Enzyme composition, method for extracting amino acid 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. The enzyme composition comprises a first enzyme, a second enzyme and a third enzyme, wherein the first enzyme is obtained by fermenting bacillus safensis with the preservation number of CGMCC No.24246; and any one of the following items: a second enzyme obtained by fermenting bacillus subtilis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24134; the third enzyme is obtained by fermenting paenibacillus amyloliquefaciens with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.24248 The preservation number of the bacillus amyloliquefaciens is CGMCC (China General Microbiological Culture Collection Center) No.24247. The bacillus amyloliquefaciens is The invention further relates to a method for extracting the amino acid from the tobacco waste, an extract containing the amino acid, a tobacco composition and a tobacco product. The enzyme composition can significantly improve the extraction rate of amino acid in tobacco waste; and / or when the cigarette is smoked, the aroma quality and the aroma harmony of the cigarette can be improved, the aftertaste comfort is improved, the offensive odor and the irritation are reduced, the smoke elasticity is improved, 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 amino acids from tobacco waste, an extract containing amino acids, a tobacco composition and a tobacco product. Background Art

[0002] Tobacco waste refers to tobacco raw materials generated during the preparation, re-roasting and cigarette production processes that 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 causing environmental pollution.

[0003] Tobacco waste, as a tobacco raw material homologous to tobacco leaves, contains protein. Protein is the source of free amino acids, which are not only an important material basis for unique flavors, but also an important nitrogen source for the Maillard reaction. However, the current technology has a low extraction rate of amino acids in tobacco waste, which is not conducive to the efficient utilization of tobacco waste and hinders the improvement of corporate economic and social benefits.

[0004] Currently, there is an urgent need for a method to effectively increase the extraction rate of amino acids 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 amino acids by preparing tobacco waste extracts with the enzyme composition; and / or, by adding the prepared tobacco waste extracts to cigarettes, the aroma quality and aroma coordination of the cigarettes can be improved when smoking, the comfort of the aftertaste can be improved, the impurities and irritation can be reduced, the elasticity of the smoke can be increased, and the overall sensory quality can be significantly improved. On this basis, another purpose of the present invention is to provide a method for extracting amino acids from tobacco waste, an extract containing amino acids, a tobacco composition and a tobacco product.

[0006] To achieve the above object, the first aspect of the present invention provides an enzyme composition, comprising a first enzyme obtained by fermentation of Bacillus saffron with a preservation number of CGMCC No. 24246; and

[0007] Any of the following:

[0008] A second enzyme obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 24134;

[0009] A third enzyme obtained by fermentation of Bacillus amyloliquefaciens with a deposit number of CGMCC No. 24248;

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

[0011] The above Bacillus saffron with a preservation number of CGMCC No. 24246, Bacillus subtilis with a preservation number of CGMCC No. 24134, Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24248 and Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24247 are all deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC), and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 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 Yongding YLC1Y-2019 tobacco leaves in Longyan, Fujian. Chinese patent applications have been filed for Bacillus saffron with CGMCC No. 24246, Bacillus amyloliquefaciens with CGMCC No. 24248, and Bacillus amyloliquefaciens with CGMCC No. 24247, and the relevant information of 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 3:0.5-3:3, for example, 3:0.5, 3:1, 3:1.5, 3:2, 3:2.5, 3:3.

[0013] In any embodiment, the ratio of the enzyme activity of the first enzyme to the second enzyme is 3:1-3:2, such as 3:1, 3:1.5, 3:2.

[0014] In any embodiment, the ratio of the enzyme activity of the first enzyme to the third enzyme is 3:0.5-3:1.5, for example, 3:0.5, 3:1, 3:1.5.

[0015] In any embodiment, the ratio of the enzyme activities of the first enzyme to the fourth enzyme is 3:2-3:3, for example, 3:2, 3:2.5, 3:3.

[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 solution; wherein the strain is Bacillus saffron with a preservation number of CGMCC No. 24246, Bacillus subtilis with a preservation number of CGMCC No. 24134, Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24248, 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, the 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, impurities are removed by filtration through a 150-250 nm (eg, 150 nm, 180 nm, 200 nm, 220 nm, 250 nm) ceramic membrane and a 100 KD-150 KD (eg, 100 KD, 150 KD) ultrafiltration membrane in sequence.

[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, for example, TSB liquid or solid culture medium, LB liquid or solid culture medium, etc.

[0027] In any embodiment, the second culture medium is a commonly used 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 commonly used 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 amino acids from tobacco waste, comprising the following steps:

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

[0030] Using the enzyme composition of the first aspect of the present invention to enzymolyze the mixture, inactivate the enzyme, and obtain an enzymolysis product;

[0031] The enzymatic hydrolysis product is separated into solid and liquid, and the liquid phase is collected as an extract.

[0032] In the present application, the enzymatic hydrolysis product refers to the mixed material obtained after enzymatic hydrolysis and enzyme inactivation.

[0033] In any embodiment, the enzymatic hydrolysis temperature is 50-65°C (e.g., 50°C, 52°C, 55°C, 57°C, 59°C, 60°C, 62°C, 64°C, 65°C), and the enzymatic hydrolysis time is 1.5-5 hours (e.g., 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours).

[0034] In any embodiment, before the solid-liquid separation, the enzymatic hydrolysate is mixed with an ethanol solution, and the resulting mixture is extracted at 50-75°C (eg, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C).

[0035] In any embodiment, the ethanol concentration in the mixture is 20% - 40%, such as 20%, 25%, 30%, 35%, 40%.

[0036] In any embodiment, the mass ratio of the ethanol solution to the enzymatic hydrolysis product is 0.25:1-0.45:1, for example, 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1.

[0037] In any embodiment, the extraction time is 0.5-4 hours (eg, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours).

[0038] In any embodiment, the extraction is carried out under stirring conditions of 40-60 r / min, for example 50 r / min.

[0039] In any embodiment, the ratio of the enzyme composition to the tobacco waste is 3500-6000U / g, for example, 3500U / g, 3700U / g, 3900U / g, 4000U / g, 4200U / g, 4500U / g, 4600U / g, 4800U / g, 5000U / g, 5100U / g, 5300U / g, 5500U / g, 5600U / g, 5800U / g, 6000U / g.

[0040] In any embodiment, the enzymatic hydrolysis is carried out under stirring conditions of 30-60 r / min, such as 30 r / min, 40 r / min, 50 r / min, 60 r / min.

[0041] In any embodiment, the mass ratio of the tobacco waste to water is 1:10-1:40, for example 1:10, 1:20, 1:25, 1:30, 1:35, 1:40.

[0042] In any embodiment, the mixing is carried out at a boil for 5 to 20 minutes, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes.

[0043] In any embodiment, the solid-liquid separation is performed by filtration and centrifugation in sequence.

[0044] In any embodiment, the filtering is performed using a filter bag or filter cloth with a mesh size of 150-300, for example, 200 mesh.

[0045] In any embodiment, the centrifugal treatment is carried out at a speed of 9000-11000 r / min (e.g., 9000 r / min, 9500 r / min, 10000 r / min, 10500 r / min, 11000 r / min) and the time is 8-15 minutes (e.g., 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes).

[0046] In any embodiment, the method further comprises: mixing the liquid phase with an additive, removing the solvent, and obtaining an extract, wherein the additive comprises propylene glycol, and the mass ratio of the additive to the tobacco waste is 0.15:1-0.35:1, for example 0.5:100, 1:100, 2:100.

[0047] In any embodiment, the solvent is removed by distillation under reduced pressure.

[0048] In any embodiment, the enzyme inactivation is performed using boiling water.

[0049] In any embodiment, before the mixing, the tobacco waste is crushed and sieved.

[0050] 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.

[0051] The third aspect of the present invention provides an extract containing amino acids, which is the extract obtained by the method of the second aspect of the present invention.

[0052] In any embodiment, the molar amount of amino acids in the extract obtained from tobacco waste per gram is 400-550 μmol, such as 420 μmol, 440 μmol, 450 μmol, 470 μmol, 480 μmol, 500 μmol, 510 μmol, 530 μmol, 550 μmol.

[0053] The fourth aspect of the present invention provides a tobacco composition, comprising the extract according to the third aspect of the present invention.

[0054] In any embodiment, the tobacco composition is a tobacco flavoring or tobacco leaf composition.

[0055] 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.

[0056] The present invention achieves one or more of the following beneficial effects:

[0057] 1. The enzyme composition of the present invention significantly improves the extraction rate of amino acids in tobacco waste and can produce an extract with a high amino acid content.

[0058] 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 aftertaste comfort can be improved, the impurities and irritation can be reduced, the smoke elasticity can be increased, and the overall sensory quality can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] 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:

[0060] Figure 1 These are the morphological characteristics of Bacillus subtilis (CGMCC No. 24134). DETAILED DESCRIPTION

[0061] 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.

[0062] Identification of Bacillus subtilis (CGMCC No. 24134)

[0063] Morphological characteristics of Bacillus subtilis (CGMCC No. 24134)

[0064] 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.

[0065] Molecular Biological Identification of Bacillus subtilis (CGMCC No. 24134)

[0066] The universal primers were synthesized by the solid phase phosphoramidite triester method, and the primer sequences were:

[0067] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1)

[0068] 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′ (SEQ ID No. 2)

[0069] 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:

[0070] The total volume of the system was 25 μL: 2× Rapid Taq MasterMix 12.0 μL, 27F and 1492R primers 1 μL each, DNA 1 μL, ddH2O 10 μL.

[0071] 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, and extension at 72 °C for 15 s, for a total of 32 cycles; extension at 72 °C for 5 min.

[0072] The amplified PCR product was sequenced after agarose gel electrophoresis (Weisburg WGS, Barns SM, Pelletier DA, et al. 16S ribosomal DNA amplification for phylogeneticstudy. [J]. Journal of Bacteriology, 1991, 173 (2): 697-703.), and the obtained 16S rDNA sequence (SEQ ID No. 3, 1483 bp) was as follows:

[0073]

[0074] After NCBI gene sequence comparison, the strain had the highest similarity with Bacillus subtilis and was therefore identified as Bacillus subtilis.

[0075] Materials used:

[0076] 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;

[0077] LB liquid medium: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028324;

[0078] LB solid medium: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028334;

[0079] Fermentation medium 1: Add sodium carboxymethyl cellulose to LB liquid medium to make the content of sodium carboxymethyl cellulose 5 g / L;

[0080] Fermentation medium 2 included 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;

[0081] Fermentation medium 3 includes 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 pH 7.0;

[0082] Fermentation medium 4 includes soluble starch 2 g / L, beef extract 13 g / L, 60-100 mesh redried tobacco leaf powder 1 g / L, MnCl2 0.2 g / L, K2HPO4 5 g / L;

[0083] Preparation Example 1 Preparation of protease preparation

[0084] 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 for culturing at 30° C. and 200 r / min for 24 hours to obtain seed solution.

[0085] The seed liquid was inoculated into the fermentation medium 1 at an inoculum volume ratio of 2%, and cultured at 37°C and 110r / min for 15 hours. The fermentation liquid was filtered with 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 protease liquid, which was freeze-dried to obtain a protease preparation.

[0086] The enzymatic 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 40 U / mg.

[0087] Preparation Example 2 Preparation of cellulase preparation

[0088] Bacillus subtilis with the accession number of CGMCC No. 24134 was rejuvenated in LB solid medium at 37°C, and 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.

[0089] 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 h, 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.

[0090] 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.

[0091] Preparation Example 3 Preparation of pectinase preparation

[0092] 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.

[0093] The seed liquid was inoculated into the fermentation medium 3 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.

[0094] 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.

[0095] Preparation Example 4 Preparation of amylase preparation

[0096] Paenibacillus amylolyticus with a preservation number of CGMCC No. 24247 was used. One loop of the strain was streaked in TSB liquid medium and activated and cultured at 30°C until single colonies were visible. One 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 solution.

[0097] 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 24h 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.

[0098] The enzyme activity of the amylase preparation was determined by referring to the literature (Guo Aiying, Qin Ling, Ling Yun, et al. Study 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. Example 1

[0099] (1) Use a crusher to crush the waste cigarette dust, then pass it through a 30-40 mesh sieve and take out the sieved material.

[0100] (2) Add purified water to the sieved material in an amount that is 20 times the mass of the sieved material, boil it for 10-15 minutes, and then cool it to obtain a mixture.

[0101] (3) Add a protease preparation (the activity of the protease preparation is 3000 U per gram of the sieved material) and a cellulase preparation (the activity of the cellulase preparation is 1500 U per gram of the sieved material) to the mixture for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 55°C and the enzymatic hydrolysis time is 3 h. During the enzymatic hydrolysis, the mixture is stirred at a speed of 40-50 r / min.

[0102] (4) After the enzymatic hydrolysis in step (3), the system was inactivated by boiling water for 20 min.

[0103] (5) Add ethanol solution to the cooled enzymatic hydrolysate after enzyme inactivation. The mass of the ethanol solution is 0.35 times the mass of the enzymatic hydrolysate. The ethanol concentration in the resulting mixture is 25% by mass. Extraction is carried out in a 65°C water bath for 1.5 h. During the extraction process, the mixture is stirred at a speed of 50 r / min and the extract is collected by filtration through a 200-mesh filter cloth.

[0104] (6) The extract obtained in step (5) is centrifuged at 10,000 r / min for 10 min, the supernatant is collected, propylene glycol is added to the supernatant, the mass of propylene glycol being 25% of the mass of the sieved material obtained in step (1), and the mixture is distilled under reduced pressure until no solvent is distilled out, thereby obtaining an extract. Example 2

[0105] (1) Use a crusher to crush the waste cigarette dust, then pass it through a 30-40 mesh sieve and take out the sieved material.

[0106] (2) Add purified water to the sieved material in an amount that is 20 times the mass of the sieved material, boil it for 10-15 minutes, and then cool it to obtain a mixture.

[0107] (3) Add a protease preparation (the activity of the protease preparation is 3000 U per gram of the sieved material) and a pectinase preparation (the activity of the pectinase preparation is 1000 U per gram of the sieved material) to the mixture for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 58°C and the enzymatic hydrolysis time is 3 h. During the enzymatic hydrolysis, the mixture is stirred at a speed of 40-50 r / min.

[0108] (4) After the enzymatic hydrolysis in step (3), the system was inactivated by boiling water for 20 min.

[0109] (5) Add ethanol solution to the cooled enzymatic hydrolysate after enzyme inactivation. The mass of the ethanol solution is 0.35 times the mass of the enzymatic hydrolysate. The ethanol concentration in the resulting mixture is 25% by mass. Extraction is carried out in a 65°C water bath for 1.5 h. During the extraction process, the mixture is stirred at a speed of 50 r / min and the extract is collected by filtration through a 200-mesh filter cloth.

[0110] (6) The extract obtained in step (5) is centrifuged at 10,000 r / min for 10 min, the supernatant is collected, propylene glycol is added to the supernatant, the mass of propylene glycol being 25% of the mass of the sieved material obtained in step (1), and the mixture is distilled under reduced pressure until no solvent is distilled out, thereby obtaining an extract. Example 3

[0111] (1) Use a crusher to crush the waste cigarette dust, then pass it through a 30-40 mesh sieve and take out the sieved material.

[0112] (2) Add purified water to the sieved material in an amount that is 20 times the mass of the sieved material, boil it for 10-15 minutes, and then cool it to obtain a mixture.

[0113] (3) Add a protease preparation (the activity of the protease preparation is 3000 U per gram of the sieved material) and an amylase preparation (the activity of the amylase preparation is 2500 U per gram of the sieved material) to the mixture for enzymolysis. The enzymolysis temperature is 60°C and the enzymolysis time is 3 h. During the enzymolysis process, the mixture is stirred at a speed of 40-50 r / min.

[0114] (4) After the enzymatic hydrolysis in step (3), the system was inactivated by boiling water for 20 min.

[0115] (5) Add ethanol solution to the cooled enzymatic hydrolysate after enzyme inactivation. The mass of the ethanol solution is 0.35 times the mass of the enzymatic hydrolysate. The ethanol concentration in the resulting mixture is 25% by mass. Extraction is carried out in a 65°C water bath for 1.5 h. During the extraction, the mixture is stirred at a speed of 40-50 r / min and the extract is collected by filtration through a 200-mesh filter cloth.

[0116] (6) The extract obtained in step (5) is centrifuged at 10,000 r / min for 10 min, the supernatant is collected, propylene glycol is added to the supernatant, the mass of propylene glycol being 25% of the mass of the sieved material obtained in step (1), and the mixture is distilled under reduced pressure until no solvent is distilled out, thereby obtaining an extract.

[0117] Comparative Example 1a

[0118] In step (3) of Example 1, the cellulase preparation was replaced with a protein enzyme preparation with equal enzyme activity, and the rest was the same as in Example 1.

[0119] Comparative Example 1b

[0120] In step (3) of Example 1, the protein enzyme preparation was replaced with a cellulase preparation with the same enzyme activity, and the rest was the same as in Example 1.

[0121] Comparative Example 2a

[0122] In step (3) of Example 2, the pectinase preparation was replaced with a protein enzyme preparation with equal enzyme activity, and the rest was the same as in Example 2.

[0123] Comparative Example 2b

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

[0125] Comparative Example 3a

[0126] In step (3) of Example 3, the amylase preparation was replaced with a protein enzyme preparation with equal enzyme activity, and the rest was the same as in Example 3.

[0127] Comparative Example 3b

[0128] In step (3) of Example 2, the protein enzyme preparation was replaced with an amylase preparation with equal enzyme activity, and the rest was the same as in Example 3.

[0129] Comparative Example 4

[0130] (1) Use a pulverizer to crush the waste cigarette dust and then pass it through a 30-40 mesh sieve to obtain the sieved material.

[0131] (2) Add purified water to the sieved material in an amount of 20 times the mass of the sieved material, boil it for 10-15 minutes, and then cool it to obtain a mixture I.

[0132] (3) Add ethanol solution to mixture I, the mass of the ethanol solution being 0.35 times the mass of mixture I, so that the ethanol concentration in the resulting mixture II is 25% by mass. Extract in a 65°C water bath for 1.5 h, stirring at a speed of 40-50 r / min during the extraction process, and collect the extract through 200 mesh filter cloth.

[0133] (4) The extract obtained in step (3) is centrifuged at 10,000 r / min for 10 min, the supernatant is collected, propylene glycol is added to the supernatant, the mass of propylene glycol is 25% of the mass of the sieved material obtained in step (1), and distilled under reduced pressure until no solvent is distilled out, thereby obtaining an extract.

[0134] test

[0135] (1) Amino acid content test method: The amino acid content detection kit (Shanghai Solebow Biotechnology Co., Ltd., catalog number: BC1575) was used to test the amino acid concentration in the extract using the ninhydrin colorimetric method according to the instructions. The total molar amount of amino acids in the extract was calculated and then divided by the mass of the waste cigarette butts used to obtain the molar amount of amino acids extracted per gram of waste cigarette butts.

[0136] The results are shown in Table 1.

[0137] Table 1 Results of the molar amount of amino acids extracted per gram of waste tobacco

[0138] Note: "*" indicates that there is a significant difference from the results of Example 1 (P < 0.05);

[0139] "**" indicates that there is a significant difference from the results of Example 2 (P < 0.05);

[0140] “#” indicates that there is a significant difference compared with the result of Example 3 (P < 0.05).

[0141] It can be seen from the above table:

[0142] The molar amount of amino acids extracted per gram of waste tobacco dust obtained by using the protease preparation and the cellulase preparation in Example 1 is significantly higher than the molar amount of amino acids extracted per gram of waste tobacco dust obtained by using only the protease preparation with equal enzyme activity in Comparative Example 1a, and is also significantly higher than the molar amount of amino acids extracted per gram of waste tobacco dust obtained by using only the cellulase preparation with equal enzyme activity in Comparative Example 1b;

[0143] The molar amount of amino acids extracted per gram of waste tobacco dust obtained by using the protease preparation and pectinase in Example 2 is significantly higher than the molar amount of amino acids extracted per gram of waste tobacco dust obtained by using only the protease preparation with equal enzyme activity in Comparative Example 2a, and is also significantly higher than the molar amount of amino acids extracted per gram of waste tobacco dust obtained by using only the pectinase preparation with equal enzyme activity in Comparative Example 2b;

[0144] The molar amount of amino acids extracted per gram of waste tobacco dust obtained by using the protease preparation and the amylase preparation in Example 3 is significantly higher than the molar amount of amino acids extracted per gram of waste tobacco dust obtained by using only the protease preparation with equal enzyme activity in Comparative Example 3a, and is also significantly higher than the molar amount of amino acids extracted per gram of waste tobacco dust obtained by using only the amylase preparation with equal enzyme activity in Comparative Example 3b;

[0145] Among Examples 1-3, Example 1 obtained the highest amount of amino acids extracted per gram of waste tobacco dust using the protease preparation and the cellulase preparation, followed by Example 2 obtained the highest amount of amino acids extracted per gram of waste tobacco dust using the protease preparation and the pectinase preparation;

[0146] The molar amount of amino acids extracted from each gram of waste tobacco dust in Comparative Example 4 is the lowest.

[0147] (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 48 h, and professional smokers were organized to conduct comparative smoking evaluation of the cigarettes in accordance with "YC / T 497-2014 Sensory Evaluation Method for Chinese Cigarette Style". The results are shown in Table 2.

[0148] Table 2 Sensory quality evaluation results

[0149] It can be seen from the above table:

[0150] Compared with the extract prepared by only protease preparation in comparative example 1a and the extract prepared by only cellulase preparation in comparative example 1b, the extract prepared by using protease preparation and cellulase preparation in Example 1 was added to the cigarette, and the aroma quality of the cigarette was better, the aroma coordination was better, the aftertaste was more comfortable, the impurities and irritation were reduced, the smoke elasticity was increased, and the overall sensory quality was significantly improved;

[0151] Compared with the extract prepared by only protease preparation in comparative example 2a and the extract prepared by only pectinase preparation in comparative example 2b, the extract prepared by using protease preparation and pectinase preparation in Example 2 was added to the cigarette, and the aroma quality of the cigarette was better, the aroma coordination was better, the aftertaste was more comfortable, the impurities and irritation were reduced, the smoke elasticity was increased, and the overall sensory quality was significantly improved;

[0152] Compared with the extract prepared by only protease preparation in comparative example 3a and the extract prepared by only amylase preparation in comparative example 3b, the extract prepared by using protease preparation and amylase preparation in Example 3 was added to the cigarette, and the aroma quality of the cigarette was better, the aroma coordination was better, the aftertaste was more comfortable, the impurities and irritation were reduced, the smoke elasticity was increased, and the overall sensory quality was significantly improved;

[0153] In Examples 1-3, the extract prepared by the protease preparation and the cellulase preparation in Example 1 was added to the cigarette. When smoking, the cigarette had the best aroma quality and aroma coordination, the most comfortable aftertaste, the most obvious reduction in impurities and irritation, the best smoke elasticity, and the best overall sensory quality.

[0154] Compared with Example 3, the extract prepared by the protease preparation and the pectinase preparation in Example 2 was added to the cigarette. When smoking, the cigarette had better aroma quality and aroma coordination, more comfortable aftertaste, less impurities, lower irritation, better smoke elasticity, and better overall sensory quality.

[0155] When the extract of Comparative Example 4 was added to the cigarette, the aroma quality, aroma coordination, and aftertaste of the cigarette were poor, there were more impurities, the irritation was greater, the smoke elasticity was poor, and the overall sensory quality was poor.

[0156] 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 sabdariffa with a deposit number of CGMCC No. 24246; and, Any of the following: A second enzyme obtained by fermentation of Bacillus subtilis with a deposit number of CGMCC No. 24134; A third enzyme obtained by fermentation of Bacillus amyloliquefaciens with a deposit number of CGMCC No. 24248; 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 3:0.5 - 3:3; 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 3:1 - 3:2; The ratio of the enzyme activity of the first enzyme to the third enzyme is 3:0.5 - 3:1.5; The ratio of the enzyme activities of the first enzyme to the fourth enzyme is 3:2-3:

3.

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 solution; wherein the strain is Bacillus saffron with a preservation number of CGMCC No. 24246, Bacillus subtilis with a preservation number of CGMCC No. 24134, Bacillus amyloliquefaciens with a preservation number of CGMCC No. 24248, 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; Impurities are removed by filtering through 150-250nm ceramic membrane and 100KD-150KD ultrafiltration membrane in sequence; 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 amino acids from tobacco waste, comprising the following steps: mixing tobacco waste with water to obtain a mixture; Using the enzyme composition according to any one of claims 1 to 4 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 enzymatic hydrolysis temperature is 50-65°C, and the enzymatic hydrolysis time is 1.5-5 hours.

7. The method according to claim 5 or 6, wherein: Before the solid-liquid separation, the enzymatic hydrolysate is mixed with an ethanol solution, and the resulting mixture is extracted at 50-75°C; Optionally, the ethanol concentration in the mixture is 20% - 40%; Optionally, the mass ratio of the ethanol solution to the enzymatic hydrolysis product is 0.25:1 – 0.45:1; Optionally, the extraction time is 0.5-4 hours; Optionally, the extraction is carried out under stirring conditions of 40 - 60 r / min.

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 to the tobacco waste is 3500 – 6000 U / g; The enzymolysis is carried out under stirring conditions of 30-60r / min; The mass ratio of the tobacco waste to water is 1:10-1:40; Mix at a boil for 5 – 20 minutes; The solid-liquid separation is carried out by filtering and centrifuging in sequence; The filtration is carried out using a filter bag or filter cloth with a mesh size of 150-300; The centrifugal treatment is performed at a speed of 9000-11000 r / min for 8-15 minutes; The method further comprises: mixing the liquid phase with an additive, removing the solvent, and obtaining an extract, wherein the additive comprises propylene glycol, and the mass ratio of the additive to the tobacco waste is 0.15:1-0.35:1; The enzyme inactivation is carried out using boiling water; 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 amino acids, which is the extract obtained by the method according to any one of claims 5 to 8.

10. The extract according to claim 9, wherein The amino acid molar amount in the extract obtained from tobacco waste is 400 - 550 μmol per gram.

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

12. The tobacco composition according to claim 11, wherein The tobacco composition is a tobacco flavoring or tobacco leaf composition.

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

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