Enzyme composition, method for extracting carbohydrates 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.24134; and any one of the following items: a second enzyme obtained by fermenting paenibacillus amyloliquefaciens with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24248; the third enzyme is obtained by fermenting bacillus subtilis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24244; the preservation number of the bacillus amyloliquefaciens is CGMCC (China General Microbiological Culture Collection Center) No.24247. The bacillus The invention also relates to a method for extracting carbohydrates from tobacco waste, an extract containing carbohydrates, a composition for cigarettes and a tobacco product. According to the enzyme composition, the extraction yield of carbohydrates in the tobacco waste can be remarkably increased; and / or the extract is added into the cigarette, so that when the cigarette is smoked, the aroma quality and aroma coordination of the cigarette can be improved, the smoke fluency and fluffy feeling are improved, offensive odor and irritation are reduced, the sweet feeling is increased, 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 sugar substances from tobacco waste, an extract containing sugar substances, 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 carbohydrates. Carbohydrates mainly include reducing sugars (such as glucose, fructose, galactose, lactose, maltose, etc.) and non-reducing sugars (such as starch, cellulose, etc.). Rich carbohydrates play an important role in balancing nitrogen-containing compounds in tobacco, improving smoke strength and irritation, and increasing the filling value and flexibility of tobacco. At the same time, carbohydrates usually undergo a series of complex chemical reactions during the combustion of cigarettes, and the generated products directly or indirectly affect the sensory quality of cigarettes. However, the existing technology has a low extraction rate for carbohydrates (especially reducing sugars and total sugars) in tobacco waste, which is not conducive to the efficient utilization of tobacco waste.

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

[0005] One of the purposes of the present invention is to provide an enzyme composition, which can significantly improve the extraction rate of sugar substances (especially reducing sugars and total sugars) by preparing tobacco waste extracts with the enzyme composition; and / or, the prepared tobacco waste extracts are added to cigarettes, which can improve the aroma quality and aroma coordination of cigarettes when smoking, improve the fluency and fluffiness of smoke, reduce impurities and irritation, increase sweetness, and significantly improve the overall sensory quality. On this basis, another purpose of the present invention is to provide a method for extracting sugar substances from tobacco waste, an extract containing sugar substances, 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 preservation number of CGMCC No. 24134; and

[0007] Any of the following:

[0008] A second enzyme obtained by fermentation of Bacillus amyloliquefaciens with a deposit number of CGMCC No. 24248;

[0009] A third enzyme obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 24244;

[0010] The fourth enzyme is obtained by fermentation of Bacillus amyloliquefaciens with a preservation number of CGMCC No.24247.

[0011] The above Bacillus subtilis with a deposit number of CGMCC No. 24134, Bacillus amyloliquefaciens with a deposit number of CGMCC No. 24248, Bacillus subtilis with a deposit number of CGMCC No. 24244, or Bacillus amyloliquefaciens with a deposit number of CGMCC No. 24247 are all deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC), and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101. Among them, the deposit date of Bacillus subtilis with a deposit number of CGMCC No. 24134 is December 20, 2021, and it was screened from Yongding YLC1Y-2019 tobacco leaves in Longyan, Fujian. Chinese patent applications have been submitted for Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24248, Bacillus subtilis with a preservation number of CGMCC No. 24244, or Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24247, and relevant information about the strains has been recorded in the public text.

[0012] In any embodiment, the ratio of the enzyme activity of the first enzyme to any one selected from the second enzyme, the third enzyme, and the fourth enzyme is 30:30-30:0.08, for example, 30:30, 30:25, 30:20, 30:15, 30:10, 30:5, 30:1, 30:0.5, 30:0.2, 30:0.1, and 30:0.08.

[0013] In any embodiment, the ratio of the enzyme activity of the first enzyme to the second enzyme is 30:30-30:20, for example, 30:30, 30:25, 30:20.

[0014] In any embodiment, the ratio of the enzyme activities of the first enzyme to the third enzyme is 30:0.5-30:0.08, for example, 30:0.5, 30:0.2, 30:0.1, 30:0.08.

[0015] In any embodiment, the ratio of the enzyme activities of the first enzyme to the fourth enzyme is 30:10-30:1, for example, 30:10, 30:5, 30:1.

[0016] In any embodiment, the first enzyme, the second enzyme, the third enzyme or the fourth enzyme is obtained by the following steps:

[0017] The strain is inoculated into the first culture medium and cultured to obtain a bacterial liquid; wherein the strain is Bacillus subtilis with a preservation number of CGMCC No. 24134, Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24248, Bacillus subtilis with a preservation number of CGMCC No. 24244, or Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24247;

[0018] 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;

[0019] The fermentation product is cleaned, concentrated, and optionally dried.

[0020] 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%, or 7%.

[0021] 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.

[0022] In any embodiment, ultrafiltration is performed through a 100KD-150KD (e.g., 100KD, 150KD) ultrafiltration membrane to remove impurities; optionally, before ultrafiltration, impurity removal is performed through a 150-250nm (e.g., 150nm, 180nm, 200nm, 220nm, 250nm) ceramic membrane.

[0023] In any embodiment, the concentration is performed by a 3KD-30KD (eg, 3KD, 3.5KD, 6KD, 10KD, 20KD, 30KD) ultrafiltration membrane.

[0024] 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).

[0025] 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.

[0026] In any embodiment, the first culture medium is a common culture medium used in the art for strain rejuvenation or proliferation, such as TSB liquid or solid culture medium, LB liquid or solid culture medium, etc.

[0027] 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, and can also include 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.

[0028] A second aspect of the present invention provides a method for extracting sugar substances from tobacco waste, wherein the sugar substances include one or more of reducing sugars and non-reducing sugars;

[0029] The method comprises the following steps:

[0030] mixing tobacco waste with water to obtain a mixture;

[0031] Using the enzyme composition or the first enzyme in the first aspect of the present invention to enzymolyze the mixture, inactivate the enzyme, and obtain an enzymolysis product;

[0032] The enzymatic hydrolysis product is subjected to solid-liquid separation, and the liquid phase is collected as an extract.

[0033] In the present application, the enzymatic hydrolysis product refers to the mixed material obtained after enzymatic hydrolysis and enzyme inactivation.

[0034] 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).

[0035] In any embodiment, before the solid-liquid separation, the enzymatic hydrolysate is maintained at 70-85°C (e.g., 70°C, 72°C, 75°C, 77°C, 78°C, 80°C, 81°C, 83°C, 84°C) for 1-5 hours (e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours).

[0036] In any embodiment, the method further comprises: removing impurities from the liquid phase and removing the solvent to obtain an extract.

[0037] In any embodiment, the impurities are removed by filtering through a 150-300 mesh (e.g., 150 mesh, 200 mesh, 250 mesh, 300 mesh) filter bag or filter cloth and a 100-150 nm (e.g., 100 nm, 120 nm, 150 nm) microfiltration membrane in sequence.

[0038] In any embodiment, the solvent is removed by distillation under reduced pressure.

[0039] In any embodiment, before removing the solvent, the liquid phase after impurities are removed is mixed with an additive, the mass ratio of the additive to the tobacco waste is 0.35:1-0.55:1 (e.g. 0.35:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1), and the additive includes propylene glycol.

[0040] In any embodiment, the solvent is removed by distillation under reduced pressure.

[0041] In any embodiment, the ratio of the enzyme composition or the first enzyme to the tobacco waste is 2800-7000U / g, for example, 2800U / g, 3000U / g, 3200U / g, 3300U / g, 3500U / g, 3600U / g, 3800U / g, 4000U / g, 4200U / g, 4400U / g, 4500U / g, 4700U / g, 4900U / g, 5000U / g, 5200U / g, 5500U / g, 5600U / g, 5800U / g, 6000U / g, 6100U / g, 6300U / g, 6500U / g, 6700U / g, 6900U / g, and 7000U / g.

[0042] In any embodiment, the enzymatic hydrolysis is carried out under stirring conditions of 20-60 r / min.

[0043] In any embodiment, the solid-liquid separation is performed by filtering through a 100-300 mesh (eg, 100 mesh, 150 mesh, 200 mesh, 250 mesh, 300 mesh) filter cloth or filter bag.

[0044] In any embodiment, in the step of preparing the mixture, the mass ratio of the tobacco waste to water is 1:20-1:40, for example, 1:20, 1:25, 1:30, 1:35, 1:40.

[0045] 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).

[0046] In any embodiment, before the mixing, the tobacco waste is crushed and sieved.

[0047] 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.

[0048] The third aspect of the present invention provides an extract containing carbohydrates, which is an extract obtained by the method of the second aspect of the present invention; the carbohydrates include one or more of reducing sugars and non-reducing sugars.

[0049] In any embodiment, the reducing sugar yield of the extract is ≥16.5%, preferably 18%-25%, such as 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%; and / or,

[0050] The total sugar yield of the extract is ≥22%, preferably 22.5%-30%, for example 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 26%, 27%, 28%, 29%, 30%.

[0051] In the present application, the reducing sugar yield of the extract can be understood as the mass of reducing sugar in the extract obtained from tobacco waste per unit mass. The definition of the total sugar yield of the extract is similar and will not be repeated here.

[0052] 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.

[0053] 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.

[0054] The present invention achieves one or more of the following beneficial effects:

[0055] 1. The enzyme composition of the present invention significantly improves the extraction rate of sugar substances, especially reducing sugar and total sugar in tobacco waste.

[0056] 2. When the tobacco waste extract prepared by the enzyme composition of the present invention is added to cigarettes, the aroma quality and aroma coordination of the cigarettes can be improved, the fluency and fluffiness of the smoke can be improved, the impurities and irritation can be reduced, the sweetness can be increased, and the overall sensory quality can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] 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:

[0058] Figure 1 It is the morphological characteristic of Bacillus subtilis (CGMCC No.24134). DETAILED DESCRIPTION

[0059] 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.

[0060] Bacillus subtilis Bacillus subtilis Identification of (CGMCC No.24134)

[0061] (1) Morphological characteristics of Bacillus subtilis (CGMCC No. 24134)

[0062] like Figure 1 As 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.

[0063] (2) Molecular biological identification of Bacillus subtilis (CGMCC No. 24134)

[0064] The universal primers were synthesized by the solid phase phosphoramidite triester method, and the primer sequences were:

[0065] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1)

[0066] 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′ (SEQ ID No. 2)

[0067] 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:

[0068] 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.

[0069] 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.

[0070] The amplified PCR products were examined by agarose gel electrophoresis and then sequenced (Weisburg W G S, Barns S M, Pelletier D A, et al. 16S ribosomal DNA amplification for phylogenetic study. [J]. Journal of Bacteriology, 1991, 173(2): 697-703.). The obtained 16S rDNA sequence (SEQ ID No. 3, 1483 bp) is as follows: TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT

[0071] After NCBI gene sequence alignment, this strain has the highest similarity with Bacillus subtilis, so it is identified as Bacillus subtilis.

[0072] Materials used:

[0073] 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;

[0074] LB liquid medium: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028324; LB solid medium: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028334;

[0075] Fermentation medium 1 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;

[0076] Fermentation medium 2 comprises 1.5 mass % pectin, 0.6 mass % yeast powder, 0.6 mass % tryptone, 0.2 mass % calcium chloride, 0.4 mass % zinc sulfate heptahydrate, 5 mass % sodium dihydrogen phosphate, and a pH of 7.0;

[0077] Fermentation medium 3: 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 sections, 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;

[0078] The fermentation medium 4 includes 2 g / L soluble starch, 13 g / L beef extract, 1 g / L 60-100 mesh redried tobacco leaf powder, 0.2 g / L MnCl2, and 5 g / L K2HPO4.

[0079] Preparation Example 1 Preparation of Cellulase Preparation

[0080] 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.

[0081] The seed liquid was inoculated into the fermentation medium 1 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.

[0082] 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.

[0083] Preparation Example 2 Preparation of pectinase preparation

[0084] Paenibacillus amylolyticus FG-1 with a preservation number of CGMCC No. 24248 was used, and a ring of Paenibacillus amylolyticus FG-1 spores was streaked into TSB liquid culture medium, and cultured at 30° C. and 200 r / min for 12 hours to obtain seed solution.

[0085] The seed liquid was inoculated into the fermentation medium 2 at a volume ratio of 1%, and the culture was constant temperature at 30°C and 130r / min for 24 hours to obtain a fermentation liquid. The fermentation liquid was filtered through a 200nm 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 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 pectinase liquid, which was freeze-dried to obtain a pectinase preparation.

[0086] The enzyme activity of the pectinase preparation was measured to be 200 U / mg according to the pectinase determination method in Chinese patent document CN117247866A.

[0087] Preparation Example 3 Preparation of ligninase

[0088] 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.

[0089] The seed liquid was inoculated into the fermentation medium 3 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.

[0090] 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.

[0091] Preparation Example 4 Preparation of amylase preparation

[0092] Paenibacillus amylolyticus with a preservation number of CGMCC No. 24247 was used, and a loop of the strain was taken and streaked in TSB liquid medium, and activated and cultured at 30°C until single colonies were visible. A loop of the activated strain was inoculated in TSB liquid medium, and cultured at 30°C and 200 r / min for 12 hours to obtain seed liquid.

[0093] The seed liquid was inoculated into the fermentation medium 4 at a volume ratio of 5%, and the culture was constant temperature at 37°C and 200r / min for 24 hours to obtain a fermentation liquid. The fermentation liquid was filtered through a ceramic membrane with a pore size of 200nm, 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 amylase liquid, which was freeze-dried to obtain an amylase preparation.

[0094] The enzyme activity of the amylase preparation was determined by referring to the literature (Guo Aiying, Qin Ling, Ling Yun, et al. Research on the fermentation conditions of low-temperature amylase-producing marine bacteria HYM-7 [J]. Food Industry Science and Technology, 2010, 31(11): 235-238.), and the value was 330 U / mg.

[0095] Example 1

[0096] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 40-50 mesh sieve and take out the sieved material.

[0097] (2) Add purified water to the sieved material and mix, wherein the amount of water added is 25 times the mass of the sieved material to obtain a mixture.

[0098] (3) adding a cellulase preparation (the cellulase preparation activity is 6000 U per gram of the sieved material) to the mixture for enzymolysis at a temperature of 60° C. for 2 h, and stirring at 50 rpm during the enzymolysis process.

[0099] (4) After the enzymatic hydrolysis in step (3), the system was inactivated at 100°C for 10 min.

[0100] (5) The enzymatic hydrolysate after enzyme inactivation was kept in a 78°C water bath for 1.2 h, filtered, and the filtrate was collected.

[0101] (6) The filtrate obtained in step (5) is filtered through a double-layer 200-mesh filter cloth, and after the filtrate is collected, it is microfiltered using a 100 nm microfiltration membrane (ceramic membrane pilot equipment, CeraMem0100-013), and the permeate is collected. Propylene glycol is added to the permeate, and the mass of propylene glycol is 45% of the mass of the sieved material obtained in step (1), and the extract is obtained by vacuum distillation until no solvent is distilled out.

[0102] Example 2

[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 and mix, wherein the amount of water added is 25 times the mass of the sieved material to obtain a mixture.

[0105] (3) Adding a cellulase preparation (the enzymatic activity of the cellulase preparation is 3000 U per gram of the sieved material) and a pectinase preparation (the enzymatic activity of the pectinase preparation is 2500 U per gram of the sieved material) to the mixture for enzymolysis. The enzymolysis temperature is 60° C., the enzymolysis time is 2 h, and the mixture is stirred at 30 rpm during the enzymolysis process.

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

[0107] (5) The enzymatic hydrolysate after enzyme inactivation was kept in a 78°C water bath for 1.2 h, filtered, and the filtrate was collected.

[0108] (6) The filtrate obtained in step (5) is filtered through a double-layer 200-mesh filter cloth, and after the filtrate is collected, it is microfiltered using a 100 nm microfiltration membrane (ceramic membrane pilot equipment, CeraMem0100-013), and the permeate is collected. Propylene glycol is added to the permeate, and the mass of propylene glycol is 45% of the mass of the sieved material obtained in step (1), and the extract is obtained by vacuum distillation until no solvent is distilled out.

[0109] Example 3

[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 and mix, wherein the amount of water added is 25 times the mass of the sieved material to obtain a mixture.

[0112] (3) Adding a cellulase preparation (the enzymatic activity of the cellulase preparation is 3000 U per gram of the sieved material) and a ligninase preparation (the enzymatic activity of the ligninase preparation is 0.12 U per gram of the sieved material) to the mixture for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40°C, the enzymatic hydrolysis time is 2 h, and the mixture is stirred at 30 rpm during the enzymatic hydrolysis process.

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

[0114] (5) The enzymatic hydrolysate after enzyme inactivation was kept in a 78°C water bath for 1.2 h, filtered, and the filtrate was collected.

[0115] (6) The filtrate obtained in step (5) is filtered through a double-layer 200-mesh filter cloth, and after the filtrate is collected, it is microfiltered using a 100 nm microfiltration membrane (ceramic membrane pilot equipment, CeraMem0100-013), and the permeate is collected. Propylene glycol is added to the permeate, and the mass of propylene glycol is 45% of the mass of the sieved material obtained in step (1), and the extract is obtained by vacuum distillation until no solvent is distilled out.

[0116] Example 4

[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 and mix, wherein the amount of water added is 25 times the mass of the sieved material to obtain a mixture.

[0119] (3) Adding a cellulase preparation (the enzymatic activity of the cellulase preparation is 3000 U per gram of the sieved material) and an amylase preparation (the enzymatic activity of the amylase preparation is 500 U per gram of the sieved material) to the mixture for enzymolysis at a temperature of 55° C. for 2 h. The mixture was stirred at 30 rpm during the enzymolysis process.

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

[0121] (5) The enzymatic hydrolysate after enzyme inactivation was kept in a 78°C water bath for 1.2 h, filtered, and the filtrate was collected.

[0122] (6) The filtrate obtained in step (5) is filtered through a double-layer 200-mesh filter cloth, and after the filtrate is collected, it is microfiltered using a 100 nm microfiltration membrane (ceramic membrane pilot equipment, CeraMem0100-013), and the permeate is collected. Propylene glycol is added to the permeate, and the mass of propylene glycol is 45% of the mass of the sieved material obtained in step (1), and the extract is obtained by vacuum distillation until no solvent is distilled out.

[0123] Example 5

[0124] In step (3) of Example 2, the pectinase preparation was replaced with a cellulase preparation with the same enzyme activity, and the rest was the same as in Example 2.

[0125] Example 6

[0126] 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 in Example 3.

[0127] Example 7

[0128] In step (3) of Example 4, the amylase preparation was replaced with a cellulase preparation with the same enzyme activity, and the rest was the same as in Example 4.

[0129] Comparative Example 1

[0130] In step (3) of Example 2, the cellulase preparation was replaced with a pectinase preparation with the same enzyme activity, and the rest was the same as in Example 2.

[0131] Comparative Example 2

[0132] In step (3) of Example 3, the cellulase preparation was replaced with a ligninase preparation with the same enzyme activity, and the rest was the same as in Example 3.

[0133] Comparative Example 3

[0134] In step (3) of Example 4, the cellulase preparation was replaced with an amylase preparation with the same enzyme activity, and the rest was the same as in Example 4.

[0135] Comparative Example 4

[0136] (1) Use a pulverizer to grind the waste cigarette dust, then pass it through a 30-mesh sieve and take out the sieved material.

[0137] (2) Add purified water to the sieved material in an amount of 25 times the mass of the sieved material, extract in a 78°C water bath for 1.2 h, filter, and collect the filtrate.

[0138] (3) The filtrate obtained in step (2) is filtered through a double-layer 200-mesh filter cloth, and after the filtrate is collected, it is microfiltered using a 100 nm microfiltration membrane (ceramic membrane pilot equipment, CeraMem0100-013), and the permeate is collected. Propylene glycol is added to the permeate, and the mass of propylene glycol is 45% of the mass of the sieved material obtained in step (1), and the extract is obtained by vacuum distillation until no solvent is distilled out.

[0139] test

[0140] (1) Test method for reducing sugar and total sugar yield:

[0141] All extracts were diluted with water, with the volume ratio of extract to water being 1:200, to obtain extracts for determining the reducing sugar yield.

[0142] 3 mol / L hydrochloric acid solution was added to all the above extracts, the volume ratio of the extract to the hydrochloric acid solution was 25:7, and acid hydrolysis was carried out at 100°C for 30 minutes. After cooling, the pH was adjusted to neutral to obtain an acid hydrolyzate for determining the total sugar yield.

[0143] Take 1 mL of the above extract, acid hydrolyzate and blank control solution (deionized water) in a centrifuge tube, add 2 mL of the color developer dinitrosalicylic acid (DNS), heat in a boiling water bath for 5 min, add 9 mL of deionized water after cooling, shake well, and measure the absorbance at 540 nm on a UV spectrophotometer.

[0144] Prepare a series of glucose standard solutions, take 1 mL of each series of glucose standard solutions in a centrifuge tube, add 2 mL of the color developing agent dinitrosalicylic acid (DNS), heat in a boiling water bath for 5 min, add 9 mL of deionized water after cooling, shake well, and measure the absorbance at 540 nm using a UV spectrophotometer. According to the concentration and absorbance values ​​of the glucose standard solutions (the difference between the absorbance values ​​measured by the glucose standard solutions and the absorbance values ​​measured by the blank control solution), establish a standard curve: y = 1.2267x-0.0379 (x represents the difference between the absorbance values ​​measured by the standard solution and the absorbance values ​​measured by the blank control solution; y represents the concentration of the glucose standard solution, mg / mL).

[0145] Calculate the difference between the absorbance of the extract or acid hydrolyzate and the absorbance of the blank control solution, substitute the difference into the above standard curve to calculate the glucose concentration, and then calculate the reducing sugar yield and total sugar yield of the extract according to the following formula.

[0146]

[0147] Wherein, c is the measured glucose concentration (mg / mL); V is the total volume of the extract or acid hydrolyzate (mL); and m is the total mass of the waste tobacco dust (mg).

[0148] The results are shown in Table 1.

[0149] Table 1 Reducing sugar yield and total sugar yield

[0150]

[0151]

[0152] Note: "*" indicates that there is a significant difference from the results of Example 2 (P<0.05);

[0153] "**" indicates that there is a significant difference from the results of Example 3 (P<0.05);

[0154] “#” indicates that the result is significantly different from that in Example 4 (P<0.05).

[0155] It can be seen from the above table:

[0156] The reducing sugar and total sugar yields of the extract prepared by using cellulase preparation and pectinase preparation in Example 2 were significantly higher than those of the extract prepared by using only cellulase preparation with equal enzyme activity in Example 5, and were also significantly higher than those of the extract prepared by using only pectinase preparation with equal enzyme activity in Comparative Example 1.

[0157] The reducing sugar and total sugar yields of the extract prepared by using cellulase preparations and ligninase preparations in Example 3 were significantly higher than those of the extract prepared by using only cellulase preparations with equal enzyme activity in Example 6, and were also significantly higher than those of the extract prepared by using only ligninase preparations with equal enzyme activity in Comparative Example 2.

[0158] The reducing sugar and total sugar yields of the extract prepared by using cellulase preparations and amylase preparations in Example 4 were significantly higher than those of the extract prepared by using only cellulase preparations with equal enzyme activity in Example 7, and were also significantly higher than those of the extract prepared by using only amylase preparations with equal enzyme activity in Comparative Example 3.

[0159] Among Examples 2-4, the extract prepared by using the cellulase preparation and the pectinase preparation in Example 2 had the highest yield of reducing sugar and total sugar.

[0160] Compared with comparative example 4 without enzymatic hydrolysis, the extracts prepared by using cellulase preparations in examples 1, 5-7 of the present invention have significantly higher reducing sugar and total sugar yields.

[0161] (2) Evaluation of sensory quality of cigarettes: The extract was uniformly added into the tobacco of the reference cigarettes at a ratio of 0.05% by mass using an essence injector. The reference cigarettes without extract were used as blank samples. The cigarettes were equilibrated 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 2.

[0162] Table 2 Sensory quality evaluation results of cigarettes

[0163]

[0164] It can be seen from the above table:

[0165] Compared with the extract prepared by using only cellulase preparation in Example 5 and the extract prepared by using only pectinase preparation in Comparative Example 1, the extract prepared by using cellulase preparation and pectinase preparation in Example 2 is added to cigarettes, which can improve the aroma quality and aroma coordination of cigarettes, improve the fluency and fluffiness of smoke, reduce impurities and irritation, increase sweetness, and significantly improve the overall sensory quality when smoking.

[0166] Compared with the extract prepared by using only the cellulase preparation in Example 6 and the extract prepared by using only the ligninase preparation in Comparative Example 2, the extract prepared by using the cellulase preparation and the ligninase preparation in Example 3 is added to the cigarette, which can improve the aroma quality and aroma coordination of the cigarette when smoking, improve the fluency and fluffiness of the smoke, reduce the impurities and irritation, increase the sweetness, and significantly improve the overall sensory quality;

[0167] Compared with the extract prepared by using only cellulase preparation in Example 7 and the extract prepared by using only amylase preparation in Comparative Example 3, the extract prepared by using cellulase preparation and amylase preparation in Example 4 is added to cigarettes, which can improve the aroma quality and aroma coordination of cigarettes, improve the fluency and fluffiness of smoke, reduce impurities and irritation, increase sweetness, and significantly improve the overall sensory quality when smoking.

[0168] In Examples 2-4, the extract prepared by using cellulase preparation and pectinase preparation in Example 2 was added to the cigarette. When smoking, the cigarette had the best aroma quality, aroma coordination, smoke smoothness and fluffiness, the best sweetness, the least impurities, the least irritation, and the best overall sensory quality.

[0169] Compared with the extract prepared without enzymatic hydrolysis in Comparative Example 4, the extracts prepared by using cellulase preparations in Examples 1, 5-7 are added to cigarettes. When smoked, the aroma quality and aroma coordination of the cigarettes can be improved, the fluency and fluffiness of the smoke can be improved, the impurities and irritation can be reduced, the sweetness can be increased, and the overall sensory quality can be significantly improved.

[0170] 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. 24134; and Any of the following: A second enzyme obtained by fermentation of Bacillus amyloliquefaciens with a deposit number of CGMCC No. 24248; A third enzyme obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 24244; The fourth enzyme is obtained by fermentation of Bacillus amyloliquefaciens with a preservation number of CGMCC No.24247.

2. The enzyme composition according to claim 1, wherein The ratio of the enzyme activity of the first enzyme to any one selected from the second enzyme, the third enzyme, and the fourth enzyme is 30:30-30:0.08; 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 30:30-30:20; The ratio of the enzyme activity of the first enzyme to the third enzyme is 30:0.5-30:0.08; The ratio of the enzyme activities of the first enzyme to the fourth enzyme is 30:10-30:

1.

3. The enzyme composition according to claim 1 or 2, wherein The first enzyme, the second enzyme, the third enzyme or the fourth enzyme is obtained by the following steps: The strain is inoculated into the first culture medium and cultured to obtain a bacterial liquid; wherein the strain is Bacillus subtilis with a preservation number of CGMCC No. 24134, Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24248, Bacillus subtilis with a preservation number of CGMCC No. 24244, or Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24247; 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; Ultrafiltration is performed through a 100KD-150KD ultrafiltration membrane to remove impurities; optionally, before ultrafiltration, impurities are removed through a 150-250nm ceramic 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 a stirring condition of 150-220 r / min.

5. A method for extracting sugar substances from tobacco waste, wherein the sugar substances include one or more of reducing sugars and non-reducing sugars; The method comprises the following steps: mixing tobacco waste with water to obtain a mixture; Using the enzyme composition described in any one of claims 1 to 4 or the first enzyme to enzymolyze the mixture, inactivate the enzyme, and obtain an enzymolysis product; The enzymatic hydrolysis product is subjected to solid-liquid separation, and the liquid phase is collected as an extract.

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, wherein: Before the solid-liquid separation, the enzymatic hydrolysate is kept at 70-85° C. for 1-5 hours.

8. The method according to any one of claims 5 to 7, further comprising: removing impurities and solvent from the liquid phase to obtain an extract; Optionally, filter and remove impurities through a 150-300 mesh filter bag or filter cloth and a 100nm-150nm microfiltration membrane in sequence; Optionally, removing the solvent by distillation under reduced pressure; Optionally, before removing the solvent, the liquid phase after impurities are removed is mixed with an additive, the mass ratio of the additive to the tobacco waste is 0.35:1-0.55:1, and the additive includes propylene glycol.

9. The method according to any one of claims 5 to 8, characterized in that One or more of the following: The ratio of the enzyme composition or the first enzyme to the tobacco waste is 2800-7000 U / g; The enzymolysis is carried out under stirring conditions of 20-60 r / min; The solid-liquid separation is carried out by filtering through a 100-300 mesh filter cloth or filter bag; In the step of preparing the mixture, the mass ratio of the tobacco waste to water is 1:20-1:40; The enzyme inactivation is performed by heat treatment at 95°C or above for more than 8 minutes; 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.

10. An extract containing carbohydrates, which is an extract obtained by the method according to any one of claims 5 to 9; the carbohydrates include one or more of reducing sugars and non-reducing sugars.

11. The extract according to claim 10, wherein The reducing sugar yield of the extract is ≥16.5%, and can be optionally 18%-25%; and / or, The total sugar yield of the extract is ≥22%, and can be optionally 22.5%-30%.

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

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

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