Method for improving tobacco mellowing efficiency through combined use of double strains

Abstract

The invention discloses a method for improving tobacco mellowing efficiency through combined use of double strains, which comprises the following steps: (1) strain activation: respectively selecting pseudomonas paraxanthoceras ZY-03 and bacillus velezensis HJ-04, inoculating the pseudomonas paraxanthoceras ZY-03 and bacillus velezensis HJ-04 into a liquid culture medium, and culturing to obtain a seed solution; (2) amplification culture: inoculating the seed solution into a liquid culture medium for amplification culture to obtain a bacterial solution; (3) preparing a crude enzyme preparation: centrifuging the bacterial liquid, collecting supernate to obtain crude enzyme mother liquor, and diluting to obtain the crude enzyme preparation; (4) applying the pseudomonas paraxanthomonas ZY-03 crude enzyme preparation to the surface of the crude tobacco, and threshing and redrying after treatment; and (5) applying the bacillus velezensis HJ-04 crude enzyme preparation to the surfaces of the tobacco leaves for treatment in a tobacco leaf gathering link after leaf-stem separation. The method has the technical advantages that the lignin content in the tobacco leaves can be effectively reduced, the content of various important flavor substances in the tobacco leaves is increased, and the mellowing efficiency of the tobacco leaves is effectively improved.

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 leaves by using two strains in combination. Background Art

[0002] The aging of tobacco leaves is a key link in improving the quality of tobacco leaves. Natural aging refers to placing tobacco leaves under natural climatic conditions. During this period, the internal chemical components of the tobacco leaves change with the seasons and the environment, and their overall quality is gradually improved. Although this process has the characteristics of simple process and easy operation, it has defects such as a long storage period of tobacco leaves, inconsistent optimal aging periods for tobacco leaves of different origins and grades, large warehouse occupancy for tobacco leaf aging, and high costs, thus limiting the effective utilization efficiency of tobacco leaves.

[0003] The cell wall substances (such as cellulose, hemicellulose, lignin, pectin, etc.) in tobacco leaves are the main factors causing a large sense of irritation and woody aroma during cigarette burning, and the cell wall substances are difficult to degrade during the natural aging process, easily leading to insufficient accumulation of aroma precursor substances in tobacco leaves, thus affecting the aroma quality after tobacco leaf aging. The microbial technology has obvious advantages in promoting the rapid degradation of cell wall substances in tobacco leaves and natural flavor enhancement, providing a feasible technical approach for rapidly improving the overall quality of tobacco leaves and shortening the natural aging cycle of tobacco leaves. 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 leaves by using two strains in combination, to solve the problems in the traditional tobacco leaf aging process such as long storage period of tobacco leaves, inconsistent aging periods for tobacco leaves of different origins and grades, large warehouse occupancy, and high costs, and to rapidly improve the aging efficiency and quality of tobacco leaves through the combined use of two strains.

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

[0006] A method for improving the aging efficiency of tobacco leaves by using two strains in combination, comprising the following steps:

[0007] (1) Strain activation: Separate single colonies of Pseudomonas pseudoalcaligenes ZY-03 and Bacillus velezensis HJ-04 are inoculated into YPD liquid medium for culture to obtain seed solutions;

[0008] (2) Subculture: The seed solutions in step (1) are respectively inoculated into YPD liquid medium according to an inoculation amount of 3% for subculture to obtain bacterial solutions;

[0009] (3) Preparation of crude enzyme preparation: The bacterial solutions after subculture are 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) Apply the crude enzyme preparation of Pseudomonas pseudoalcaligenes ZY-03 to the surface of the raw tobacco in the form of spraying according to the tobacco leaf quality ratio of 2%, and carry out leaf threshing and redrying after treatment;

[0011] (5) At the link of aggregating the tobacco leaves after separating the leaf stalks, apply the crude enzyme preparation of Bacillus velezensis HJ-04 to the surface of the tobacco leaves in the form of spraying for treatment according to the tobacco leaf quality ratio of 5%.

[0012] Preferably, in the above technical solution, the Pseudomonas pseudoalcaligenes ZY-03 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC NO. 24422; the Bacillus velezensis HJ-04 strain is currently deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20231959.

[0013] Preferably, in the above technical solution, step (1) is specifically:

[0014] Pick a single colony of Pseudomonas pseudoalcaligenes ZY-03 and inoculate it into YPD liquid medium. Under the conditions of 22-37 °C and a shaking table speed of 100-250 r / min, culture for 12-72 h to obtain a seed liquid;

[0015] Pick a single colony of Bacillus velezensis HJ-04 and inoculate it into YPD liquid medium. Shake and culture at 25-35 °C and 100-150 r / min for 24-48 h to obtain a seed liquid.

[0016] Preferably, in the above technical solution, the conditions for the enlarged culture in step (2) are: temperature 37 °C, shaking table speed 200 r / min, shaking culture for 72 h, and culturing until the OD600 of the bacterial liquid is 2.0.

[0017] Preferably, in the above technical solution, in step (3), the centrifugation conditions are: 8000-12000 r / min, 4 °C, and the centrifugation time is 10-20 min; the volume ratio of the crude enzyme mother liquid to sterile water is 1:9.

[0018] Preferably, in the above technical solution, in step (4), the treatment conditions are: adjust the moisture content of the tobacco leaves to 23%, temperature 35 °C, humidity 75%, and the placement time is 48 h.

[0019] Preferably, in the above technical solution, in step (5), the treatment conditions are: temperature 30 °C, humidity 70%, and the placement time is 4 h.

[0020] Preferably, in the above technical solution, in step (4), the Pseudomonas pseudoalcaligenes ZY-03 enzyme is inactivated by leaf moistening at 100 °C, and in step (5), the Bacillus velezensis HJ-04 enzyme is inactivated by baking at 80 °C.

[0021] A strain combination for improving the aging efficiency of tobacco leaves, comprising Pseudomonas parafulva ZY-03 and Bacillus velezensis HJ-04.

[0022] An application of a strain combination in improving the aging efficiency of tobacco leaves, wherein the strain combination is used to prepare a crude enzyme preparation, and the crude enzyme preparation is used to treat tobacco leaves to accelerate the aging process of tobacco leaves and improve their sensory quality.

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

[0024] 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 lignin content in tobacco leaves, increase the content of various important flavor substances in tobacco leaves, and effectively improve the aging efficiency of tobacco leaves. Therefore, it has good popularization and application value in the field of cigarette manufacturing technology. Detailed implementation manners

[0025] 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 of components and steps, numerical expressions and values set forth in these embodiments do not limit the scope of the present invention.

[0026] In the following embodiments, the experimental methods used 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.

[0027] Pseudomonas parafulva ZY-03 was deposited at 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.

[0028] Bacillus velezensis HJ-04 was deposited at the China Center for Type Culture Collection (CCTCC) on October 20, 2023. Preservation address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M20231959.

[0029] Example 1

[0030] A method for improving the aging efficiency of tobacco leaves by using two strains in combination, using Pseudomonas parafulva ZY-03 strain and Bacillus velezensis HJ-04 strain. The specific steps are as follows:

[0031] 1. Strain activation:

[0032] Pick a single colony of Pseudomonas parafulva ZY-03 and inoculate it into YPD liquid medium. Incubate at 22 - 37 °C with a shaker speed of 100 - 250 r / min for 12 - 72 h to obtain a seed solution.

[0033] Pick a single colony of Bacillus velezensis HJ-04 and inoculate it into YPD liquid medium. Incubate with shaking at 25 - 35 °C and 100 - 150 r / min for 24 - 48 h to obtain a seed solution.

[0034] 2. Subculture:

[0035] Inoculate the seed solutions of Pseudomonas parafulva ZY-03 and Bacillus velezensis HJ-04 obtained in step 1 into YPD liquid medium at an inoculation amount of 3% respectively. Incubate with shaking at 37 °C and a shaker speed of 200 r / min for 72 h. At this time, OD600 = 2.0.

[0036] 3. Preparation of crude enzyme preparation:

[0037] Centrifuge the bacterial solutions of Pseudomonas parafulva ZY-03 and Bacillus velezensis HJ-04 after subculture in step 2 at 8000 - 12000 r / min and 4 °C for 10 - 20 min. Collect the supernatant to obtain a crude enzyme mother solution, and then dilute it with sterile water at a dilution ratio of 1:9 to obtain a crude enzyme preparation.

[0038] 4. Apply the crude enzyme preparation of Pseudomonas parafulva ZY-03 obtained in step 3 to the surface of raw tobacco by spraying according to 2% of the tobacco leaf quality ratio. Then adjust the moisture of the tobacco leaves to 23%, place them at 35 °C and 75% humidity for 48 h, and then use the high-temperature environment (100 °C) in the leaf moistening stage to inactivate the enzyme.

[0039] 5. At the stage of collecting tobacco leaves after leaf stalk separation, apply the crude enzyme preparation of Bacillus velezensis HJ-04 obtained in step 3 to the surface of the tobacco leaves by spraying according to 5% of the tobacco leaf quality ratio. Then place them at 30 °C and 70% humidity for 4 h, and then use the high-temperature environment (80 °C) in baking to inactivate the enzyme preparation.

[0040] Application Example 1

[0041] Detect the chemical components of the tobacco leaves treated with the crude enzyme preparation of ZY-03.

[0042] Table 1 Chemical component content table of tobacco leaves (unit: %)

[0043]

[0044] As can be seen from Table 1, compared with the control tobacco leaves, after treatment with the crude enzyme preparation of ZY-03, the contents of starch and lignin in the tobacco leaves decreased, while the contents of total sugar and reducing sugar increased, and the contents of various chemical components in the tobacco leaves were close to those after natural aging for 12 months.

[0045] The flavor components of the tobacco leaves treated with the crude enzyme preparation of Bacillus velezensis HJ-04 were detected.

[0046] Table 2 Contents of flavor components in tobacco leaves

[0047]

[0048]

[0049] As can be seen from Table 2, compared with the control tobacco leaves, after treatment with the crude enzyme preparation of HJ-04, the contents of various flavor substances such as furfural, 2-acetylfuran, 4-hydroxy-β-damascenone, and phytol in the tobacco leaves increased significantly, and the contents of the main flavor substances in the tobacco leaves were close to those after natural aging for 12 months.

[0050] The sensory quality evaluation was carried out on the tobacco leaves treated with the crude enzyme preparation of ZY-03 and HJ-04 in combination, the control tobacco leaves, and the tobacco leaves naturally aged for 12 months. The results showed that: compared with the control tobacco leaves, the woody and miscellaneous odors of the tobacco leaves treated with the crude enzyme preparation of ZY-03 and Bacillus velezensis HJ-04 in combination were significantly reduced, the quality of the aroma was significantly improved, the aroma quantity and the smoke concentration were increased, and the smoke was softer and more delicate. It was basically equivalent to the sensory quality of the tobacco leaves naturally aged for 12 months.

[0051] Comparative Example 1

[0052] No. 1#: The tobacco leaves treated with the crude enzyme preparation of ZY-03 and HJ-04 in combination (the method refers to Example 1 and Application Example 1);

[0053] No. 2#: The tobacco leaves treated with the crude enzyme preparation of ZY-03 alone;

[0054] No. 3#: The tobacco leaves treated with the crude enzyme preparation of HJ-04 alone.

[0055] Table 3 Results of sensory quality evaluation of different tobacco leaves

[0056] Type Aroma quality Aroma quantity Concentration Softness and fineness Aftertaste Off-odor Irritation Total score Control group 5.65 5.65 5.65 6.07 5.67 5.68 6.00 40.37 1# 5.91 5.88 5.79 6.27 5.78 5.75 6.09 41.47 2# 5.67 5.65 5.65 6.19 5.71 5.75 6.10 40.72 3# 5.83 5.70 5.70 6.05 5.65 5.70 6.05 40.68

[0057] As can be seen from Table 3, compared with the control tobacco leaves, after the combined treatment with the crude enzyme preparation of ZY-03 and the crude enzyme preparation of HJ-04, the aroma quality of the tobacco leaves was significantly improved, the smoke concentration increased, the smoke was more delicate, the aftertaste was improved, and the off-flavors and irritation were reduced; compared with the control tobacco leaves, after the single treatment with the crude enzyme preparation of ZY-03, the aftertaste of the tobacco leaves was improved, and the off-flavors and irritation were reduced; compared with the control tobacco leaves, after the single treatment with the crude enzyme preparation of HJ-04, the aroma quality of the tobacco leaves was improved and the smoke concentration increased. Generally speaking, after the combined treatment with the crude enzyme preparation of ZY-03 and the crude enzyme preparation of HJ-04, the overall sensory quality of the tobacco leaves was improved most significantly, and the total score of the sensory quality was the highest; after the single treatment with the crude enzyme preparation of ZY-03 and the crude enzyme preparation of HJ-04, the sensory quality scores of the tobacco leaves were all improved compared with the control tobacco leaves, but were lower than those of the tobacco leaves treated with the combined crude enzyme preparation of ZY-03 and HJ-04.

[0058] The present invention relates to two specific strains, namely Pseudoxanthomonas paraflava strain ZY-03 and Bacillus velezensis strain HJ-04. By using the two strains in combination during the threshing and redrying stage, the lignin in the tobacco leaves is efficiently degraded, the contents of various important flavor substances in the tobacco leaves are effectively increased, and the overall quality of the tobacco leaves at the initial stage of natural aging is rapidly improved, thereby effectively increasing the aging speed of the tobacco leaves.

[0059] 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 method for improving the aging efficiency of tobacco leaves by using two strains in combination, characterized in that It includes the following steps: (1) Strain activation: Single colonies of Pseudomonas pseudoalcaligenes ZY-03 and Bacillus velezensis HJ-04 were separately picked and inoculated into YPD liquid medium for cultivation to obtain seed solutions. (2) Subculture: The seed solutions from step (1) were inoculated into YPD liquid medium at an inoculation amount of 3% respectively for subculture to obtain bacterial solutions. (3) Preparation of crude enzyme preparation: The bacterial solutions after subculture 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) According to the proportion of 2% of the tobacco leaf mass, the crude enzyme preparation of Pseudomonas pseudoalcaligenes ZY-03 was applied to the surface of raw tobacco by spraying, and after treatment, it was threshed and redried. (5) At the link of tobacco leaf aggregation after leaf-stem separation, according to the proportion of 5% of the tobacco leaf mass, the crude enzyme preparation of Bacillus velezensis HJ-04 was applied to the surface of tobacco leaves by spraying for treatment.

2. The method according to claim 1, wherein The Pseudomonas pseudoalcaligenes ZY-03 is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO. 24422; the Bacillus velezensis HJ-04 strain is currently preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20231959.

3. The method according to claim 1, wherein Step (1) is specifically as follows: Pick a single colony of Pseudomonas pseudoalcaligenes ZY-03 and inoculate it into YPD liquid medium. Under the conditions of 22 - 37°C and a shaker speed of 100 - 250 r / min, cultivate for 12 - 72 h to obtain a seed solution. Pick a single colony of Bacillus velezensis HJ-04 and inoculate it into YPD liquid medium. Under the conditions of 25 - 35°C and 100 - 150 r / min, shake and cultivate for 24 - 48 h to obtain a seed solution.

4. The method according to claim 1, characterized in that, The conditions for the subculture in step (2) are: temperature 37°C, shaker speed 200 r / min, shake and cultivate for 72 h until the OD600 of the bacterial solution = 2.

0.

5. The method according to claim 1, characterized in that, In step (3), the centrifugation conditions are: 8000 - 12000 r / min, 4°C, centrifugation time 10 - 20 min; the volume ratio of the crude enzyme mother liquor to sterile water is 1:

9.

6. The method according to claim 1, characterized in that In step (4), the treatment conditions are: adjust the moisture content of tobacco leaves to 23%, temperature 35°C, humidity 75%, and placement time 48 h.

7. The method according to claim 1, characterized in that In step (5), the treatment conditions are: temperature 30°C, humidity 70%, and placement time 4 h.

8. The method according to claim 7, characterized in that, In step (4), the Pseudomonas pseudoalcaligenes ZY-03 enzyme preparation is inactivated by leaf moistening at 100°C, and in step (5), the Bacillus velezensis HJ-04 enzyme preparation is inactivated by baking at 80°C.

9. A strain combination for improving the aging efficiency of tobacco leaves, characterized in that, It includes the Pseudomonas pseudoalcaligenes ZY-03 and Bacillus velezensis HJ-04 as described in any one of claims 1 - 8.

10. Use of the strain combination according to claim 9 in improving the aging efficiency of tobacco leaves, characterized in that, The strain combination is used for preparing a crude enzyme preparation, and the crude enzyme preparation is used for treating tobacco leaves to accelerate the aging process of tobacco leaves and improve their sensory quality.

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