, 0.8 μg / g;

[0060] The linalool yield of the extract is ≥0.33 μg / g, preferably ≥0.37 μg / g, for example, 0.33 μg / g, 0.34 μg / g, 0.35 μg / g, 0.36 μg / g, 0.37 μg / g, 0.38 μg / g, 0.4 μg / g, 0.41 μg / g, 0.42 μg / g, 0.45 μg / g, 0.47 μg / g, 0.48 μg / g, 0.5 μg / g;

[0061] The benzyl alcohol yield of the extract is ≥0.36μg / g, preferably ≥0.37μg / g, for example, 0.36μg / g, 0.37μg / g, 0.38μg / g, 0.39μg / g, 0.4μg / g, 0.41μg / g, 0.42μg / g, 0.43μg / g, 0.45μg / g, 0.47μg / g, 0.48μg / g, 0.49μg / g, and 0.5μg / g.

[0062] In the present application, the aroma component yield of the extract can be understood as the mass of the aroma components in the extract obtained from the tobacco waste per unit mass.

[0063] The fourth aspect of the present invention provides a tobacco composition, comprising the extract according to the third aspect of the present invention; preferably, the tobacco composition is a tobacco flavoring or tobacco leaf composition.

[0064] The fifth aspect of the present invention provides a tobacco product, comprising the extract according to the third aspect of the present invention or the tobacco composition according to the fourth aspect of the present invention.

[0065] The present invention achieves one or more of the following beneficial effects:

[0066] 1. The enzyme composition of the present invention significantly improves the extraction rate of aroma components, especially main aroma components, in tobacco waste.

[0067] 2. When the tobacco waste extract prepared by the enzyme composition of the present invention is added to cigarettes, the original tobacco aroma and sweet aroma of the cigarettes can be increased, the softness and roundness of the smoke can be increased, the comfort of the aftertaste can be improved, the aroma coordination can be improved, the impurities and irritation can be reduced, and the overall sensory quality can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0069] Figure 1 It is the morphological characteristic of Bacillus subtilis (CGMCC No.24134). DETAILED DESCRIPTION

[0070] The embodiments of the present invention will be described clearly and completely below in conjunction with the examples. Obviously, the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0071] Bacillus subtilis Bacillus subtilis Identification of (CGMCC No.24134)

[0072] (1) Morphological characteristics of Bacillus subtilis (CGMCC No. 24134)

[0073] like Figure 1As shown, the colony morphology of the strain verified by streaking is grayish white, with a dry and raised surface, which can be seen to collapse and form wrinkles, and the edges are irregular and burr-like.

[0074] (2) Molecular biological identification of Bacillus subtilis (CGMCC No. 24134)

[0075] The universal primers were synthesized by the solid phase phosphoramidite triester method, and the primer sequences were:

[0076] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1)

[0077] 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′ (SEQ ID No. 2)

[0078] Using total DNA of Bacillus subtilis (CGMCC No. 24134) as template [extracted by bacterial DNA kit of Bioengineering (Shanghai) Co., Ltd.] and 27F / 1492R as primers, 16S rDNA sequence was amplified. The PCR reaction system was as follows:

[0079] The total volume of the system is 25 μL: 2× Rapid Taq MasterMix 12.0 μL, 27F and 1492R primers 1 μL each, DNA 1 μL, ddH2O 10 μL.

[0080] PCR reaction program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 15 s, for a total of 32 cycles; extension at 72°C for 5 min.

[0081]

[0082] After NCBI gene sequence comparison, the strain had the highest similarity with Bacillus subtilis and was therefore identified as Bacillus subtilis.

[0083] Materials used:

[0084] LB liquid medium: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028324;

[0085] LB solid medium: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028334;

[0086] TSB liquid medium: 17 g / L tryptone, 3 g / L soy peptone, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, 2.5 g / L glucose, pH 7.3 ± 0.2;

[0087] Fermentation medium 1: NaNO3 2.5g / L, KH2PO4 1g / L, MgSO4·7H2O 0.5g / L, NaCl 0.5g / L, CaCl2 0.1g / L and 30g / L alkali-treated tobacco stems; the preparation method of the alkali-treated tobacco stems is as follows: 100g of tobacco stems are cut into 3cm small segments, soaked in a 1% NaOH aqueous solution for 24h, washed with 1% hydrochloric acid for 3-4h, and then washed with running water until the tobacco stems are neutral and dried to constant weight;

[0088] Fermentation medium 2 includes 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium carboxymethyl cellulose, 10.0 g of sodium chloride, and 1000 mL of distilled water, with a pH of 7.0;

[0089] Fermentation medium 3: add sodium carboxymethyl cellulose to LB liquid medium to make the content of sodium carboxymethyl cellulose 5 g / L;

[0090] Preparation Example 1 Preparation of ligninase preparation

[0091] Bacillus subtilis with a deposit number of CGMCC No. 24244 was used, a ring of spores was picked out and cultured in LB solid medium at 37° C. for 24 hours, and then a single colony was picked out and inoculated into LB liquid medium, and cultured in a constant temperature shaker at 37° C. and 180 rpm for 12 hours to obtain a seed solution.

[0092] The seed liquid was inoculated into the fermentation medium 1 at an inoculum volume ratio of 1%, and the culture was carried out in a constant temperature shaker at 37°C and 130 rpm for 18 hours. The fermentation liquid was filtered with a 200 nm pore size ceramic membrane, and the permeate was collected to obtain a crude enzyme liquid. The crude enzyme liquid was filtered through an ultrafiltration membrane with a molecular weight cutoff of 150 KD to collect the permeate, and then the permeate was concentrated with an ultrafiltration membrane with a molecular weight cutoff of 10 KD to obtain a refined lignin enzyme liquid, which was freeze-dried to obtain a lignin enzyme preparation.

[0093] According to the lignin peroxidase detection method in Example 2 of Chinese patent document CN117343867A, the enzyme activity of the lignin enzyme preparation was measured to be 9 U / g.

[0094] Preparation Example 2 Preparation of Cellulase Preparation

[0095] Bacillus subtilis with the accession number of CGMCC No. 24134 was taken and rejuvenated in LB solid medium at 37°C. A single colony was scraped into 50 mL of LB liquid medium and cultured at 37°C and 180 r / min for 12 h to obtain seed solution.

[0096] The seed liquid was inoculated into the fermentation medium 2 at an inoculum volume ratio of 1%, and fermented at 37°C and 150 r / min for 48 hours, and the fermentation liquid was collected to obtain a crude enzyme liquid. The crude enzyme liquid was filtered through an ultrafiltration membrane with a molecular weight cutoff of 150KD to collect the permeate, and then the permeate was concentrated with an ultrafiltration membrane with a molecular weight cutoff of 10KD to obtain a refined cellulase liquid, which was freeze-dried to obtain a cellulase preparation.

[0097] The enzymatic activity of the cellulase preparation was measured to be 35 U / mg according to the cellulase detection method in Chinese patent document CN117247866A.

[0098] Preparation Example 3 Preparation of protease preparation

[0099] Bacillus safensis FD-1 with a preservation number of CGMCC No. 24246 was used, and a ring of spores of Bacillus safensis FD-1 was taken and inoculated into TSB liquid culture medium at 30° C. and 200 r / min for 24 hours to obtain seed solution.

[0100] The seed liquid was inoculated into the fermentation medium 3 at an inoculum volume ratio of 2%, and cultured at 37°C and 110 r / min for 15 hours, and the fermentation liquid was filtered through a ceramic membrane with a pore size of 200 nm, and the permeate was collected to obtain a crude enzyme liquid. The crude enzyme liquid was filtered through an ultrafiltration membrane with a molecular weight cutoff of 150KD to collect the permeate, and then the permeate was concentrated with an ultrafiltration membrane with a molecular weight cutoff of 10KD to obtain a refined protease liquid, which was freeze-dried to obtain a protease preparation.

[0101] The enzyme activity of the protease preparation was determined according to the method in the literature (Cai Wen, Wu Xinying, Zhang Qianying, et al. Optimization of neutral protease production conditions by Bacillus Gaussianus and its effect on tobacco fermentation [J]. Food and Fermentation Technology, 2022, 58(3): 92-98), and the value was 40U / mg.

[0102] Example 1

[0103] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 30-mesh sieve and take out the sieved material.

[0104] (2) Add purified water to the sieved material in an amount of 25 times the mass of the sieved material to obtain a mixture.

[0105] (3) Adding lignin enzyme preparation (the enzyme activity of lignin enzyme preparation is 0.003U per gram of sieved material) to the mixture for enzymolysis at a temperature of 50° C. for 2 h. During the enzymolysis process, stirring is performed at a speed of 50 rpm.

[0106] (4) After the enzymatic hydrolysis in step (3), the system was inactivated at 100°C for 10 min.

[0107] (5) The enzymatic hydrolysis product of step (4) was placed in a round-bottom flask, distilled at 100° C. for 2 h, and the condensed distillate was collected.

[0108] (6) The distillate was concentrated by reverse osmosis through a spiral membrane multifunctional pilot test device (RNF0460-001, Xiamen Fumei Technology Co., Ltd.), wherein the sodium chloride retention rate of the reverse osmosis membrane was 99.3%, and the retentate was collected as an extract.

[0109] Example 2

[0110] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 30-mesh sieve and take out the sieved material.

[0111] (2) Add purified water to the sieved material in an amount that is 30 times the mass of the sieved material to obtain a mixture.

[0112] (3) Adding lignin enzyme preparation (the enzyme activity of lignin enzyme preparation is 0.0015U per gram of sieved material) and protease preparation (the enzyme activity of protease preparation is 5U per gram of sieved material) to the mixture for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 3h, and the mixture is stirred at 30rpm during the enzymatic hydrolysis.

[0113] (4) After the enzymatic hydrolysis in step (3), the system was inactivated at 100°C for 10 min.

[0114] (5) The enzymatic hydrolysis product of step (4) was placed in a round-bottom flask, distilled at 100° C. for 2 h, and the condensed distillate was collected.

[0115] (6) The distillate was concentrated by reverse osmosis through a spiral membrane multifunctional pilot test device (RNF0460-001, Xiamen Fumei Technology Co., Ltd.), wherein the sodium chloride retention rate of the reverse osmosis membrane was 99.3%, and the retentate was collected as an extract.

[0116] Example 3

[0117] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 30-mesh sieve and take out the sieved material.

[0118] (2) Add purified water to the sieved material in an amount of 25 times the mass of the sieved material to obtain a mixture.

[0119] (3) Adding ligninase preparation (the enzyme activity of ligninase preparation is 0.0015U per gram of sieved material) and cellulase preparation (the enzyme activity of cellulase preparation is 5U per gram of sieved material) to the mixture for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 3h, and the mixture is stirred at 30rpm during the enzymatic hydrolysis.

[0120] (4) After the enzymatic hydrolysis in step (3), the system was inactivated at 100°C for 10 min.

[0121] (5) The enzymatic hydrolysis product of step (4) was placed in a round-bottom flask, distilled at 100° C. for 2 h, and the condensed distillate was collected.

[0122] (6) The distillate was concentrated by reverse osmosis through a spiral membrane multifunctional pilot test device (RNF0460-001, Xiamen Fumei Technology Co., Ltd.), wherein the sodium chloride retention rate of the reverse osmosis membrane was 99.3%, and the retentate was collected as an extract.

[0123] Example 4

[0124] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 30-mesh sieve and take out the sieved material.

[0125] (2) Add purified water to the sieved material in an amount of 20 times the mass of the sieved material to obtain a mixture.

[0126] (3) Add ligninase preparation (the enzyme activity of ligninase preparation is 0.001U per gram of sieved material), cellulase preparation (the enzyme activity of cellulase preparation is 3U per gram of sieved material) and protease preparation (the enzyme activity of protease preparation is 3U per gram of sieved material) to the mixture for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 3h, and the mixture is stirred at 30rpm during the enzymatic hydrolysis.

[0127] (4) After the enzymatic hydrolysis in step (3), the system was inactivated at 100°C for 10 min.

[0128] (5) The enzymatic hydrolysis product of step (4) was placed in a round-bottom flask, distilled at 100° C. for 2 h, and the condensed distillate was collected.

[0129] (6) The distillate was concentrated by reverse osmosis through a spiral membrane multifunctional pilot test device (RNF0460-001, Xiamen Fumei Technology Co., Ltd.), wherein the sodium chloride retention rate of the reverse osmosis membrane was 99.3%, and the retentate was collected as an extract.

[0130] Example 5

[0131] In step (3) of Example 2, the protease preparation was replaced with a ligninase preparation with equal enzyme activity, and the rest was the same as Example 2.

[0132] Example 6

[0133] In step (3) of Example 4, the cellulase preparation and the protease preparation were replaced with a ligninase preparation with the same enzyme activity, and the rest was the same as Example 4.

[0134] Comparative Example 1

[0135] In step (3) of Example 2, the lignin enzyme preparation was replaced with a protease preparation with equal enzyme activity, and the rest was the same as Example 2.

[0136] Comparative Example 2

[0137] In step (3) of Example 3, the ligninase preparation was replaced with a cellulase preparation with the same enzyme activity, and the rest was the same as Example 3.

[0138] Comparative Example 3

[0139] In step (3) of Example 4, the ligninase preparation is replaced by a cellulase preparation and a protease preparation, the total enzyme activity of the cellulase preparation and the protease preparation is equal to the enzyme activity of the ligninase preparation, and the enzyme activity ratio of the cellulase preparation and the protease preparation is 1:1, and the rest is the same as Example 4.

[0140] Comparative Example 4

[0141] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 30-mesh sieve and take out the sieved material.

[0142] (2) Add purified water to the sieved material, the amount of water added being 20 times the mass of the sieved material.

[0143] (3) The mixture obtained in step (2) was placed in a round-bottom flask, distilled at 100° C. for 2 h, and the condensed distillate was collected.

[0144] (4) The distillate was concentrated by reverse osmosis through a spiral membrane multifunctional pilot test device (RNF0460-001, Xiamen Fumei Technology Co., Ltd.), wherein the sodium chloride retention rate of the reverse osmosis membrane was 99.3%, and the retentate was collected as an extract.

[0145] test

[0146] (1) Test method for the content of volatile aroma components and main aroma components:

[0147] Take 6 mL of the extract into a headspace bottle, add 10 μL of 2-octanol and 2.0 g of sodium chloride, seal it, put it into a constant temperature heated magnetic stirrer at 60 ° C for 15 minutes, insert the aged extraction needle, adsorb in the headspace for 45 minutes, and analyze it at 250 ° C for 5 minutes at the gas chromatography injection port for GC-MS analysis.

[0148] GC operating conditions: chromatographic column, Agilent HP-INNOWAX (30m×0.25mm×0.25μm) flexible quartz capillary column; injection port temperature, 250°C; heating program: initial column temperature 40°C, hold for 5min, increase to 240°C at 5°C / min, hold for 5min, post-run time 5min; carrier gas is He (purity>99.999%), column flow rate 1.0mL / min; non-split injection.

[0149] MS operating conditions: ionization source is EI; ionization energy is 70 eV; ion source temperature is 230°C; mass spectrometer quadrupole temperature is 150°C; mass scanning range (m / z) is 35-450.

[0150] Qualitative analysis: After GC-MS detection and analysis, the volatile components were matched with the NIST.11 library for similarity and characteristic peak analysis. Compounds with similarity greater than 60 were retained, and qualitative analysis of the detected components was performed in combination with relevant literature.

[0151] Quantitative analysis: The quantitative analysis of volatile components adopts the internal standard method, with 2-octanol as the internal standard to calculate the content of volatile aroma components in the extract, and then calculate the total amount of each volatile aroma component in the extract, and then divide it by the mass of the discarded tobacco powder to obtain the yield of each volatile aroma component in the extract.

[0152] The results are shown in Tables 1-2.

[0153] Table 1 Yield of different types of volatile aroma components of the extracts of Examples 1-6 and Comparative Examples 1-4 (μg / 10g)

[0154] serial number Alcohols Aldehydes Ketones Esters Olefins other Total rate Example 1 18.00±0.15 21.83±0.8 355.56±0.64 34.07±0.28 46.2±1.00 61.54±1.24 539.39±2.54 Example 2 23.42±0.05 27.37±0.79 394.12±10.52 39.66±3.75 74.83±3.01 79.63±1.23 639.03±7.71 Example 3 21.95±0.19 26.72±1.28 348.03±5.80 52.67±3.91 55.54±1.16 66.67±2.96 571.60±12.35 Example 4 21.58±1.16 28.02±1.08 363.77±4.79 33.63±3.39 70.09±4.71 76.69±0.69 593.78±4.25 Example 5 19.24±0.08ab 21.53±0.61ab 265.21±2.14ab 35.64±0.84b 46.14±0.95a 62.52±0.82a 450.28±3.24ab Example 6 18.85±0.12c 20.67±1.15c 266.36±3.12c 34.28±2.67 43.67±1.14c 61.29±1.15c 445.12±4.13c Comparative Example 1 15.26±1.01a 18.37±1.02a 254.95±1.17a 32.82±2.54 40.59±0.89a 58.37±0.67a 420.36±5.14a Comparative Example 2 16.29±0.07b 17.36±0.96b 241.82±0.93b 31.45±2.12b 39.46±1.25b 52.69±0.72b 399.07±7.16b Comparative Example 3 15.39±0.08c 19.21±0.81c 262.39±1.02c 30.26±1.97 42.23±0.46c 59.34±1.97c 428.82±4.19c Comparative Example 4 17.10±0.38c 16.85±1.68c 235.92±9.17c 27.89±0.51c 28.54±0.10c 46.36±7.60c 372.65±18.42c

[0155] Note: "a" indicates that there is a significant difference from the result of Example 2 (P<0.05);

[0156] "b" indicates that there is a significant difference from the result of Example 3 (P<0.05);

[0157] "c" indicates that there is a significant difference from the result of Example 4 (P<0.05).

[0158] It can be seen from the above table:

[0159] Compared with the extract prepared by using only lignin enzyme preparations with equal enzyme activity in Example 5 and the extract prepared by using only protease preparations with equal enzyme activity in Comparative Example 1, the yield of volatile aroma components other than esters in the extract prepared by using lignin enzyme preparations and protease preparations in Example 2 of the present invention is significantly improved.

[0160] Compared with the extract prepared by using only the ligninase preparation of equal enzyme activity in Example 5, the yield of volatile aroma components of alcohols, aldehydes, ketones, and esters in the extract prepared by using the ligninase preparation and the cellulase preparation in Example 3 of the present invention was significantly improved, and the total yield of volatile aroma components was significantly improved. Compared with the extract prepared by using only the cellulase preparation of equal enzyme activity in Comparative Example 2, the yield of various volatile aroma components in the extract prepared by using the ligninase preparation and the cellulase preparation in Example 3 of the present invention was significantly improved.

[0161] Compared with the extract prepared by using only ligninase preparations with equal enzyme activity in Example 6 and the extract prepared by using cellulase preparations and protease preparations with equal total enzyme activity in Comparative Example 3, the yield of volatile aroma components other than esters in the extract prepared by using ligninase preparations, cellulase preparations and protease preparations in Example 4 of the present invention is significantly improved.

[0162] In Examples 2-4, the extract prepared by using the lignin enzyme preparation and the protease preparation in Example 2 has the highest yield of alcohols, ketones, alkenes and other volatile aroma components, and the highest total yield of volatile aroma components.

[0163] Compared with comparative example 4 without enzymatic hydrolysis, the yields of various volatile aroma components of the extracts prepared by using lignin enzyme preparations in examples 1, 5-6 of the present invention are significantly higher.

[0164] Table 2 Main aroma component yields of extracts from Examples 1-6 and Comparative Examples 1-4 (μg / 10g)

[0165]

[0166]

[0167] Note: "a" indicates that there is a significant difference from the result of Example 2 (P<0.05);

[0168] "b" indicates that there is a significant difference from the result of Example 3 (P<0.05);

[0169] "c" indicates that there is a significant difference from the result of Example 4 (P<0.05).

[0170] It can be seen from the above table:

[0171] Compared with the extract prepared by using only the ligninase preparation of equal enzyme activity in Example 5, the yield of the main aroma components except β-damascenone and benzyl alcohol in the extract prepared by using the ligninase preparation and the protease preparation in Example 2 of the present invention is significantly improved. The yield of various main aroma components of the extract in Example 2 of the present invention is significantly higher than the yield of various main aroma components of the extract prepared by using only the protease preparation of equal enzyme activity in Comparative Example 1.

[0172] Compared with the extract prepared by using only the ligninase preparation of equal enzyme activity in Example 5, the yield of the main aroma components except megastigmatrienone, β-damascenone and benzyl alcohol in the extract prepared by using the ligninase preparation and the cellulase preparation in Example 3 of the present invention is significantly improved. The yield of various main aroma components of the extract of Example 3 of the present invention is significantly higher than the yield of various main aroma components of the extract prepared by using only the cellulase preparation of equal enzyme activity in Comparative Example 2.

[0173] Compared with the extract prepared by using only ligninase preparations with equal enzyme activity in Example 6, the yield of the main aroma components except β-damascenone, linalool and benzyl alcohol in the extract prepared by using ligninase preparations, cellulase preparations and protease preparations in Example 4 of the present invention is significantly improved. The yields of various main aroma components of the extract in Example 4 of the present invention are significantly higher than the yields of various main aroma components of the extract prepared by using cellulase preparations and protease preparations with equal total enzyme activity in Comparative Example 3.

[0174] In Examples 2-4, the extract prepared by using the lignin enzyme preparation and the protease preparation in Example 2 had the highest yields of megastigmatrienone, damascena, solanone, geranylacetone, benzaldehyde, linalool and benzyl alcohol.

[0175] Compared with comparative example 4 without enzymatic hydrolysis, the yields of various main aroma components of the extracts prepared by using lignin enzyme preparations in examples 1, 5-6 of the present invention are significantly higher.

[0176] (2) Evaluation of sensory quality of cigarettes: The extract was uniformly added into the tobacco of the reference cigarettes at a ratio of 0.08% by mass using an essence injector. The reference cigarettes without extract were used as blank samples. The cigarettes were balanced in an environment of 22±1℃ and RH (relative humidity) of 60±2% for 48h, and professional smokers were organized to conduct comparative smoking evaluation of the cigarettes according to "YC / T 497-2014 Sensory Evaluation Method for Chinese Cigarette Style". The results are shown in Table 3.

[0177] Table 3 Sensory quality evaluation results of cigarettes

[0178] serial number Gloss (5) Aroma(32) Coordination (6) Miscellaneous gas (12) Irritant(20) Aftertaste (25) Total score (100) Blank 4.50 26.26 4.21 9.35 16.23 20.03 80.6 Example 1 4.50 27.13 4.46 9.93 16.92 20.39 83.3 Example 2 4.50 27.41 4.61 10.18 17.19 20.51 84.4 Example 3 4.50 27.25 4.52 10.09 17.11 20.42 83.9 Example 4 4.50 27.32 4.56 10.12 17.15 20.45 84.1 Example 5 4.50 27.01 4.42 9.78 16.77 20.32 82.8 Example 6 4.50 26.97 4.41 9.77 16.75 20.31 82.7 Comparative Example 1 4.50 26.89 4.36 9.7 16.66 20.27 82.4 Comparative Example 2 4.50 26.85 4.35 9.66 16.63 20.25 82.2 Comparative Example 3 4.50 26.91 4.38 9.72 16.68 20.29 82.5 Comparative Example 4 4.50 26.63 4.32 9.56 16.49 20.16 81.7

[0179] It can be seen from the above table:

[0180] Compared with the extract prepared only with lignin enzyme preparation in Example 5 and the extract prepared only with protease preparation in Comparative Example 1, the extract prepared with lignin enzyme preparation and protease preparation in Example 2 is added to cigarettes, which can increase the tobacco aroma and sweet aroma of the cigarettes when smoking, increase the softness and roundness of the smoke, make the aftertaste more comfortable, improve the coordination of the aroma, reduce the impurities and irritation, and significantly improve the overall sensory quality.

[0181] Compared with the extract prepared only with ligninase preparation in Example 5 and the extract prepared only with cellulase preparation in Comparative Example 2, the extract prepared with ligninase preparation and cellulase preparation in Example 3 is added to cigarettes, which can increase the tobacco aroma and sweet aroma of the cigarettes when smoking, increase the softness and roundness of the smoke, make the aftertaste more comfortable, improve the aroma coordination, reduce the impurities and irritation, and significantly improve the overall sensory quality.

[0182] Compared with the extract prepared only with ligninase preparation in Example 6 and the extract prepared with cellulase preparation and protease preparation in Comparative Example 3, the extract prepared with ligninase preparation, cellulase preparation and protease preparation in Example 4 is added to cigarettes, which can increase the tobacco aroma and sweet aroma of the cigarettes when smoking, increase the softness and roundness of the smoke, make the aftertaste more comfortable, improve the coordination of the aroma, reduce the impurities and irritation, and significantly improve the overall sensory quality.

[0183] In Examples 2-4, the extract prepared by the lignin enzyme preparation and the protease preparation in Example 2 was added to the cigarette. When smoking, the tobacco aroma and sweet aroma of the cigarette were the most obvious, the smoke had the best softness and roundness, the best aftertaste comfort, the best aroma coordination, the least impurities, the least irritation, and the best overall sensory quality.

[0184] Compared with the extract prepared without enzymatic hydrolysis in Comparative Example 4, the extracts prepared by using lignin enzyme preparations in Examples 1, 5-6 are added to cigarettes. When smoking, the tobacco aroma and sweet aroma of the cigarettes are more obvious, the smoke has a softer and rounder feeling, the aftertaste is more comfortable, the aroma is better coordinated, the impurities and irritation are reduced, and the overall sensory quality is improved.

[0185] The main aroma components in the extract have a certain effect on improving the smoking quality of cigarettes. For example, megastigmatrienone has a sweet ketone aroma, which is beneficial to improving the aroma quality; β-damascenone has a fragrance similar to grape juice with a cool smell, and has the aroma of plum, grapefruit, raspberry, tea and tobacco, which is beneficial to increase the sweetness of the smoke, set off the smoke, reduce the smell, improve the texture, and improve the aftertaste; solanone has a faint carrot aroma and grass flavor, with a slight medicinal aroma, which can improve the softness and permeability of the smoke, increase the sweetness, improve the texture, and help reduce the smell and irritation of the smoke; damascenone has a strong rose-like aroma, which can enhance the aroma of the smoke. The main aroma components in Table 2 are as follows: 1. Benzyl alcohol has an almond-like aroma and a light sweet flowery aroma, which is conducive to improving the aroma quality of the smoke; 2. Benzaldehyde has a strong almond flavor and a baking and nutty aroma, which is conducive to increasing the sweetness of the smoke, making the smoke delicate and soft, and reducing the stray smell and irritation of the smoke; 3. Linalool has a typical floral aroma, a fresh and elegant aroma, a weak citrus fruity aroma, a soft and transparent aroma, which is conducive to making the smoke rise and bright, with green fragrance, floral fragrance, and woody characteristics, covering up stray smells (especially burnt stray smells), improving the aftertaste, and having a more obvious aftertaste; 4. Benzyl alcohol has an almond-like aroma and a light sweet flower aroma, which is conducive to improving the softness of the smoke. Therefore, the joint action of the main aroma components in Table 2 is conducive to the improvement of the sensory quality of cigarettes.

[0186] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An enzyme composition comprising a first enzyme obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 24244; and One or both of the following: A second enzyme obtained by fermentation of Bacillus saffron with a preservation number of CGMCC No. 24246; The third enzyme is obtained by fermentation of Bacillus subtilis with a preservation number of CGMCC No.24134.

2. The enzyme composition according to claim 1, wherein The ratio of the enzyme activity of the first enzyme to one or both of the second enzyme and the third enzyme is 0.00005:1-0.0005:1; Optionally, it is characterized by any one of the following: The ratio of the enzyme activity of the first enzyme to the second enzyme is 0.0001:1-0.0005:1; The ratio of the enzyme activity of the first enzyme to the third enzyme is 0.0001:1-0.0005:1; The ratio of the enzyme activities of the first enzyme, the second enzyme and the third enzyme is 0.0001:1:1-0.0005:1:

1.

3. The enzyme composition according to claim 1 or 2, wherein The first enzyme, the second enzyme or the third enzyme is obtained by the following steps: Inoculate the strain into the first culture medium and culture it to obtain a bacterial solution; wherein the strain is Bacillus subtilis with a preservation number of CGMCC No. 24244, Bacillus safovici with a preservation number of CGMCC No. 24246, or Bacillus subtilis with a preservation number of CGMCC No. 24134; Inoculating the bacterial liquid into a second culture medium and fermenting at 29-40° C. for 10-60 hours to obtain a fermentation product; The fermentation product is cleaned, concentrated, and optionally dried.

4. The enzyme composition according to claim 3, characterized in that One or more of the following: In the step of preparing the fermentation product, the volume ratio of the bacterial liquid to the second culture medium is 0.5%-7%; Fermentation under stirring conditions of 100-220r / min; Remove impurities by filtering with 100KD-150KD ultrafiltration membrane; optionally, filter with 150-250nm ceramic membrane before filtering with ultrafiltration membrane; Concentrate through 3KD-30KD ultrafiltration membrane; In the step of preparing the bacterial solution, the culture temperature is 29-40° C. and the culture time is 10-36 hours; In the step of preparing the bacterial solution, the culture is carried out under stirring conditions of 150-220 r / min.

5. A method for extracting aroma components from tobacco waste, wherein: The aroma components include one or more compounds of alcohols, aldehydes, ketones, esters, and olefins; optionally, the aroma components include one or more of megastigmatrienone, damascenone, solanone, β-damascenone, geranylacetone, methyl palmitate, benzaldehyde, linalool, and benzyl alcohol; The method comprises the following steps: mixing tobacco waste with water to obtain a mixture; The mixture is enzymatically hydrolyzed by using the enzyme composition described in any one of claims 1 to 4 or the first enzyme to inactivate the enzyme and obtain an enzymatic hydrolysis product.

6. The method according to claim 5, wherein: The temperature of the enzymolysis is 35-65° C., and the time of the enzymolysis is 1-7 hours.

7. The method according to claim 5 or 6, further comprising: distilling the enzymatic hydrolysis product, collecting the distillate, and concentrating the distillate; Optionally, the distillation temperature is 80-120°C; Optionally, the distillation time is 1-4 hours.

8. The method according to any one of claims 5 to 7, characterized in that One or more of the following: The ratio of the enzyme composition or the first enzyme to the tobacco waste is 0.001-0.01 U / g or 3-10 U / g; The enzymolysis is carried out under stirring conditions of 20-60 r / min; In the step of preparing the mixture, the mass ratio of the tobacco waste to water is 1:15-1:40; The enzyme inactivation is performed by heat treatment at 95°C or above for more than 8 minutes; Use reverse osmosis membrane for concentration; Before the mixing, the tobacco waste is crushed and sieved; The tobacco waste is selected from one or more of waste tobacco leaves, waste tobacco shreds, waste cigarette butts, and waste cigarette butt rods.

9. An extract containing aroma components, which is an extract obtained by the method according to any one of claims 5 to 8; the aroma components include one or more compounds of alcohols, aldehydes, ketones, esters, and olefins; optionally, the aroma components include one or more of megastigmatrienone, damascenone, solanone, β-damascenone, geranylacetone, methyl palmitate, benzaldehyde, linalool, and benzyl alcohol.

10. The extract according to claim 9, characterized in that One or more of the following: The alcohol compound yield of the extract is ≥1.75 μg / g, preferably ≥2 μg / g; The yield of aldehyde compounds in the extract is ≥2 μg / g, preferably ≥2.4 μg / g; The ketone compound yield of the extract is ≥26.5 μg / g, preferably ≥30 μg / g; The ester compound yield of the extract is ≥3.3 μg / g, preferably ≥3.6 μg / g; The olefin compound yield of the extract is ≥4.3 μg / g, preferably ≥5 μg / g; The total yield of aroma components of the extract is ≥44 μg / g, preferably ≥50 μg / g; The yield of megastigmatrienone in the extract is ≥20 μg / g, preferably ≥25 μg / g; The damascene yield of the extract is ≥4 μg / g, preferably ≥4.5 μg / g; The solanone yield of the extract is ≥3.4 μg / g, preferably ≥5 μg / g; The β-damascenone yield of the extract is ≥1.9 μg / g, preferably ≥2.0 μg / g; The geranylacetone yield of the extract is ≥1.2 μg / g, preferably ≥1.6 μg / g; The palmitic acid methyl ester yield of the extract is ≥0.95 μg / g, preferably ≥1.1 μg / g; The benzaldehyde yield of the extract is ≥0.55 μg / g, preferably ≥0.6 μg / g; The linalool yield of the extract is ≥0.33 μg / g, preferably ≥0.37 μg / g; The benzyl alcohol yield of the extract is ≥0.36 μg / g, preferably ≥0.37 μg / g.

11. A tobacco composition, comprising the extract according to claim 9 or 10; preferably, the tobacco composition is a tobacco flavor or tobacco leaf composition.

12. A tobacco product comprising the extract according to claim 9 or 10 or the tobacco composition according to claim 11.

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