Method for improving tobacco stem alcoholization efficiency through combined use of multiple strains

Abstract

The invention discloses a method for improving tobacco stem alcoholization efficiency through combined use of multiple strains, the method uses a composite crude enzyme preparation to treat tobacco stems, and the composite crude enzyme preparation is prepared by the following steps: (1) strain activation: respectively activating pseudomonas paraxanthoceras ZY-03, bacillus subtilis XC-19-1 and sphingobacterium neosphingobacterium Y3-1 to prepare a seed solution; (2) amplification culture: respectively inoculating the seed liquid in the step (1) into a culture medium for amplification culture to obtain bacterial liquid; (3) preparing a crude enzyme preparation: respectively centrifuging the bacterial liquid obtained in the step (2), collecting supernatant, and diluting to obtain the crude enzyme preparation; and (4) compounding the crude enzyme preparations: compounding the three crude enzyme preparations obtained in the step (3) according to a certain proportion to obtain the compound crude enzyme preparation. Through combined use of multiple strains, cell wall substances are quickly degraded, and the content of neutral flavor substances is increased, so that the tobacco stem alcoholization efficiency and quality are remarkably improved, and the tobacco stem storage cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cigarette raw material production and processing, and particularly relates to a method for improving the aging efficiency of tobacco stems by using multiple strains in combination. Background Art

[0002] Tobacco stems are the thick and hard leaf veins of tobacco leaves. They can not only improve the filling power of cut tobacco, improve the combustibility of cigarettes, reduce the consumption of tobacco leaf raw materials, but also effectively reduce the content of harmful substances such as cigarette tar, nicotine, and CO. After being separated by threshing and redrying, tobacco stems often need to undergo a natural aging process for a period of time to improve the overall quality. However, the content of cell wall substances (such as cellulose, hemicellulose, lignin, pectin, etc.) in tobacco stems is relatively high, which is likely to produce a large sense of irritation and woody off-flavors during smoking, thus affecting the overall smoking quality. Moreover, cell wall substances are difficult to degrade during the natural aging process, easily leading to insufficient accumulation of aroma precursors in tobacco stems, thereby affecting the aroma quality after aging of tobacco stems.

[0003] Microbial technology has obvious advantages in promoting the rapid degradation of cell wall substances and natural flavor enhancement, providing a feasible technical approach for rapidly improving the overall quality of tobacco stems, shortening the natural aging cycle of tobacco stems, and reducing the storage cost of tobacco stems. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the aging efficiency of tobacco stems by using multiple strains in combination, to solve the problems existing in the traditional natural aging process of tobacco stems, such as difficult degradation of cell wall substances, long aging cycle, high cost, and insufficient aroma quality. By using multiple strains in combination, the cell wall substances can be rapidly degraded, and the content of neutral flavor substances can be increased, thereby significantly improving the aging efficiency and quality of tobacco stems and reducing the storage cost of tobacco stems.

[0005] The technical problem to be solved by the present invention is realized through the following technical solutions:

[0006] A composite crude enzyme preparation for improving the aging efficiency of tobacco stems, and the composite crude enzyme preparation is prepared by the following method:

[0007] (1) Strain activation: Pseudomonas parafulva ZY-03, Bacillus subtilis XC-19-1, and Novosphingobium sp. Y3-1 are respectively activated to prepare seed solutions;

[0008] (2) Subculture: The seed solutions obtained in step (1) are respectively inoculated into a culture medium for subculture to obtain bacterial solutions;

[0009] (3) Preparation of crude enzyme preparation: The bacterial solutions after subculture in step (2) are respectively centrifuged, and the supernatant is collected to obtain a crude enzyme mother liquor, which is then diluted with sterile water to obtain a crude enzyme preparation;

[0010] (4) Compound crude enzyme preparation: The crude enzyme preparation of Pseudomonas pseudoaurantiaca ZY-03, the crude enzyme preparation of Bacillus subtilis XC-19-1, and the crude enzyme preparation of Novosphingobium sp. Y3-1 obtained in step (3) are compounded in a certain proportion to obtain a compound crude enzyme preparation.

[0011] Preferably, in the above technical solution, the Pseudomonas pseudoaurantiaca ZY-03 is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC NO.24422; the Bacillus subtilis XC-19-1 is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M2017351; the Novosphingobium sp. Y3-1 is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC NO.13436.

[0012] Preferably, in the above technical solution, the specific steps of strain activation in step (1) include:

[0013] Pick a single colony of Pseudomonas pseudoaurantiaca ZY-03 and inoculate it into an LB liquid medium, and shake culture at 22-37°C and 100-250 r / min for 12-15 h to obtain a seed solution;

[0014] Pick a single colony of Bacillus subtilis XC-19-1 and inoculate it into an LB liquid medium, and shake culture at 25-35°C and 150-200 r / min for 12-20 h to obtain a seed solution;

[0015] Pick a single colony of Novosphingobium sp. Y3-1 and inoculate it into an LB liquid medium, and shake culture at 30-35°C and 150-220 r / min for 12-18 h to obtain a seed solution.

[0016] Preferably, in the above technical solution, the specific steps of scale-up culture in step (2) include:

[0017] Inoculate the seed solution of Pseudomonas pseudoaurantiaca ZY-03 into an NA liquid medium at an inoculation amount of 3%, and shake culture at a temperature of 37°C and a shaker speed of 200 r / min for 48-72 h to make OD600 = 2.0;

[0018] Inoculate the seed solution of Bacillus subtilis XC-19-1 into an NA liquid medium at an inoculation amount of 3%, and shake culture at a temperature of 37°C and a shaker speed of 200 r / min for 48-72 h to make OD600 = 2.0;

[0019] Inoculate the seed solution of Sphingobacterium novum Y3-1 at an inoculation amount of 3% into NA liquid medium, and culture it with shaking at a temperature of 37°C and a shaker speed of 200 r / min for 48 - 72 h until OD600 = 2.0.

[0020] Preferably, in the above technical solution, step (3) specifically includes:

[0021] Centrifuge the cultured Pseudoxanthomonas paraflava ZY-03 bacterial liquid at 8000 - 12000 r / min and 4°C for 10 - 20 min, collect the supernatant to obtain the crude enzyme mother liquor of ZY-03, add sterile water for dilution, and the volume ratio of the crude enzyme mother liquor to sterile water is 1:5 to obtain the crude enzyme preparation.

[0022] Centrifuge the cultured Bacillus subtilis XC-19-1 bacterial liquid at 8000 - 12000 r / min and 4°C for 10 - 20 min, collect the supernatant to obtain the crude enzyme mother liquor, add sterile water for dilution, and the volume ratio of the crude enzyme mother liquor to sterile water is 1:5 to obtain the XC-19-1 crude enzyme preparation.

[0023] Centrifuge the cultured Sphingobacterium novum Y3-1 bacterial liquid at 8000 - 12000 r / min and 4°C for 10 - 20 min, collect the supernatant to obtain the crude enzyme mother liquor, add sterile water for dilution, and the volume ratio of the crude enzyme mother liquor to sterile water is 1:5 to obtain the Y3-1 crude enzyme preparation.

[0024] Preferably, in the above technical solution, in the composite crude enzyme preparation of step (4), the mass ratio of the crude enzyme preparation of Pseudoxanthomonas paraflava ZY-03, the crude enzyme preparation of Bacillus subtilis XC-19-1, and the crude enzyme preparation of Sphingobacterium novum Y3-1 is 4:5:3.

[0025] Application of a composite crude enzyme preparation in shortening the natural aging period of tobacco stems and improving the overall quality of tobacco stems.

[0026] A method for improving the aging efficiency of tobacco stems by using multiple strains in combination, which uses the above composite crude enzyme preparation to treat tobacco stems, including the following steps:

[0027] (1) Strain activation: Activate Pseudoxanthomonas paraflava ZY-03, Bacillus subtilis XC-19-1, and Sphingobacterium novum Y3-1 respectively to prepare seed solutions.

[0028] (2) Subculture: Inoculate the seed solutions obtained in step (1) into the medium for subculture to obtain bacterial liquids.

[0029] (3) Preparation of crude enzyme preparation: Centrifuge the subcultured bacterial liquids in step (2), collect the supernatant to obtain the crude enzyme mother liquor, and then dilute it with sterile water to obtain the crude enzyme preparation.

[0030] (4) Compound crude enzyme preparation: The crude enzyme preparation of Pseudomonas parafulva ZY-03, the crude enzyme preparation of Bacillus subtilis XC-19-1, and the crude enzyme preparation of Novosphingobium sp. Y3-1 obtained in step (3) are compounded in a certain proportion to obtain a compound crude enzyme preparation;

[0031] (5) Treatment of compound crude enzyme preparation: The compound crude enzyme preparation in step (4) is applied to the surface of tobacco stems, the moisture of the tobacco stems is adjusted to 25%, and it is placed for a certain period of time under the set temperature and humidity conditions, and then redried, and the compound crude enzyme preparation is inactivated by using the high-temperature environment of baking.

[0032] Preferably, in the above technical solution, the set temperature and humidity conditions are: temperature 35°C - 37°C, humidity 65% - 75%, placed for 48 hours, and then redried, and the redrying temperature is 80°C.

[0033] Preferably, in the above technical solution, the crude enzyme preparation of Pseudomonas parafulva ZY-03, the crude enzyme preparation of Bacillus subtilis XC-19-1, and the crude enzyme preparation of Novosphingobium sp. Y3-1 respectively account for 4%, 5%, and 3% of the mass of the tobacco stems.

[0034] The above technical solution of the present invention has the following beneficial effects:

[0035] The present invention has the technical advantages of simple and efficient process flow, low cost of tobacco leaf treatment, short treatment cycle, safety and reliability, can effectively reduce the content of cell wall substances such as lignin and cellulose in tobacco stems, improve the content of neutral flavor substances in tobacco stems, thereby improving the overall quality of tobacco stems before the start of natural aging, and effectively improving the aging efficiency of tobacco stems. Therefore, it has good popularization and application value in the field of cigarette manufacturing technology. Detailed Embodiments

[0036] Now, various exemplary embodiments of the present invention will be described in detail. It should be noted that: unless otherwise specifically stated, the relative arrangements, numerical expressions, and numerical values of the components and steps set forth in these embodiments do not limit the scope of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials and reagents used, unless otherwise specified, can all be obtained from commercial channels. The equipment used in the experiments, unless otherwise specified, are all well-known to those skilled in the art.

[0038] Pseudomonas parafulva ZY-03 was deposited at the General Microbiological Center of the China General Microbiological Culture Collection Center (CGMCC) on February 23, 2022. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 24422.

[0039] Bacillus subtilis XC-19-1 was deposited at the China Center for Type Culture Collection (CCTCC) on June 19, 2017. Address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M 2017351.

[0040] Novosphingobium panipatense Y3-1 was deposited at the General Microbiological Center of the China General Microbiological Culture Collection Center (CGMCC) on December 8, 2016. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No: 13436.

[0041] Example 1 Preparation of compound crude enzyme preparation

[0042] 1. Strain activation:

[0043] Pick a single colony of Pseudomonas parafulva ZY-03 and inoculate it into LB liquid medium. Culture it at 22 - 37 °C with a shaker speed of 100 - 250 r / min for 12 - 15 h to obtain a seed solution.

[0044] Pick a single colony of Bacillus subtilis XC-19-1 and inoculate it into LB liquid medium. Shake and culture it at 25 - 35 °C and 150 - 200 r / min for 12 - 20 h to obtain a seed solution.

[0045] Pick a single colony of Novosphingobium panipatense Y3-1 and inoculate it into LB liquid medium. Shake and culture it at 30 - 35 °C and 150 - 220 r / min for 12 - 18 h to obtain a seed solution.

[0046] 2. Subculture:

[0047] Inoculate the seed solution of Pseudomonas parafulva ZY-03 in step 1 into NA liquid medium at an inoculation amount of 3%. Shake and culture it at a temperature of 37 °C and a shaker speed of 200 r / min for 48 - 72 h until OD600 = 2.0.

[0048] Inoculate the Bacillus subtilis XC-19-1 seed liquid in step 1 into the NA liquid medium at an inoculation amount of 3%, and culture it with shaking at a temperature of 37°C and a shaker speed of 200 r / min for 48 - 72 h until OD600 = 2.0.

[0049] Inoculate the Novosphingobium sp. Y3-1 seed liquid in step 1 into the NA liquid medium at an inoculation amount of 3%, and culture it with shaking at a temperature of 37°C and a shaker speed of 200 r / min for 48 - 72 h until OD600 = 2.0.

[0050] 3. Preparation of crude enzyme preparation:

[0051] Centrifuge the Pseudoxanthomonas paraflava ZY-03 bacterial liquid after the enlarged culture in step 2 at 8000 - 12000 r / min and 4°C for 10 - 20 min, collect the supernatant to obtain the crude enzyme mother liquid, and dilute it with sterile water. The volume ratio of the crude enzyme mother liquid to sterile water is 1:5 to obtain the crude enzyme preparation;

[0052] Centrifuge the Bacillus subtilis XC-19-1 bacterial liquid after the enlarged culture in step 2 at 8000 - 12000 r / min and 4°C for 10 - 20 min, collect the supernatant to obtain the crude enzyme mother liquid, and dilute it with sterile water. The volume ratio of the crude enzyme mother liquid to sterile water is 1:5 to obtain the crude enzyme preparation;

[0053] Centrifuge the Novosphingobium sp. Y3-1 bacterial liquid after the enlarged culture in step 2 at 8000 - 12000 r / min and 4°C for 10 - 20 min, collect the supernatant to obtain the crude enzyme mother liquid, and dilute it with sterile water. The volume ratio of the crude enzyme mother liquid to sterile water is 1:5 to obtain the crude enzyme preparation.

[0054] 4. Preparation of composite crude enzyme preparation: At the leaf-stem separation node in the threshing and redrying stage, compound the Pseudoxanthomonas paraflava ZY-03 crude enzyme preparation, Bacillus subtilis XC-19-1 crude enzyme preparation, and Novosphingobium sp. Y3-1 crude enzyme preparation according to the mass ratio of 4%, 5%, and 3% of the tobacco stem to obtain the composite crude enzyme preparation.

[0055] Among them, the ratio of the three crude enzyme preparations is obtained through the following method: Taking the total content of neutral flavor components in the tobacco stem as the core index, optimize the compounding ratio of the crude enzyme preparations by using orthogonal experimental design, and the results are shown in Table 1.

[0056] Table 1 Optimization of the compounding ratio of crude enzyme preparations

[0057]

[0058] Note: A is the application ratio of the crude enzyme preparation of Pseudoxanthomonas paraflava ZY-03; B is the application ratio of the crude enzyme preparation of Bacillus subtilis XC-19-1; C is the application ratio of the crude enzyme preparation of Novosphingobium sp. Y3-1; the content of neutral flavor components in the control tobacco stems (CK) is 67.67 μg / g.

[0059] The optimal level treatment condition is A1B2C2, that is, the addition amount of the crude enzyme preparation of Pseudoxanthomonas paraflava ZY-03 is 4% of the tobacco stem mass ratio, the addition amount of the crude enzyme preparation of Bacillus subtilis XC-19-1 is 5% of the tobacco stem mass ratio, and the addition amount of the crude enzyme preparation of Novosphingobium sp. Y3-1 is 3% of the tobacco stem mass ratio. Under this condition, the content of neutral flavor components in the treated tobacco stems is 76.63 μg / g, which is significantly higher than that of the control tobacco stems.

[0060] Application Example 1 Application of the composite crude enzyme preparation

[0061] A method for improving the aging efficiency of tobacco stems by using multiple strains in combination:

[0062] The composite crude enzyme preparation prepared in Example 1 was applied to the surface of tobacco stems by spraying, and then the moisture of the tobacco stems was adjusted to 25%. It was placed for 48 h under the conditions of temperature 35°C - 37°C and humidity 65% - 75%, and then re-dried, and the baking high-temperature environment (80°C) was used to inactivate the composite crude enzyme preparation.

[0063] The chemical components of the tobacco stems treated with the composite crude enzyme preparation were detected, and the results are shown in Table 2.

[0064] Table 2 Content table of chemical components of tobacco stems (unit: %)

[0065]

[0066] It can be seen from Table 2 that compared with the control group of tobacco stems, after treatment with the composite crude enzyme preparation, the content of cell wall substances such as cellulose and lignin in the tobacco stems decreased significantly, while the content of cellulose and lignin in the tobacco stems naturally aged for 9 months changed little compared with the control group; and after treatment with the composite crude enzyme preparation, the total sugar and reducing sugar contents in the tobacco stems both increased slightly compared with the control group.

[0067] The sensory quality evaluation was carried out on the tobacco stems treated with the composite crude enzyme preparation, the control group of tobacco stems, and the tobacco stems naturally aged for 9 months. The results showed that: compared with the control tobacco stems, the woody off-flavors of the tobacco stems treated with the composite crude enzyme preparation were significantly reduced, the quality of the aroma was significantly improved, the aftertaste of sweetness was obvious, the sensory quality was significantly improved, and the overall sensory quality was slightly better than that of the tobacco stems naturally aged for 9 months.

[0068] Comparative Example 1

[0069] 1#: Treated with a composite crude enzyme preparation of Pseudomonas paraflava ZY-03 crude enzyme preparation (the addition amount of the crude enzyme preparation is 4% of the mass ratio of tobacco stems), Bacillus subtilis XC-19-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 5% of the mass ratio of tobacco stems), and Novosphingobium sp. Y3-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 3% of the mass ratio of tobacco stems) (i.e., the compound crude enzyme preparation in Example 1 of the present application and the treatment method in Application Example 1);

[0070] 2#: Treated with Pseudomonas paraflava ZY-03 crude enzyme preparation (the addition amount of the crude enzyme preparation is 4% of the mass ratio of tobacco stems);

[0071] 3#: Treated with Bacillus subtilis XC-19-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 5% of the mass ratio of tobacco stems);

[0072] 4#: Treated with Novosphingobium sp. Y3-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 3% of the mass ratio of tobacco stems);

[0073] 5#: Treated with a mixture of Pseudomonas paraflava ZY-03 crude enzyme preparation (the addition amount of the crude enzyme preparation is 4% of the mass ratio of tobacco stems) and Novosphingobium sp. Y3-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 3% of the mass ratio of tobacco stems);

[0074] 6#: Treated with a mixture of Pseudomonas paraflava ZY-03 crude enzyme preparation (the addition amount of the crude enzyme preparation is 4% of the mass ratio of tobacco stems) and Bacillus subtilis XC-19-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 5% of the mass ratio of tobacco stems);

[0075] 7#: Treated with a mixture of Bacillus subtilis XC-19-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 5% of the mass ratio of tobacco stems) and Novosphingobium sp. Y3-1 crude enzyme preparation (the addition amount of the crude enzyme preparation is 3% of the mass ratio of tobacco stems).

[0076] Control group: Tobacco stems without any treatment, that is, tobacco stems aged under natural conditions.

[0077] The sensory quality of the tobacco stems treated in the above 1# - 7# and the control group was evaluated, and the results are shown in Table 3.

[0078] Table 3 Results of sensory quality evaluation of different tobacco stems

[0079] Type Aroma quality Aroma quantity Concentration Softness fineness Aftertaste Off-odor Irritation Total score Control group 4.50 4.50 4.50 4.50 4.53 4.52 4.51 31.56 1# 4.70 4.75 4.75 4.85 4.70 4.70 4.75 33.20 2# 4.57 4.50 4.50 4.75 4.71 4.67 4.65 32.35 3# 4.46 5.50 4.50 4.60 4.60 4.61 4.60 32.87 4# 4.66 4.60 4.55 4.50 4.55 4.51 4.52 31.89 5# 4.57 4.53 4.53 4.65 4.61 4.57 4.65 32.11 6# 4.56 4.56 4.56 4.55 4.55 4.61 4.55 31.94 7# 4.56 4.55 4.55 4.50 4.55 4.55 4.55 31.81

[0080] As can be seen from Table 3, compared with the control tobacco stems, after the tobacco stems of Example 1 of the present application were treated with a composite crude enzyme preparation (1#) of crude enzyme preparation of Pseudomonas paracitica ZY-03 (the addition amount of the crude enzyme preparation was 4% of the mass ratio of tobacco stems), crude enzyme preparation of Bacillus subtilis XC-19-1 (the addition amount of the crude enzyme preparation was 5% of the mass ratio of tobacco stems), and crude enzyme preparation of Novosphingobium sp. Y3-1 (the addition amount of the crude enzyme preparation was 3% of the mass ratio of tobacco stems), the aroma quality of the tobacco stems was significantly improved, the smoke concentration was increased, the smoke was more delicate, the aftertaste was improved, the off-flavors and irritation were reduced, and the overall sensory quality score was the highest, all higher than those of the tobacco stems treated with a single one of the 3 crude enzyme preparations and those treated with any mixture of 2 crude enzyme preparations.

[0081] The present invention uses 3 specific strains, namely Pseudomonas paracitica ZY-03, Bacillus subtilis XC-19-1, and Novosphingobium sp. Y3-1. By using the 3 strains in combination, while reducing the content of cell wall substances such as lignin and cellulose in tobacco stems, the content of neutral flavor substances in tobacco stems is effectively increased, the overall quality of tobacco stems is rapidly improved, thereby effectively improving the aging rate of tobacco stems and reducing the storage cost of tobacco stems.

[0082] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections 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 equivalent forms.

Claims

1. A compound crude enzyme preparation for improving the aging efficiency of tobacco stems, characterized in that, The compound crude enzyme preparation is prepared by the following method: (1) Strain activation: Pseudomonas parafulva ZY-03, Bacillus subtilis XC-19-1, and Novosphingobium sp. Y3-1 are respectively activated to prepare seed solutions; (2) Subculture: The seed solutions obtained in step (1) are respectively inoculated into a culture medium for subculture to obtain bacterial solutions; (3) Preparation of crude enzyme preparation: The bacterial solutions after subculture in step (2) are respectively centrifuged, and the supernatant is collected to obtain a crude enzyme mother liquor, which is then diluted with sterile water to obtain a crude enzyme preparation; (4) Compound of crude enzyme preparation: The crude enzyme preparations of Pseudomonas parafulva ZY-03, Bacillus subtilis XC-19-1, and Novosphingobium sp. Y3-1 obtained in step (3) are compounded in a certain proportion to obtain a compound crude enzyme preparation.

2. The composite crude enzyme preparation according to claim 1, wherein The Pseudomonas parafulva ZY-03 is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO.24422; the Bacillus subtilis XC-19-1 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M2017351; the Novosphingobium sp. Y3-1 is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO.13436.

3. The composite crude enzyme preparation according to claim 1, wherein The specific steps of the strain activation in step (1) include: Pick a single colony of Pseudomonas parafulva ZY-03 and inoculate it into an LB liquid medium, and shake culture at 22-37 °C and 100-250 r / min for 12-15 h to obtain a seed solution; Pick a single colony of Bacillus subtilis XC-19-1 and inoculate it into an LB liquid medium, and shake culture at 25-35 °C and 150-200 r / min for 12-20 h to obtain a seed solution; Pick a single colony of Novosphingobium sp. Y3-1 and inoculate it into an LB liquid medium, and shake culture at 30-35 °C and 150-220 r / min for 12-18 h to obtain a seed solution.

4. The composite crude enzyme preparation according to claim 1, wherein The specific steps of the subculture in step (2) include: Inoculate the seed solution of Pseudomonas parafulva ZY-03 into an NA liquid medium at an inoculation amount of 3%, and shake culture at a temperature of 37 °C and a shaker speed of 200 r / min for 48-72 h to make OD600 = 2.0; Inoculate the seed solution of Bacillus subtilis XC-19-1 into an NA liquid medium at an inoculation amount of 3%, and shake culture at a temperature of 37 °C and a shaker speed of 200 r / min for 48-72 h to make OD600 = 2.0; Inoculate the seed solution of Novosphingobium sp. Y3-1 into an NA liquid medium at an inoculation amount of 3%, and shake culture at a temperature of 37 °C and a shaker speed of 200 r / min for 48-72 h to make OD600 = 2.

0.

5. The composite crude enzyme preparation according to claim 1, characterized in that, Step (3) specifically includes: The cultured Pseudoxanthomonas paraflava ZY-03 bacterial liquid was centrifuged at 8000 - 12000 r / min for 10 - 20 min at 4°C, and the supernatant was collected to obtain a crude enzyme mother liquor, which was diluted with sterile water at a volume ratio of the crude enzyme mother liquor to sterile water of 1:5 to obtain the ZY-03 crude enzyme preparation; The cultured Bacillus subtilis XC-19-1 bacterial liquid was centrifuged at 8000 - 12000 r / min for 10 - 20 min at 4°C, and the supernatant was collected to obtain a crude enzyme mother liquor, which was diluted with sterile water at a volume ratio of the crude enzyme mother liquor to sterile water of 1:5 to obtain the XC-19-1 crude enzyme preparation; The cultured Sphingobacterium novum Y3-1 bacterial liquid was centrifuged at 8000 - 12000 r / min for 10 - 20 min at 4°C, and the supernatant was collected to obtain a crude enzyme mother liquor, which was diluted with sterile water at a volume ratio of the crude enzyme mother liquor to sterile water of 1:5 to obtain the Y3-1 crude enzyme preparation.

6. The composite crude enzyme preparation according to claim 1, characterized in that, In the composite crude enzyme preparation of step (4), the mass ratio of the Pseudoxanthomonas paraflava ZY-03 crude enzyme preparation, the Bacillus subtilis XC-19-1 crude enzyme preparation, and the Sphingobacterium novum Y3-1 crude enzyme preparation is 4:5:

3.

7. Use of the composite crude enzyme preparation according to any one of claims 1 - 6 in shortening the natural aging period of tobacco stems and improving the overall quality of tobacco stems.

8. A method for improving the aging efficiency of tobacco stems by using multiple strains in combination, characterized in that, It uses the composite crude enzyme preparation according to any one of claims 1 - 7 to treat tobacco stems, including the following steps: (1) Strain activation: Pseudoxanthomonas paraflava ZY-03, Bacillus subtilis XC-19-1, and Sphingobacterium novum Y3-1 were respectively activated to prepare seed liquids; (2) Subculture: The seed liquids obtained in step (1) were respectively inoculated into a culture medium for subculture to obtain bacterial liquids; (3) Crude enzyme preparation: The cultured bacterial liquids in step (2) were centrifuged, and the supernatant was collected to obtain a crude enzyme mother liquor, which was then diluted with sterile water to obtain a crude enzyme preparation; (4) Compound of crude enzyme preparations: The Pseudoxanthomonas paraflava ZY-03 crude enzyme preparation, the Bacillus subtilis XC-19-1 crude enzyme preparation, and the Sphingobacterium novum Y3-1 crude enzyme preparation obtained in step (3) were compounded in a certain proportion to obtain a composite crude enzyme preparation; (5) Treatment with the composite crude enzyme preparation: The composite crude enzyme preparation of step (4) was applied to the surface of tobacco stems, the moisture of the tobacco stems was adjusted to 25%, and after being placed for a certain time under the set temperature and humidity conditions, it was redried and the composite crude enzyme preparation was inactivated using the drying temperature.

9. The method according to claim 8, wherein The set temperature and humidity conditions are: temperature 35°C - 37°C, humidity 65% - 75%, placed for 48 hours, and then redried, and the redrying temperature is 80°C.

10. The method according to claim 9, wherein The Pseudoxanthomonas paraflava ZY-03 crude enzyme preparation, the Bacillus subtilis XC-19-1 crude enzyme preparation, and the Sphingobacterium novum Y3-1 crude enzyme preparation respectively account for 4%, 5%, and 3% of the mass of tobacco stems.

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