Method for improving quality of low-grade tobacco leaves through combined use of multiple strains

Abstract

The invention discloses a method for improving the quality of low-grade tobacco leaves through combined use of multiple strains, which comprises the following steps: (1) strain activation: inoculating a culture medium with pseudomonas aeruginosa ZY-03, bacillus subtilis XC-19-1 and stenotrophomonas maltophilia H3-1, and culturing to obtain a seed solution; (2) amplification culture: inoculating the seed solution into a culture medium for culture to obtain a bacterial solution; (3) preparing a crude enzyme preparation: centrifuging the bacterial liquid, collecting supernatant, and diluting to obtain the crude enzyme preparation; (4) at a raw tobacco sorting node, mixing the crude enzyme preparation of the pseudomonas paraxanthomonas ZY-03 and the crude enzyme preparation of the bacillus subtilis XC-19-1, and applying the mixture to the surface of the low-grade tobacco leaves for treatment; and (5) applying the stenotrophomonas maltophilia H3-1 crude enzyme preparation to the surface of the low-grade tobacco leaves at a tobacco leaf moisture regaining node for treatment. The method has the technical advantages that the technological process is simple, convenient and efficient, 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 overall quality of the low-grade 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 quality of low-grade tobacco leaves by using multiple strains in combination. Background Art

[0002] In low-grade tobacco leaves, there are often defects such as incomplete conversion of macromolecular compound components and insufficient aroma precursor substances, which easily lead to various miscellaneous gas stimuli during combustion and poor aroma quality, thus affecting the overall industrial usability of tobacco leaves. Microbial technology has obvious advantages in promoting the rapid degradation of the internal substance components of tobacco leaves and natural flavor enhancement, providing a feasible technical approach for rapidly improving the overall quality of low-grade tobacco leaves. However, the cell wall substances (such as cellulose, hemicellulose, lignin, pectin, etc.) in tobacco leaves have a stable structure and are difficult to degrade in the natural state, which affects the penetration efficiency of microorganisms-enzymes and directly limits their effect. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for improving the quality of low-grade tobacco leaves by using multiple strains in combination, aiming to solve the defects such as strong miscellaneous gas stimulation, poor aroma quality, and low industrial usability caused by incomplete conversion of macromolecular compounds and insufficient aroma precursor substances in low-grade tobacco leaves. By using multiple strains in combination, the cell wall substances such as lignin and cellulose in tobacco leaves can be efficiently degraded, and the content of flavor substances can be increased, thereby significantly improving the overall quality of low-grade tobacco leaves.

[0004] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0005] A method for improving the quality of low-grade tobacco leaves by using multiple strains in combination, comprising the following steps:

[0006] (1) Strain activation: Inoculate single colonies of Pseudomonas pseudoalcaligenes ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 into LB medium for culture respectively to obtain seed solutions;

[0007] (2) Subculture: Inoculate the seed solutions in step (1) into NA liquid medium according to an inoculation amount of 3% respectively for subculture to obtain bacterial solutions;

[0008] (3) Preparation of crude enzyme preparation: Centrifuge the bacterial solutions after subculture respectively, collect the supernatant to obtain a crude enzyme mother liquor, and then dilute it with sterile water to obtain a crude enzyme preparation;

[0009] (4) At the original tobacco sorting node, mix the crude enzyme preparation of Pseudomonas pseudoalcaligenes ZY-03 and the crude enzyme preparation of Bacillus subtilis XC-19-1 in proportion and apply them to the surface of low-grade tobacco leaves, and enter the threshing and redrying process after treatment under specific conditions;

[0010] (5) At the tobacco leaf rewetting node, apply the crude enzyme preparation of Stenotrophomonas maltophilia H3-1 to the surface of low-grade tobacco leaves, and after treatment under specific conditions, perform normal leaf threshing and redrying.

[0011] Preferably, in the above technical solution, the Pseudomonas pseudoalcaligenes ZY-03 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO.24422; the Bacillus subtilis XC-19-1 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2017351; the Stenotrophomonas maltophilia H3-1 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO.13905.

[0012] Preferably, in the above technical solution, the culture conditions of the seed liquid in step (1) are: temperature 22 - 37 °C, shaker speed 100 - 250 r / min, and culture time 12 - 48 h.

[0013] Preferably, in the above technical solution, the conditions for the scale-up culture in step (2) are: temperature 37 °C, shaker speed 200 r / min, shaking culture for 48 - 72 h, and culturing until the OD600 of the bacterial liquid = 2.0.

[0014] Preferably, in the above technical solution, in step (3), the centrifugation conditions are: 8000 - 12000 r / min, 4 °C, and centrifugation time 10 - 20 min; the volume ratio of the crude enzyme mother liquid to sterile water is 1:5.

[0015] Preferably, in the above technical solution, in step (4), the volume ratio of the crude enzyme preparation of Pseudomonas pseudoalcaligenes ZY-03 to the crude enzyme preparation of Bacillus subtilis XC-19-1 is 1:1, and the application amount is 3% of the tobacco leaf quality; the treatment conditions are: temperature 28 °C - 35 °C, humidity 65% - 75%, and treatment time 48 h.

[0016] Preferably, in the above technical solution, in step (5), the application amount of the crude enzyme preparation of Stenotrophomonas maltophilia H3-1 is 3% of the tobacco leaf quality; the treatment conditions are: temperature 37 °C, humidity 75%, and treatment time 6 h.

[0017] Preferably, in the above technical solution, after step (5), inactivate the enzyme by leaf conditioning and baking, where the leaf conditioning temperature is 100 °C and the baking temperature is 80 °C.

[0018] A strain combination for improving the quality of low-grade tobacco leaves, comprising Pseudomonas pseudoalcaligenes ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1.

[0019] Application of a strain combination in improving the quality of low - grade tobacco leaves. The strain combination is used for preparing a crude enzyme preparation, and the crude enzyme preparation is used for treating low - grade tobacco leaves to improve the quality of the tobacco leaves.

[0020] The above - mentioned technical solution of the present invention has the following beneficial effects:

[0021] The present invention has the technical advantages of simple and efficient process flow, low cost of tobacco leaf treatment, short treatment period, safety and reliability. It can effectively reduce the lignin and cellulose content in tobacco leaves, increase the content of various important flavor substances in tobacco leaves, and effectively improve the overall quality of low - grade tobacco leaves. Therefore, it has good popularization and application value in the field of cigarette manufacturing technology. Detailed implementation manners

[0022] Now, various exemplary embodiments of the present invention will be described in detail. It should be noted that: unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and values set forth in these embodiments do not limit the scope of the present invention.

[0023] 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.

[0024] Pseudomonas parafulva ZY - 03 was deposited on February 23, 2022 at the China General Microbiological Culture Collection Center (CGMCC), 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.

[0025] Bacillus subtilis XC - 19 - 1 was deposited on June 19, 2017 at the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M 2017351.

[0026] Stenotrophomonas maltophilia H3 - 1 was deposited on March 20, 2017 at the China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is CGMCC NO: 13905.

[0027] Example 1

[0028] A method for improving the quality of low-grade tobacco leaves by the combined use of multiple strains. The strains consist of Pseudomonas pseudoaurantiaca ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1, and it includes the following steps:

[0029] 1. Strain activation:

[0030] Pick single colonies of Pseudomonas pseudoaurantiaca ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 and inoculate them into LB liquid medium. Incubate at 22 - 37°C with a shaker speed of 100 - 250 r / min for 12 - 48 h to obtain seed solutions.

[0031] 2. Scale-up culture:

[0032] Respectively inoculate the seed solutions of Pseudomonas pseudoaurantiaca ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 obtained in step 1 into NA liquid medium at an inoculation amount of 3%. Incubate with shaking at 37°C and a shaker speed of 200 r / min for 48 - 72 h until the OD600 of each bacterial solution reaches 2.0.

[0033] 3. Preparation of crude enzyme preparation:

[0034] Centrifuge the bacterial solutions of Pseudomonas pseudoaurantiaca ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 after scale-up culture 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 1:5 to obtain a crude enzyme preparation.

[0035] 4. At the original tobacco sorting node, mix the crude enzyme preparation of Pseudomonas pseudoaurantiaca ZY-03 and the crude enzyme preparation of Bacillus subtilis XC-19-1 in step 3 at a ratio of 1:1. Then, apply the mixed crude enzyme preparation to the surface of low-grade original tobacco at a ratio of 3% of the tobacco leaf mass by spraying. Adjust the moisture content of the tobacco leaves to 19%, place them at 28°C - 35°C and a humidity of 65% - 75% for 48 h, and then enter the threshing and redrying process.

[0036] 5. At the tobacco leaf rewetting node, apply the crude enzyme preparation of Stenotrophomonas maltophilia H3-1 in step 3 to the surface of low-grade tobacco leaves at a ratio of 3% of the tobacco leaf mass by spraying. Adjust the moisture content of the tobacco leaves to 25%, then place them at 37°C and a humidity of 75% for 6 h, and then perform normal threshing and redrying. Use high-temperature environments such as leaf conditioning (temperature 100°C) and tobacco leaf baking (80°C) to inactivate all enzyme preparations.

[0037] Application Example 1

[0038] The crude enzyme preparation of Pseudoxanthomonas paraflava ZY-03 and the crude enzyme preparation of Bacillus subtilis XC-19-1 were mixed in a ratio of 1:1. Then, at a ratio of 3% of the tobacco leaf mass, the mixed crude enzyme preparation was applied to the surface of low-grade raw tobacco leaves by spraying. The moisture content of the tobacco leaves was adjusted to 19%, and they were placed for 48 h under the conditions of a temperature of 28 °C - 35 °C and a humidity of 65% - 75%. The chemical components of the treated tobacco leaves were detected, and the results are shown in Table 1.

[0039] Table 1 Chemical component contents of tobacco leaves (unit: %)

[0040]

[0041] As can be seen from Table 1, compared with the control tobacco leaves, after treatment with the mixed crude enzyme preparation, the contents of cellulose and lignin in the tobacco leaves decreased significantly, and the degradation rates were 20.47% and 20.23% respectively; the contents of total sugar and reducing sugar both increased.

[0042] At the tobacco leaf rewetting node, the crude enzyme preparation of Stenotrophomonas maltophilia H3-1 in Step 3 of Example 1 was applied to the surface of low-grade tobacco leaves by spraying at a ratio of 3% of the tobacco leaf mass, and the moisture content of the tobacco leaves was adjusted to 25%. Taking the total content of neutral flavor components in the tobacco leaves as the core index, the tobacco leaf treatment conditions were optimized using orthogonal experimental design, and the results are shown in Table 2.

[0043] Table 2 Orthogonal experimental design and result analysis

[0044]

[0045] Note: A is the treatment time; B is the treatment temperature; C is the treatment humidity; the content of neutral flavor components in the control tobacco leaves (CK) is 178.88 μg / g.

[0046] The optimal level treatment conditions were A2B3C2, that is, the treatment time was 6 h, the treatment temperature was 37 °C, and the treatment humidity was 75%. Since A2B3C2 was not within the nine groups of orthogonal design combinations, a verification test was carried out. It was found that under these conditions, the content of neutral flavor components in the treated tobacco leaves was 198.31 μg / g. This shows that after the previous cell wall material degradation treatment, it may be beneficial to the improvement of the action efficiency of the crude enzyme preparation of Stenotrophomonas maltophilia H3-1, and the content of neutral flavor components in the treated tobacco leaves increased significantly.

[0047] The sensory quality of the tobacco leaves co-treated with Pseudoxanthomonas paraflava ZY-03 strain, Bacillus subtilis XC-19-1 strain, and Stenotrophomonas maltophilia H3-1 strain was evaluated with normal control tobacco leaves. It was found that the woody off-odor in the treated tobacco leaves was significantly reduced, the quality of the aroma was significantly improved, the aroma quantity and smoke concentration were increased, and the smoke was softer and more delicate, and the overall quality of the tobacco leaves was significantly improved.

[0048] Comparative Example 1

[0049] No. 1: The tobacco leaves were treated with the crude enzyme preparations of Pseudomonas paraflava ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 in combination (the method refers to Example 1 and Application Example 1).

[0050] No. 2: The tobacco leaves were treated with the crude enzyme preparations of Pseudomonas paraflava ZY-03 and Stenotrophomonas maltophilia H3-1 in combination.

[0051] No. 3: The tobacco leaves were treated with the crude enzyme preparations of Bacillus subtilis XC-19-1 and Stenotrophomonas maltophilia H3-1 in combination.

[0052] Table 3 Results of sensory quality evaluation of different tobacco leaves

[0053] Type Aroma quality Aroma quantity Concentration Softness and fineness Aftertaste Off-odor Irritation Total score Control group 5.50 5.50 5.50 5.50 5.53 5.52 5.51 38.56 1# 5.70 5.75 5.75 5.80 5.75 5.70 5.70 40.15 2# 5.57 5.55 5.50 5.77 5.71 5.70 5.65 39.45 3# 5.60 5.60 5.60 5.55 5.55 5.51 5.65 39.06

[0054] As can be seen from Table 3, compared with the control tobacco leaves, after the tobacco leaves were treated with the crude enzyme preparations of Pseudomonas paraflava ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 in combination, 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-odor and irritation were reduced; compared with the control tobacco leaves, after being treated with the crude enzyme preparations of Pseudomonas paraflava ZY-03 and Stenotrophomonas maltophilia H3-1 in combination, the fineness of the smoke and the aftertaste were improved, and the off-odor and irritation were reduced; compared with the control tobacco leaves, after being treated with the crude enzyme preparations of Bacillus subtilis XC-19-1 and Stenotrophomonas maltophilia H3-1 in combination, the aroma quality of the tobacco leaves was improved and the smoke concentration increased. Generally speaking, after the tobacco leaves were treated with the crude enzyme preparations of Pseudomonas paraflava ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 in combination, the overall sensory quality of the tobacco leaves was improved most significantly, the total score of the sensory quality was the highest, and both were higher than those of the tobacco leaves treated with the crude enzyme preparations of Pseudomonas paraflava ZY-03 and Stenotrophomonas maltophilia H3-1 in combination, and those of the tobacco leaves treated with the crude enzyme preparations of Bacillus subtilis XC-19-1 and Stenotrophomonas maltophilia H3-1 in combination.

[0055] The present invention relates to the application of multiple specific strains, namely Pseudoxanthomonas paraflava strain ZY-03, Bacillus subtilis strain XC-19-1, and Stenotrophomonas maltophilia strain H3-1, in improving the overall quality of low-grade tobacco leaves. The aim is to efficiently degrade cell wall substances such as lignin and cellulose in tobacco leaves through the combined use of the three strains during the leaf threshing and redrying stage, and effectively increase the total amount of neutral flavor substances in tobacco leaves, thereby effectively improving the overall quality of low-grade tobacco leaves.

[0056] 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 quality of low-grade tobacco leaves by using multiple strains in combination, characterized in that, It includes the following steps: (1) Strain activation: Single colonies of Pseudomonas parafulva ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 were respectively inoculated into LB medium for cultivation to obtain seed solutions. (2) Subculture: The seed solutions from step (1) were respectively inoculated into NA liquid medium according to an inoculation amount of 3% for subculture to obtain bacterial solutions. (3) Preparation of crude enzyme preparation: The bacterial solutions after subculture were centrifuged respectively, 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) At the original tobacco sorting node, the crude enzyme preparation of Pseudomonas parafulva ZY-03 and the crude enzyme preparation of Bacillus subtilis XC-19-1 were mixed in proportion and applied to the surface of low-grade tobacco leaves. After treatment, it entered the threshing and redrying process. (5) At the tobacco leaf moistening node, the crude enzyme preparation of Stenotrophomonas maltophilia H3-1 was applied to the surface of low-grade tobacco leaves. After treatment, normal threshing and redrying were carried out.

2. The method according to claim 1, wherein The Pseudomonas parafulva ZY-03 is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO.24422; the Bacillus subtilis XC-19-1 is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2017351; the Stenotrophomonas maltophilia H3-1 is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCCNO.13905.

3. The method according to claim 1, characterized in that, The culture conditions of the seed solutions in step (1) are: temperature 22 - 37°C, shaker speed 100 - 250 r / min, and culture time 12 - 48 h.

4. The method according to claim 1, characterized in that The conditions for subculture in step (2) are: temperature 37°C, shaker speed 200 r / min, shaking culture for 48 - 72 h, and culturing until the OD600 of the bacterial solution is 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, and centrifugation time 10 - 20 min; the volume ratio of the crude enzyme mother liquor to sterile water is 1:

5.

6. The method according to claim 1, wherein In step (4), the volume ratio of the crude enzyme preparation of Pseudomonas parafulva ZY-03 to the crude enzyme preparation of Bacillus subtilis XC-19-1 is 1:1, and the application amount is 3% of the tobacco leaf quality; the treatment conditions are: temperature 28°C - 35°C, humidity 65% - 75%, and treatment time 48 h.

7. The method according to claim 1, characterized in that, In step (5), the application amount of the crude enzyme preparation of Stenotrophomonas maltophilia H3-1 is 3% of the tobacco leaf quality; the treatment conditions are: temperature 37°C, humidity 75%, and treatment time 6 h.

8. The method according to claim 7, wherein After step (5), the enzyme is inactivated by leaf moistening and baking, where the leaf moistening temperature is 100°C and the baking temperature is 80°C.

9. A strain combination for improving the quality of low-grade tobacco leaves, characterized in that, It includes Pseudomonas parafulva ZY-03, Bacillus subtilis XC-19-1, and Stenotrophomonas maltophilia H3-1 as described in any one of claims 1 - 8.

10. Use of the strain combination according to claim 9 in improving the quality of low-grade 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 low-grade tobacco leaves to improve the quality of tobacco leaves.

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