Complex enzyme preparation for directionally enhancing sticky rice aroma characteristic of tobacco leaves and application of complex enzyme preparation

Abstract

The invention discloses a compound enzyme preparation for directionally enhancing the sticky rice aroma characteristic of tobacco leaves and application of the compound enzyme preparation. The compound enzyme preparation comprises 800-1200 U / g of nicotine dehydrogenase, 1000-1500 U / g of 6-hydroxynicotine oxidase, 1000-1500 U / g of ketone dehydrogenase, 1200-1600 U / g of 2, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4 The enzyme is prepared from the following raw materials in percentage by weight: 1,500 to 1,600 U / g of gamma-N-methyl aminobutyrate oxidase, 1,600 to 2,000 U / g of aldehyde dehydrogenase and 10 to 50 U / g of enzyme activity protective agent. The compound enzyme preparation can effectively reduce the irritation and strength of cigarettes, has a good aroma enhancement effect, high enzymatic efficiency and thermal stability, can significantly improve the quality of tobacco raw materials and increase the content of aroma substances, and has a wide application prospect. The compound enzyme preparation is applied to oriented aroma enhancement of flue-cured tobacco, the problem that cost is increased due to addition of other spices is solved, the content of sticky rice aroma characteristic aroma substances in the flue-cured tobacco is increased, offensive odor of tobacco leaves is improved, the aroma quality is improved, and meanwhile the compound enzyme preparation is good in aroma enhancement effect, high in enzymatic efficiency, good in heat stability and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of active composite enzyme preparations, and in particular to a composite enzyme preparation for directionally enhancing the glutinous rice aroma characteristics of tobacco leaves and an application thereof. Background Art

[0002] With the deepening of the concept of healthy consumption, the market demand for low-hazard, high-aroma, and good-tasting cigarettes is growing. To meet this demand, the tobacco industry is realizing product upgrades through technological innovation throughout the entire industry chain. Modern tobacco leaf processing has formed a technical system covering raw material planting, biological treatment, and process optimization. Among them, bio-enzyme catalysis technology has become an important means to improve tobacco leaf quality due to its high efficiency and environmental protection. In the tobacco processing process, bio-enzymes act as a catalyst. Bio-enzymes can selectively degrade macromolecular substances (such as starch, protein, etc.) in tobacco leaves and convert them into small molecular sugars, amino acids and other potential aroma components, thereby reducing impurities and improving aroma quality.

[0003] Patent (CN201210377503.1) proposes a method for improving the sensory quality of tobacco leaves by using a composite enzyme preparation, which can increase the richness of aroma and reduce impurities to a certain extent. However, the chemical components contained in the composite enzyme preparation may bring complexity to the subsequent process flow, which is a shortcoming of the patented technology. Patent (CN202411073377) proposes a method for increasing tobacco aroma by using a composite enzyme preparation, which can significantly increase the total sugar content, flavor components and nicotine content by enzymolysis of glycoside substances in tobacco flake concentrate or tobacco extract, thereby improving the sensory quality of cigarettes. However, this method does not directly process tobacco, but processes tobacco processed materials and further applies them to cigarette production, which increases the complexity of the process and limits its large-scale application. Patent (CN202310415185) proposes a composite enzyme system for degrading tobacco polysaccharides and its application. This method effectively improves the degradation ability of tobacco polysaccharides, but its system is relatively complex, and its stability and cost control in industrial production need to be optimized. Although compound enzyme treatment has the characteristics of fast catalytic speed, natural and environmentally friendly, the existing enzyme treatment technology still has problems such as poor process compatibility and insufficient enhancement of characteristic aroma.

[0004] Therefore, there is an urgent need to develop more efficient and targeted enzyme solutions. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a composite enzyme preparation for directionally enhancing the glutinous rice fragrance characteristics of tobacco leaves, aiming at directional flavor enhancement of tobacco, shortening the aging time and expanding the application value of the composite enzyme preparation. The composite enzyme preparation can effectively reduce the irritation and strength of cigarettes, has good flavor enhancement effect, high enzyme-catalyzing efficiency and thermal stability, can significantly improve the quality of tobacco raw materials, increase the content of aroma substances, and has broad application prospects.

[0006] The technical problem to be solved by the present invention is realized through the following technical solutions:

[0007] A composite enzyme preparation for directionally enhancing the glutinous rice fragrance characteristics of tobacco leaves, comprising: 800 - 1200 U / g nicotine dehydrogenase, 1000 - 1500 U / g 6-hydroxy nicotine oxidase, 1000 - 1500 U / g ketone dehydrogenase, 1200 - 1600 U / g 2,6-dihydroxypseudooxynicotine hydratase, 1500 - 1600 U / g γ-N-methylaminobutyrate oxidase, 1600 - 2000 U / g aldehyde dehydrogenase and 10 - 50 U / g enzyme activity protector.

[0008] Preferably, in the above technical solution, it comprises: 1000 U / g of nicotine dehydrogenase, 1250 U / g of 6-hydroxy nicotine oxidase, 1250 U / g of ketone dehydrogenase, 1400 U / g of 2,6-dihydroxypseudooxynicotine hydratase, 1550 U / g of γ-N-methylaminobutyrate oxidase, 1800 U / g of aldehyde dehydrogenase and 30 U / g of enzyme activity protector.

[0009] Preferably, in the above technical solution, the amino acid sequence of the nicotine dehydrogenase is as shown in SEQ ID NO:1, the amino acid sequence of the 6-hydroxy nicotine oxidase is as shown in SEQ ID NO:2, the amino acid sequence of the ketone dehydrogenase is as shown in SEQ ID NO:3, the amino acid sequence of the 2,6-dihydroxypseudooxynicotine hydratase is as shown in SEQ ID NO:4, the amino acid sequence of the γ-N-methylaminobutyrate oxidase is as shown in SEQ ID NO:5, and the amino acid sequence of the aldehyde dehydrogenase is as shown in SEQ ID NO:6.

[0010] Preferably, in the above technical solution, the enzyme activity protector is protein protector S15.

[0011] Preferably, in the above technical solution, the volume ratio of nicotine dehydrogenase, 6-hydroxy nicotine oxidase, ketone dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, aldehyde dehydrogenase is 1:10:10:10:10:10 - 10:10:10:10:10:1, preferably 5:3:3:2:2:2.

[0012] Preferably, in the above technical solution, the expression vector of the complex enzyme system is pET-28a(+), and the expression strain of the complex enzyme system is Escherichia coli BL21(DE3).

[0013] A preparation method of a complex enzyme preparation for directionally enhancing the waxy aroma characteristics of tobacco leaves includes the following steps:

[0014] (1) Inoculate Escherichia coli BL21(DE3) into LB medium containing 50 μg / mL kanamycin sulfate, and culture at 37 °C and 220 RPM until OD 600 is 0.6 - 0.8;

[0015] (2) Add 0.2 - 1.0 mM IPTG and induce at 16 - 30 °C for 8 - 24 h;

[0016] (3) After the induction ends, centrifuge to collect the thalli and break the cells, pass the supernatant through a gel column chromatography, and separate to obtain nicotine dehydrogenase, 6-hydroxy nicotine oxidase, ketone dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase;

[0017] (4) Mix the enzymes in step (3) according to the ratio of 1:10:10:10:10:10 - 10:10:10:10:10:1 by volume, and add a protein protectant to obtain the complex enzyme preparation.

[0018] An application of a complex enzyme preparation in improving the quality of tobacco, specifically for directionally enhancing the aroma of tobacco raw materials.

[0019] Preferably, in the above technical solution, the characteristic aroma substance produced by the complex enzyme preparation through enzymatic hydrolysis is 1-acetyl-2-pyrroline.

[0020] Preferably, in the above technical solution, spray the complex enzyme preparation on the surface of flue-cured tobacco leaves, and place them in a constant temperature and humidity environment for aging treatment.

[0021] Preferably, in the above technical solution, spray the complex enzyme preparation on the surface of flue-cured tobacco leaves, and place them in an environment with a temperature of 30 °C and a humidity of 30% - 40% for aging treatment.

[0022] Preferably, in the above technical solution, the dosage of the complex enzyme preparation is 0.1 - 2% of the weight of the flue-cured tobacco, and the aging time is 48 - 120 h.

[0023] The above technical solution of the present invention has the following beneficial effects:

[0024] (1) The present invention provides a process for the directional aroma enhancement of flue-cured tobacco by applying an enzyme preparation with high enzyme activity and high stability, which not only solves the problems such as increasing costs by adding other substances, but also directionally increases the content of aroma substances in flue-cured tobacco.

[0025] (2) The process for the directional aroma enhancement of flue-cured tobacco by applying a composite enzyme preparation with high enzyme activity and high stability provided by the present invention can directionally metabolize nicotine in tobacco leaves into aroma substances with the characteristics of sticky rice fragrance, thereby playing a role in improving the off-odor of tobacco leaves and enhancing the aroma quality. The composite enzyme preparation prepared by the present invention has good aroma enhancement effect, high enzymatic efficiency and good thermal stability, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings incorporated in and constituting a part of this specification illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0027] Figure 1 Schematic diagram for constructing the composite enzyme preparation.

[0028] Figure 2 Graph of the determination result of nicotine content after treatment with the composite enzyme preparation.

[0029] Figure 3 Graph of the determination result of 2-acetyl-1-pyrroline content after treatment with the composite enzyme preparation. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] Now, various exemplary embodiments of the present invention will be described in detail with reference to the drawings. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions and values set forth in these embodiments do not limit the scope of the present invention.

[0031] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used can be obtained from commercial channels unless otherwise specified. The equipment used in the experiments is well-known to those skilled in the art unless otherwise specified.

[0032] Example 1 Strain Construction

[0033] A composite enzyme preparation for directional aroma enhancement mainly includes nicotine dehydrogenase, 6-hydroxynicotine oxidase, ketone dehydrogenase, 2,6-dihydroxynicotine pseudooxidase hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase.

[0034] (1) Construction of genetically engineered Escherichia coli producing nicotine dehydrogenase: The gene fragment of NDH was amplified using primers NDH-F and NDH-R. The purified gene fragment was ligated to the linearized vector pET-28a(+) digested with BamHΙ and EcoRΙ by homologous recombination to obtain the recombinant plasmid pET28a-NDH. The recombinant plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR. The strain with correct PCR verification was subjected to sequencing analysis. After correct sequencing, the recombinant plasmid was extracted using a plasmid extraction kit. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. After picking the transformed colonies for overnight culture, positive clones were verified again by colony PCR. The recombinant strain verified by PCR was named BL21-pET28a-NDH. The primers are as follows:

[0035] NDH-F: ATGGATAACCCACACTATTACTCTCCTCCCA.

[0036] NDH-R: TTACTCCCTGTCCATAGGGCCGCGATCAA.

[0037] (2) Construction of genetically engineered Escherichia coli producing 6-hydroxy nicotine oxidase: The gene fragment of 6HLNO was amplified using primers 6HLNO-F and 6HLNO-R. The purified gene fragment of 6HLNO was ligated to the linearized vector pET-28a(+) digested with BamHΙ and EcoRΙ by homologous recombination to obtain the recombinant plasmid pET28a-6HLNO. The recombinant plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR. The strain with correct PCR verification was subjected to sequencing analysis. After correct sequencing, the recombinant plasmid was extracted using a plasmid extraction kit. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. After picking the transformed colonies for overnight culture, positive clones were verified again by colony PCR. The recombinant strain verified by PCR was named BL21-pET28a-6HLNO. The primers are as follows:

[0038] 6HLNO-F: ATGTATGACGCGATTGTGGTTGGTGG.

[0039] 6HLNO-R: CTACGAATGCAGAATTGCGTTCACCGCG.

[0040] (3) Construction of engineered Escherichia coli producing ketone dehydrogenase: The KDH gene fragment was amplified with primers KDH-F and KDH-R. The purified KDH gene fragment was ligated by homologous recombination with the linearized vector pET-28a(+) digested with BamHΙ and EcoRΙ to obtain the recombinant plasmid pET28a-KDH. The recombinant plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR. The strain with correct PCR verification was subjected to sequencing analysis. After correct sequencing, the recombinant plasmid was extracted using a plasmid extraction kit. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. After picking the transformed colonies for overnight culture, positive clones were verified again by colony PCR. The recombinant strain verified by PCR was named BL21-pET28a-KDH. Among them, the primers are as follows:

[0041] KDH-F: ATGATGGCAAAGGCTAAAGCGCTCATC.

[0042] KDH-R: TTACCGTTCTGCTTTGTTCAAACCTTGACAGCGG.

[0043] (4) Construction of engineered Escherichia coli producing 2,6-dihydroxypseudoxynicotine hydratase: The DHPONH gene fragment was amplified with primers DHPONH-F and DHPONH-R. The purified DHPONH gene fragment was ligated by homologous recombination with the linearized vector pET-28a(+) digested with BamHΙ and EcoRΙ to obtain the recombinant plasmid pET28a-DHPONH. The recombinant plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR. The strain with correct PCR verification was subjected to sequencing analysis. After correct sequencing, the recombinant plasmid was extracted using a plasmid extraction kit. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. After picking the transformed colonies for overnight culture, positive clones were verified again by colony PCR. The recombinant strain verified by PCR was named BL21-pET28a-DHPONH. Among them, the primers are as follows:

[0044] DHPONH-F: ATGACTGTAACTTCGCAGGTGAAACCTGAAG.

[0045] DHPONH-R: TTATCCATTGAGGGGCCACCCTTTCATTGTT.

[0046] (5) Construction of engineered Escherichia coli producing γ-N-methylaminobutyrate oxidase: The MABO gene fragment was amplified using primers MABO-F and MABO-R. The purified MABO gene fragment was ligated by homologous recombination with the pET-28a(+) linearized vector digested with BamHΙ and EcoRΙ to obtain the recombinant plasmid pET28a-MABO. The recombinant plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR. The strain with correct PCR verification was subjected to sequencing analysis. After correct sequencing, the recombinant plasmid was extracted using a plasmid extraction kit. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. After picking the transformed colonies for overnight culture, positive clones were verified again by colony PCR. The recombinant strain verified by PCR was named BL21-pET28a-MABO. Among them, the primers are as follows:

[0047] MABO-F: ATGGACAGGCTTGTCGACCGCGA.

[0048] MABO-R: CTAACCGCGGAGCCGTTCGCCCTTT.

[0049] (6) Construction of engineered Escherichia coli producing aldehyde dehydrogenase: The ADH gene fragment was amplified using primers ADH-F and ADH-R. The purified ADH gene fragment was ligated by homologous recombination with the pET-28a(+) linearized vector digested with BamHΙ and EcoRΙ to obtain the recombinant plasmid pET28a-ADH. The recombinant plasmid was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR. The strain with correct PCR verification was subjected to sequencing analysis. After correct sequencing, the recombinant plasmid was extracted using a plasmid extraction kit. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. After picking the transformed colonies for overnight culture, positive clones were verified again by colony PCR. The recombinant strain verified by PCR was named BL21-pET28a-ADH. Among them, the primers are as follows:

[0050] ADH-F: ATGGCAATCGCGACCATCGACCCCA.

[0051] ADH-R: TCAGGAGGTCCAGACCGTCTTGATGTTTACG.

[0052] Example 2 Purification and preparation of nicotine dehydrogenase, 6-hydroxy nicotine oxidase, ketone dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, aldehyde dehydrogenase:

[0053] Respectively pick the E.coli BL21-pET28a-NDH, BL21-pET28a-6HLNO, BL21-pET28a-6HNO, BL21-pET28a-KDH, BL21-pET28a-DHPONH, BL21-pET28a-MABO, and BL21-pET28a-ADH strains with successful verification and inoculate them in 5 mL of LB medium (containing 50 μg / mL kanamycin sulfate) at 37 °C for overnight culture to obtain seed solutions. Then inoculate the seed solutions into 100 mL of LB medium at an inoculation amount of 2%, and culture them at 37 °C and 220 RPM until the OD 600 = 0.6 - 0.8. Add IPTG with a final concentration of 0.2 - 1 mM and induce for 8 - 24 h at 16 - 30 °C and 200 RPM. After the induction ends, use a refrigerated centrifuge to centrifuge at 4 °C and 6000 RPM for 10 min to collect the bacterial cells. The collected bacterial cells are resuspended in a buffer containing 30 mM imidazole, and the bacterial cells are disrupted for 30 min under the conditions of a working time of 2 s and an intermittent time of 3 s. Use a refrigerated centrifuge to centrifuge at 4 °C and 6000 RPM for 10 min to collect the upper-layer crude enzyme solution. Pass the crude enzyme solution through gel column chromatography, and elute the impurity proteins and target proteins with 100 mM and 500 mM imidazole buffer solutions respectively. After concentration by an ultrafiltration centrifugal tube, nicotine dehydrogenase, 6-hydroxy nicotine oxidase, ketone dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase are obtained.

[0054] Enzyme activity determination: Respectively take a certain amount of the enzyme solutions after induction and purification of the E.coli BL21-pET28a-NDH, BL21-pET28a-6HLNO, BL21-pET28a-KDH, BL21-pET28a-DHPONH, BL21-pET28a-MABO, and BL21-pET28a-ADH strains, add 100 mM substrate, react at 30 °C and 200 RPM for 1 h, and then detect the concentration of the product. Among them, the enzyme activity is defined as: the amount of enzyme required to produce 1 μmol of the corresponding product per minute is 1 U.

[0055] Example 3 Effect of the composite enzyme preparation on the directional flavor enhancement of tobacco raw materials

[0056] In order to measure the effect of the composite enzyme preparation provided in the present invention on the directional flavor enhancement of tobacco raw materials, the following steps are carried out for directional flavor-enhancing fermentation:

[0057] (1) Preparation of composite enzyme: The purified enzyme solution was obtained from the strain in Example 2 under the above conditions, and was concentrated using a refrigerated centrifuge at 4°C and 6000 RPM to prepare nicotine dehydrogenase, 6-hydroxy nicotine oxidase, keto dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase.

[0058] (2) Composite enzyme system: Nicotine dehydrogenase, 6-hydroxy nicotine oxidase, keto dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase were mixed in a ratio of 5:3:3:2:2:2 by mass concentration, and a protein protection solution was added.

[0059] (3) Pretreatment of tobacco leaves: Flue-cured tobacco was pulverized with a pulverizer, and 10 g of flue-cured tobacco was added to a 250 mL shaking flask.

[0060] (4) Fermentation of tobacco leaves: The composite enzyme preparation in step (1) was added to the pretreated tobacco leaves, and added in a ratio of 5% of the volume of the composite enzyme preparation to the weight of the flue-cured tobacco, and sprayed on the surface of the flue-cured tobacco in a spraying manner.

[0061] (5) The tobacco leaves treated in step (3) were placed in a constant temperature and humidity environment for aging, and the aging was controlled to incubate in a shaking table at 30°C and 150 RPM for 48 h - 120 h.

[0062] Comparative Example 1

[0063] The difference between the method of this Comparative Example 1 and Example 3 is that: PBS buffer was used to treat the tobacco leaves instead of the composite enzyme, and other steps and parameters were the same as those in Example 3.

[0064] Comparative Example 2

[0065] The method provided in this Comparative Example 2 includes the following steps:

[0066] (1) Preparation of composite enzyme: The purified enzyme solution was obtained from the strain in Example 2 under the above conditions, and was concentrated using an ultrafiltration centrifugal tube at 4°C and 6000 RPM to prepare nicotine dehydrogenase, 6-hydroxy nicotine oxidase, keto dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase.

[0067] (2) Composite enzyme system: Nicotine dehydrogenase, 6-hydroxy nicotine oxidase, keto dehydrogenase, 2,6-dihydroxypseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase were mixed in a ratio of 5:3:3:2:2:2 by mass concentration, and a protein protectant was added.

[0068] (3) Pretreatment of tobacco leaves: After pulverizing flue-cured tobacco with a pulverizer, add 10 g of flue-cured tobacco to a 250 mL shaking flask.

[0069] (4) Fermentation of tobacco leaves: Add the composite enzyme preparation in step (1) to the pretreated tobacco leaves, and add it according to the ratio of the volume of the composite enzyme preparation to the weight of flue-cured tobacco being 1%, and spray it on the surface of the flue-cured tobacco in a spraying manner.

[0070] (5) Place the tobacco leaves treated in step (3) in a constant temperature and humidity environment for aging, and control the aging to incubate in a constant temperature air bath shaker at 30 °C and 150 RPM for 48 h - 120 h.

[0071] Comparative Example 3

[0072] The method provided in this Comparative Example 3 includes the following steps:

[0073] (1) Preparation of composite enzyme: Obtain the purified enzyme solution from the strain in Example 2 under the above conditions, and concentrate it using a refrigerated centrifuge at 4 °C and 6000 RPM to prepare nicotine dehydrogenase, 6-hydroxy nicotine oxidase, keto dehydrogenase, 2,6-dihydroxy pseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase.

[0074] (2) Composite enzyme system: Mix nicotine dehydrogenase, 6-hydroxy nicotine oxidase, keto dehydrogenase, 2,6-dihydroxy pseudooxynicotine hydratase, γ-N-methylaminobutyrate oxidase, and aldehyde dehydrogenase in a ratio of 1:1:1:1:1:1 by mass concentration, and add a protein protectant.

[0075] (3) Pretreatment of tobacco leaves: After pulverizing flue-cured tobacco with a pulverizer, add 10 g of flue-cured tobacco to a 250 mL shaking flask.

[0076] (4) Fermentation of tobacco leaves: Add the composite enzyme preparation in step (1) to the pretreated tobacco leaves, and add it according to the ratio of the composite enzyme preparation to the weight of flue-cured tobacco being 5%, and spray it on the surface of the flue-cured tobacco in a spraying manner.

[0077] (5) Place the tobacco leaves treated in step (3) in a constant temperature and humidity environment for aging, and control the aging to incubate in a constant temperature air bath shaker at 30 °C and 150 RPM for 48 h - 120 h.

[0078] As shown in Table 1 and Table 2, divide the same batch of tobacco leaves into 4 groups, and treat the tobacco leaves using the methods provided in Example 3 and Comparative Examples 1 - 3 respectively, where Comparative Example 1 is the control group. Analyze the content of the aroma substance 2-acetyl-1-pyrroline in the tobacco leaves, extract the aroma substances using solid-phase microextraction technology, and determine the content of flavor components using gas chromatography-mass spectrometry.

[0079] As can be seen from Table 1, compared with Comparative Example 1, the nicotine content of tobacco leaves treated by the method of the present invention is significantly reduced, the degradation rate reaches more than 40%, the aroma substance content is relatively increased, and the internal components tend to be balanced, which is conducive to improving the intrinsic quality of tobacco leaves. Compared with the tobacco leaves treated by the method provided in Comparative Examples 2 and 3, the nicotine content of tobacco leaves treated by the method provided in Comparative Examples 2 and 3 is higher, while the aroma substance content is lower, indicating that the composite enzyme has a synergistic effect, and the addition of the composite enzyme ratio in Example 3 can achieve the best nicotine degradation effect in tobacco leaves. Similarly, the results in Comparative Examples 2 and 3 can also illustrate that the ratio of the composite enzyme preparation to the weight of flue-cured tobacco plays an important role in tobacco leaf processing. Although the ratio of 1% has a certain effect, the aroma substance content is low, and the nicotine in the tobacco leaves can be degraded to a lower level by the ratio of 5%, and the aroma substance content increases with the increase.

[0080] Table 1 Changes in aroma components of tobacco leaves before and after treatment

[0081] Group Nicotine (wt.%) 2-Acetyl-1-pyrroline (wt.%) Example 3 2.02 0.62％ Comparative Example 1 3.12 0.12％ Comparative Example 2 2.38 0.36％ Comparative Example 3 2.18 0.43％

[0082] It can be seen from Table 2 that tobacco leaves treated by the method of the present invention show significant advantages in sensory evaluation. Specifically, the tobacco leaves treated by Example 3 are superior to the tobacco leaves treated by Comparative Examples 1-3 in various indicators such as aroma quality, aroma content, miscellaneous odor, and aftertaste, and its overall score is the highest, reaching 40.1 points. This shows that the composite enzyme preparation of the present invention can effectively improve the aroma quality of tobacco leaves, making the aroma more intense and pure, thereby significantly improving the overall sensory quality of tobacco leaves. In comparison, Comparative Example 1 replaces the composite enzyme with ultrapure water to treat tobacco leaves, and the aroma content in the tobacco leaves is less, and the sensory evaluation effect is poor. It shows that biological enzymes can significantly improve the quality of tobacco leaves, improve tobacco aroma, reduce miscellaneous odors, etc. Although Comparative Examples 2 and 3 also use composite enzymes, due to improper proportions or dosages, they fail to achieve the treatment effect of Example 3, further proving the importance of the proportions and dosages of the composite enzyme preparations in the present invention to the improvement of tobacco leaf quality.

[0083] Table 2 shows the sensory evaluation analysis of tobacco leaves before and after treatment

[0084]

[0085] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalents.

Claims

1. A composite enzyme preparation for directionally enhancing the Nuomixiang characteristics of tobacco leaves, characterized in that, Comprising: 800 - 1200 U / g nicotine dehydrogenase, 1000 - 1500 U / g 6 - hydroxy nicotine oxidase, 1000 - 1500 U / g ketone dehydrogenase, 1200 - 1600 U / g 2,6 - dihydroxy pseudooxynicotine hydratase, 1500 - 1600 U / g γ - N - methylaminobutyrate oxidase, 1600 - 2000 U / g aldehyde dehydrogenase and 10 - 50 U / g enzyme activity protectant.

2. The composite enzyme preparation for directionally enhancing the waxy rice fragrance characteristics of tobacco leaves according to claim 1, wherein, Comprising: Composed of 1000 U / g nicotine dehydrogenase, 1250 U / g 6 - hydroxy nicotine oxidase, 1250 U / g ketone dehydrogenase, 1400 U / g 2,6 - dihydroxy pseudooxynicotine hydratase, 1550 U / g γ - N - methylaminobutyrate oxidase, 1800 U / g aldehyde dehydrogenase and 30 U / g enzyme activity protectant.

3. The composite enzyme preparation for directionally enhancing the waxy aroma characteristics of tobacco leaves according to claim 2, wherein The amino acid sequence of the said nicotine dehydrogenase is as shown in SEQ ID NO:1, the amino acid sequence of the said 6 - hydroxy nicotine oxidase is as shown in SEQ ID NO:2, the amino acid sequence of the said ketone dehydrogenase is as shown in SEQ ID NO:3, the amino acid sequence of the said 2,6 - dihydroxy pseudooxynicotine hydratase is as shown in SEQ ID NO:4, the amino acid sequence of the said γ - N - methylaminobutyrate oxidase is as shown in SEQ ID NO:5, and the amino acid sequence of the said aldehyde dehydrogenase is as shown in SEQ ID NO:

6.

4. The composite enzyme preparation for directionally enhancing the waxy aroma characteristics of tobacco leaves according to claim 2, characterized in that, The said enzyme activity protectant is protein protectant S15.

5. The composite enzyme preparation for directionally enhancing the waxy aroma characteristics of tobacco leaves according to claim 1, wherein The volume ratio of nicotine dehydrogenase, 6 - hydroxy nicotine oxidase, ketone dehydrogenase, 2,6 - dihydroxy pseudooxynicotine hydratase, γ - N - methylaminobutyrate oxidase, aldehyde dehydrogenase is 1:10:10:10:10:10 - 10:10:10:10:10:1, preferably 5:3:3:2:2:

2.

6. The composite enzyme preparation for directionally enhancing the nuomixiang characteristics of tobacco leaves according to claim 1, wherein The expression vector of the complex enzyme preparation is pET - 28a(+), and the expression strain of the complex enzyme preparation is Escherichia coli BL21(DE3).

7. The preparation method of the composite enzyme preparation for directionally enhancing the waxy rice fragrance characteristics of tobacco leaves according to any one of claims 1-6, characterized in that, Including the following steps: (1) Inoculate Escherichia coli BL21(DE3) into LB medium containing 50 μg / mL kanamycin sulfate respectively, and culture it at 37 °C and 220 RPM until OD 600 reaches 0.6 - 0.8; (2) Add 0.2 - 1.0 mM IPTG and induce at 16 - 30 °C for 8 - 24 h; (3) After the induction ends, centrifuge to collect the thallus and break the cells respectively, and pass the supernatant through a gel column chromatography to separate nicotine dehydrogenase, 6 - hydroxy nicotine oxidase, ketone dehydrogenase, 2,6 - dihydroxy pseudooxynicotine hydratase, γ - N - methylaminobutyrate oxidase, aldehyde dehydrogenase; (4) Mix the enzymes in step (3) according to the volume ratio of 1:10:10:10:10:10 - 10:10:10:10:10:1, and add a protein protectant to obtain the complex enzyme preparation.

8. Use of the complex enzyme preparation according to any one of claims 1 - 6 or the complex enzyme preparation prepared by the preparation method according to claim 7 in improving the quality of tobacco.

9. The application according to claim 8, wherein The characteristic aroma substance produced by the said complex enzyme preparation through enzymatic hydrolysis is 1 - acetyl - 2 - pyrroline.

10. The application according to claim 8, wherein Apply the complex enzyme preparation on the surface of flue-cured tobacco leaves and place them in a constant temperature and humidity environment for aging treatment. The dosage of the complex enzyme preparation is 0.1-5% of the weight of the flue-cured tobacco, and the aging time is 48-120h.

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