High-temperature-resistant active composite biological agent for improving tobacco quality and application thereof

Abstract

The invention relates to the technical field of tobacco processing, and particularly discloses a high-temperature-resistant active composite biological agent for improving tobacco quality and application thereof, and is characterized in that the active composite biological agent comprises the following components in parts by weight: 1-3 parts of amylase; 15 to 20 parts of cellulase; 35 to 40 parts of glucose oxidase; according to the present invention, enterobacter hormaechei is added, wherein the enterobacter hormaechei comprises enterobacter hormaechei C4, and the preservation number of the enterobacter hormaechei C4 is CGMCC No.26480; the high-temperature-resistant active composite biological agent for improving the tobacco quality is applied to the tobacco surface, conditions are mild, no side effect exists, operation is easy and convenient, the component content is low, offensive odor can be effectively reduced, tobacco irritation is reduced, and the aroma quality and the overall quality of tobacco are remarkably improved.

Description

Technical Field

[0001] This application relates to the technical field of tobacco processing, and specifically relates to a high-temperature resistant active composite biological agent for improving tobacco quality and its application. Background Art

[0002] Flue-cured tobacco is one of the important cash crops in China. Due to its complex chemical composition, it is impossible to effectively control its quality during the baking process. The quality of tobacco leaves determines the value and income of tobacco, and plays a crucial role in the development of the tobacco economy. The aging process of flue-cured tobacco will determine the quality of tobacco leaves. At present, the industrial method often uses natural fermentation to improve the quality of tobacco, but this method has a long fermentation cycle, which increases the economic cost of flue-cured tobacco, and at the same time, the improvement of tobacco quality is limited. Artificial fermentation accelerates the physical and chemical changes in the tobacco aging process by artificially controlling the temperature, humidity, moisture, etc. required for tobacco fermentation, so as to shorten the fermentation time. However, certain aroma substances will be lost during the process of artificially fermenting tobacco, and the improvement degree in terms of color, fragrance, and irritation is not enough, resulting in low tobacco quality.

[0003] Therefore, there is an urgent need for a product that is mild, has no side effects, and no harmful substances to assist tobacco during the baking process to improve its aroma quality, aroma quantity, concentration, irritation, aftertaste, sweetness and other qualities, while simplifying the operation steps and efficiently improving tobacco quality. Summary of the Invention

[0004] The purpose of this application is to overcome the deficiencies of the prior art. To solve the current situation, this application selects a high-temperature resistant active composite biological agent, which is the combined action of biological enzymes and microbial agents. Biological enzymes have the advantages of mild conditions, no obvious side effects, and no production of harmful substances. The application of biological enzymes on the surface of tobacco leaves can destroy the surface structure of tobacco leaves, increase the pores on the surface of tobacco leaves, and decompose macromolecules into soluble substances; the selected microorganism Enterobacter hormaechei C4 of the present invention has the function of catalytically degrading flavor precursors and can degrade substances such as carotene, carotenoids, and polysaccharides, so as to achieve the effect of increasing aroma and improving quality. This application combines biological enzymes with beneficial microorganisms to better improve the quality of tobacco leaves, provides a high-temperature resistant active composite biological agent for improving tobacco quality and its application, reduces miscellaneous odors and irritating odors while improving the fermentation efficiency, improves the aroma quality of tobacco, and thus improves the quality of tobacco leaves.

[0005] To solve the above problems, this application adopts the following technical solutions:

[0006] On the one hand, this application provides a high-temperature resistant active composite biological agent for improving tobacco quality, specifically including:

[0007] The three enzymes are respectively by weight:

[0008]

[0009] Among them, the Enterobacter hormaechei preparation includes Enterobacter hormaechei C4, with the preservation number of CGMCC No. 26480;

[0010] Among them, the enzyme activity of amylase is 40000 U / g; the enzyme activity of cellulase is 10000 U / g; the enzyme activity of glucose oxidase is 10000 U / g;

[0011] Among them, the bacterial content of the Enterobacter hormaechei preparation is 1.25×10 8 ~1.75×10 8 CFU / g;

[0012] Furthermore, the method for preparing the high-temperature resistant active compound biological preparation includes the following steps:

[0013] 1) Mix amylase, cellulase, and glucose oxidase evenly at a weight ratio of (1-3:15-20:35-40) to obtain a high-temperature resistant active compound enzyme preparation;

[0014] 2) Mix the Enterobacter hormaechei preparation and the high-temperature resistant active compound enzyme preparation at a mass ratio of 1:1 to obtain the high-temperature resistant active compound biological preparation.

[0015] Among them, the storage condition is: store the active high-temperature resistant compound enzyme preparation at 4°C to 10°C, protected from light, moisture-proof, airtight, and dry.

[0016] On the other hand, the application method of the high-temperature resistant active biological preparation includes the following steps:

[0017] 1) Prepare the high-temperature resistant active compound biological preparation according to the optimal addition amount, and spray it onto the surface of tobacco leaves to obtain tobacco leaves treated with the high-temperature resistant active compound biological preparation;

[0018] 2) Place the tobacco leaves treated with the high-temperature resistant active compound biological preparation at 25°C in a place protected from light, ventilated, and dry for one month to obtain aged tobacco leaves.

[0019] Among them, when the addition amount of the high-temperature resistant active biological preparation is 5‰, it can still maintain its activity after baking at 70°C for 5-10 minutes;

[0020] Among them, the tobacco varieties applied in this application include Yunyan 87, Honghua Dajinyuan, and Yibin K326. The applicability to other tobacco varieties needs to be further explored.

[0021] Among them, the mass ratio of the composite biological agent to the tobacco leaf sample is 1: (800-1200) under laboratory conditions, while in industrial production, the ratio is 5: (800-1200).

[0022] Among them, the tobacco quality that can be improved by using the high temperature resistant active composite biological agent developed in this application includes aroma quality, aroma quantity, concentration, irritation, aftertaste and sweetness.

[0023] Furthermore, the time for spraying the high temperature resistant active composite biological agent on the leaf surface is before the tobacco leaves are re-roasted, and the agent is sprayed on the leaf surface.

[0024] The beneficial effects of this application are:

[0025] 1. The present application provides a high temperature resistant active composite biological agent for improving tobacco quality, which is applied to the surface of tobacco under mild conditions, has no side effects, and is safe and reliable.

[0026] 2. The present application provides a high-temperature-resistant active composite biological agent for improving tobacco quality. When applied to the surface of tobacco, it can effectively reduce the generation of impurities, reduce the irritation of tobacco leaves, enhance the aroma of tobacco leaves, and improve the quality of tobacco leaves.

[0027] 3. The present application provides a high-temperature-resistant active composite biological agent for improving tobacco quality, which can shorten the tobacco aging and fermentation cycle, and the spraying operation of the agent is simple, which helps to reduce the application cost. DETAILED DESCRIPTION

[0028] The technical solution of the present application is described in further detail below, but the protection scope of the present application is not limited to the following.

[0029] Example 1

[0030] Screening of enzyme preparations with the function of improving tobacco leaf quality from amylase, saccharifying enzyme, pectinase, cellulase, protease and glucose oxidase

[0031] The test samples were YQ2 grade Yunyan 87 varieties collected from Liangshan, Sichuan in 2023, and the samples were provided by Sichuan China Tobacco Industry Co., Ltd. The test tobacco leaves were baked and stored separately according to the varieties, graded according to the national 42-grade grading standard, and tobacco samples of the same grade were selected for testing. The samples were dried, the moisture content was adjusted to 8%, and packaged in 200g bags for subsequent experiments.

[0032] The specific preparation steps for preparing enzyme preparations of different types and concentrations are as follows:

[0033] 1) The amylase used in this experiment was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 40,000 U / g. The amylase was mixed with sterile water to set 4 concentration gradients, namely 0.2 g / L, 0.4 g / L, 0.8 g / L, and 1.6 g / L. It was sprayed on the surface of tobacco leaves at 1‰ of the tobacco leaf quality. After being placed at 25°C for one month, smoking evaluation and chemical composition detection were carried out.

[0034] 2) The glucoamylase used in this experiment was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. The glucoamylase was mixed with sterile water to set 4 concentration gradients, namely 0.2 g / L, 0.4 g / L, 0.8 g / L, and 1.6 g / L. It was sprayed on the surface of tobacco leaves at 1‰ of the tobacco leaf quality. After being placed at 25°C for one month, smoking evaluation and chemical composition detection were carried out.

[0035] 3) The pectinase used in this experiment was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 40,000 U / g. The pectinase was mixed with sterile water to set 4 concentration gradients, namely 1.0 g / L, 2.0 g / L, 4.0 g / L, and 8.0 g / L. It was sprayed on the surface of tobacco leaves at 1‰ of the tobacco leaf quality. After being placed at 25°C for one month, smoking evaluation and chemical composition detection were carried out.

[0036] 4) The cellulase used in this experiment was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g. The cellulase was mixed with sterile water to set 4 concentration gradients, namely 2.0 g / L, 4.0 g / L, 8.0 g / L, and 16.0 g / L. It was sprayed on the surface of tobacco leaves at 1‰ of the tobacco leaf quality. After being placed at 25°C for one month, smoking evaluation and chemical composition detection were carried out.

[0037] 5) The protease used in this experiment was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g. The protease was mixed with sterile water to set 4 concentration gradients, namely 1.0 g / L, 2.0 g / L, 4.0 g / L, and 8.0 g / L. It was sprayed on the surface of tobacco leaves at 1‰ of the tobacco leaf quality. After being placed at 25°C for one month, smoking evaluation and chemical composition detection were carried out.

[0038] 6) The glucose oxidase used in this experiment was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g. The glucose oxidase was mixed with sterile water to set 4 concentration gradients, namely 2.0 g / L, 4.0 g / L, 8.0 g / L, and 16.0 g / L. It was sprayed on the surface of tobacco leaves at 1‰ of the tobacco leaf quality. After being placed at 25°C for one month, smoking evaluation and chemical composition detection were carried out.

[0039] The sensory evaluation of this experiment was responsible by the Technology Center of Sichuan China Tobacco Industry Co., Ltd. Seven experts were organized to conduct smoking evaluations on the tobacco leaves treated differently. The evaluation indicators included aroma quality, aroma quantity, offensive odor, concentration, irritation, aftertaste, sweetness, etc. The evaluation basis was the "Sensory Evaluation Method for Chinese Cigarette Style" QJ / 02.J.001-2016.A standard. For the detailed smoking evaluation results, please refer to Table 1.

[0040] Table 1 Smoking Evaluation Results of Tobacco Leaves under Different Treatments

[0041]

[0042] Note: The numbers after the enzyme names represent different concentration gradients. For example, protease 1 represents the first concentration gradient of protease, and the same applies hereinafter.

[0043] As can be seen from Table 1, through the smoking evaluations by seven experts, it was found that the smoking quality of tobacco leaves was improved to a certain extent after being treated with five concentration enzymes, namely amylase 1, glucoamylase 2, cellulase 1, protease 2, and glucose oxidase 2. After the enzyme treatment, the aroma quality and sweetness of the tobacco leaves were significantly improved, while the concentration and body decreased to a certain extent.

[0044] The chemical composition analysis of this experiment was carried out by the Technology Center of Sichuan China Tobacco Industry Co., Ltd., using Fourier transform near-infrared spectroscopy analysis technology. The detection indicators covered the main chemical components including total alkaloids, reducing sugars, total sugars, total nitrogen, potassium, chlorine, starch, neophytadiene, etc. For the detailed results, please refer to Table 2.

[0045] Table 2 Contents of Various Chemical Substances in Tobacco Leaves under Different Treatments

[0046]

[0047]

[0048] After being treated with five enzyme concentrations, namely amylase 1, glucoamylase 2, cellulase 1, protease 2, and glucose oxidase 2, the chemical composition inside the tobacco leaves changed. After the enzyme treatment, the contents of alkaloids, starch, and total nitrogen in the tobacco leaves decreased, while the contents of substances such as potassium, chlorine, and neophytadiene increased, which would help improve the smoking taste and quality of the tobacco leaves.

[0049] In summary, amylase, glucose oxidase, cellulase, glucoamylase, and protease all improve the overall smoking quality of tobacco leaves to a certain extent. Protease 2, amylase 1, glucoamylase 2, cellulase 1, and glucose oxidase 2 have the best effects among various enzymes; enzyme preparations can improve the aroma quality, aroma quantity, off-odors, concentration, body, irritation, aftertaste, and sweetness of tobacco leaves, and their effectiveness is as follows: amylase > glucose oxidase > cellulase > glucoamylase > protease; after treatment with amylase 1, glucoamylase 2, cellulase 1, protease 2, and glucose oxidase 2, the contents of alkaloids, starch, and total nitrogen in tobacco leaves decrease, while the contents of substances such as potassium, chlorine, and neophytadiene increase, which helps to improve the smoking taste and quality of tobacco leaves; low levels of enzyme preparations have a more obvious effect on improving tobacco leaves, and as the enzyme dosage increases, the quality of tobacco leaves gradually decreases. Further experiments are needed to verify the appropriate dosage.

[0050] Example 2

[0051] Combined with Example 1 to explore the addition amount of enzymes. It was found in Example 1 that amylase, glucose oxidase, cellulase, glucoamylase, and protease can improve the quality of tobacco leaves at low addition amounts, and the effectiveness relationship is: amylase > glucose oxidase > cellulase > glucoamylase > protease. The application ranges of different enzymes were initially determined, but further determination is still needed. In this example, amylase, glucose oxidase, and cellulase, which have a better effect on improving the quality of tobacco, were selected for further experiments.

[0052] Enzyme preparations: Amylase (concentrations: 0.1 g / L, 0.2 g / L, 0.3 g / L, and 0.4 g / L); Cellulase (concentrations: 0.5 g / L, 1 g / L, 2 g / L, 3 g / L); Glucose oxidase (concentrations: 2 g / L, 3 g / L, 4 g / L, and 5 g / L). The specific test method is as follows: Sprinkle different concentrations of amylase, cellulase, and glucose oxidase on the leaf surface at an addition amount of 1‰, and place them at 25°C for one month, then conduct smoking evaluation and chemical composition detection. The specific results are shown in Table 3 and Table 4.

[0053] Table 3 Smoking evaluation results of tobacco leaves under different treatments

[0054]

[0055] As shown in Table 3, Cellulase 3 has higher scores in terms of aroma quality, off-odors, irritation, aftertaste, and sweetness, indicating that when the concentration of cellulase is 2 g / L, the quality of tobacco leaves is higher; Glucose Oxidase 3 has higher scores in terms of aroma quantity, off-odors, irritation, and aftertaste, indicating that when the concentration of glucose oxidase is 4 g / L, the quality of tobacco leaves is higher; Amylase 2 has higher scores in terms of aroma quality, aroma quantity, off-odors, concentration, aftertaste, and sweetness, indicating that when the concentration of amylase is 2 g / L, the quality of tobacco leaves is higher.

[0056] Table 4 Contents of various chemical substances in tobacco leaves under different treatments

[0057]

[0058] As can be seen from Table 4, cellulase 3 significantly increased the total potassium content in tobacco leaves, which helped to improve the combustibility of tobacco leaves. At the same time, it reduced the starch content in tobacco leaves, effectively reducing the miscellaneous gas generated during the combustion of tobacco leaves. Glucose oxidase 3 significantly increased the total sugar content in tobacco leaves and reduced the starch content. The application of glucose oxidase reduced the generation of miscellaneous gas in tobacco leaves while ensuring the sweetness of tobacco leaves. Amylase 2 significantly increased the total sugar and potassium ion contents in tobacco leaves, ensuring both the sweetness and the combustibility of tobacco leaves.

[0059] Example 3

[0060] It can be seen from Example 2 that the application of cellulase, glucose oxidase, and amylase mainly affects the quality of tobacco leaves. The enzyme activities of the three enzymes are as follows: cellulase activity: 10000 U / g, amylase activity: 40000 U / g, glucose oxidase activity: 10000 U / g. To explore the optimal combination method of the three enzymes, in this example, the addition amounts of the three enzymes were used as variables, and the comprehensive evaluation score of experts was used as the evaluation index. An orthogonal experiment was carried out. The experimental factors and levels are shown in Table 5, and the experimental results are shown in Table 6.

[0061] Table 5 Factors and levels of orthogonal experiment

[0062]

[0063] Table 6 Factors of orthogonal experiment and comprehensive score

[0064]

[0065] As can be seen from Table 6, the best application scheme in the indicators comprehensively evaluated by experts is A 1 B 1 C 1 , that is, the addition amount of amylase is 0.1 g / L, the addition amount of cellulase is 1.5 g / L, and the addition amount of glucose oxidase is 3.5 g / L.

[0066] Example 4

[0067] To verify the inactivation of the complex enzyme preparation during the tobacco leaf baking process and determine the final addition amount, a complex enzyme preparation was formulated according to a mass ratio of glucose oxidase: cellulase amylase: amylase of 35:15:1 (dissolve 6.25 g of the enzyme preparation in 1 L of water), and sprayed onto tobacco leaf samples (100 g each) with a moisture content of about 8% at addition amounts of 1‰, 2‰, and 5‰ respectively. After standing for 10 min, after baking at 70°C for a certain period of time (10 min, 15 min, and 20 min), the sample treatment conditions are shown in Table 7. The baked tobacco leaves were crushed, bagged, and the enzyme activity was tested. The specific results are shown in Table 8.

[0068] Table 7 Design of test groups for addition amount, baking time, and enzyme activity

[0069]

[0070] Table 8 Test results of addition amount, time, and enzyme activity

[0071]

[0072] The results of the heat resistance test of the enzyme preparation are shown in Table 8. The addition amount of the enzyme preparation and the baking time both have an impact on the activity of the enzyme preparation in tobacco leaves. When the addition amount of the enzyme preparation is 5‰ and the baking time is 10 min, the enzyme activity is similar to the enzyme activity before baking with an addition amount of 1‰. The existing tobacco leaf aging preparation will be baked at about 70°C for about 10 min during use. Therefore, an addition amount of 5‰ is used in the subsequent industrial application of the enzyme preparation.

[0073] Example 5

[0074] To verify the effect of the active complex biological preparation on improving the quality of tobacco, control treatment, high-temperature resistant active complex enzyme preparation (formulate a high-temperature resistant active complex enzyme preparation according to a mass ratio of glucose oxidase: cellulase amylase: amylase of 35:15:1), and active complex biological preparation (formulate a complex biological preparation according to a mass ratio of 1:1 of the high-temperature resistant active complex enzyme preparation to the Enterobacter hormaechei preparation or the bacterial agent, where the Enterobacter hormaechei preparation has a bacterial content of 1.25×10 8 ~1.75×10 8 CFU / g) were set up.

[0075] Table 9 Analysis of smoking evaluation results of tobacco leaves under different treatments

[0076]

[0077]

[0078] Table 10 Component contents of tobacco leaves under different treatments

[0079]

[0080] According to Tables 9 and 10, the active composite biological agent has the best effect. From the analysis of the smoking evaluation results, the total score of applying the high-temperature resistant active composite biological agent is the highest, significantly improving the aroma quality and irritation of the tobacco leaves, reducing the off-flavors during the smoking process of the tobacco leaves, and increasing the aftertaste and sweetness of the tobacco leaves. From the analysis of the results of the chemical components in the tobacco leaves, applying the high-temperature resistant active composite biological agent helps to increase the contents of reducing sugar, potassium ion, chloride ion, and neophytadiene in the tobacco leaves, enhance the combustibility of the tobacco leaves, increase the sweetness, and improve the aroma quality of the tobacco leaves.

[0081] Example 6

[0082] Pilot-scale application tests of the composite biological agent were carried out in the tobacco leaf re-drying factories in Huili and Yibin. The tobacco leaves used in the tests included the tobacco leaves of the YQ2 module in Huili in 2024 and the tobacco leaves of the No. 2 module of Y2C3-HH(2103JXHHMK) in Yibin in 2024, which were provided by the tobacco leaf re-drying factories in Huili and Yibin respectively. The specific process of the experiment is as follows:

[0083] 1) Prepare a composite enzyme preparation by mixing glucose oxidase: cellulase amylase: amylase according to a mass ratio of 35:15:1, and then mix it with the bacterial agent at a ratio of 1:1 to obtain a high-temperature resistant active composite biological agent, where the bacterial content of the Enterobacter hormaechei preparation is 1.25×10 8 ~1.75×10 8 CFU / g;

[0084] 2) Set up experimental groups and control groups in different regions respectively. Dissolve the high-temperature resistant active composite biological agent at a concentration of 12.5 g / L and spray it evenly on the surface of the tobacco leaves after primary baking, with an addition amount of 5‰. Subsequently, the tobacco leaves are aged and re-dried according to the actual situation of the tobacco barns in each tobacco-growing area. Except for the addition of the biological agent, the experimental groups and control groups in the same region adopt the same treatment steps.

[0085] 3) Conduct smoking evaluations and chemical component detections on the experimental groups and control groups in different regions respectively.

[0086] The specific evaluation results and chemical component detections are shown in Table 11.

[0087] Table 11 Smoking evaluation results of tobacco leaves under different treatments in different regions

[0088]

[0089] As shown in Table 11, after the treatment with enzyme preparations, the quality of Huili tobacco leaves and Yibin tobacco leaves has been significantly improved. The comprehensive score of Huili tobacco leaves has increased by 6.94% compared with the control group, while the comprehensive score of Yibin tobacco leaves has increased by 9.46%. In terms of sensory evaluation indicators, including aroma quality, aroma quantity, off-flavor, concentration, irritation, aftertaste and sweetness, etc., they have all been improved compared with the control group.

[0090] In summary, this application has no side effects, is safe and reliable, can effectively reduce the generation of off-flavor, reduce the irritation of tobacco leaves, and enhance the aroma quality of tobacco leaves. The high-temperature-resistant active composite biological preparation developed in this application has a significant effect on promoting the improvement of tobacco leaf quality.

[0091] The above are only the preferred embodiments of this application. It should be understood that this application is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be changed within the scope of the concept described herein through the above teachings or the technology or knowledge in related fields. Any changes and modifications made by those skilled in the art without departing from the spirit and scope of this application shall fall within the protection scope of the appended claims of this application.

Claims

1. A high temperature resistant active composite biological agent for improving tobacco quality, characterized in that: include: By weight, they are: 1-3 parts of amylase; 15-20 parts of cellulase; 35-40 parts of glucose oxidase; 51-63 portions of Enterobacter holmesii preparation; Wherein, the Enterobacter hormaechei preparation includes Enterobacter hormaechei C4, and its preservation number is CGMCC No.26480.

2. The high temperature resistant active composite biological agent according to claim 1, characterized in that: The enzymatic activity of the amylase is 40000U / g; And / or, the enzymatic activity of the cellulase is 10000 U / g; And / or, the enzyme activity of the glucose oxidase is 10000 U / g.

3. The high temperature resistant active composite biological agent according to claim 1, characterized in that: The bacteria content of the Enterobacter holmesii preparation is 1.25×10 8 ~1.75×10 8 CFU / g.

4. Preparation of the high temperature resistant active composite biological agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: The high temperature resistant active composite enzyme preparation is obtained by mixing amylase, cellulase and glucose oxidase in a weight ratio of (1-3:15-20:35-40). The Enterobacter hallii preparation and the thermostable active composite enzyme preparation are mixed in a mass ratio of 1:1 to obtain the thermostable active composite biological preparation.

5. The method for applying the high temperature resistant active composite biological agent according to claim 4, characterized in that: The steps include: The high temperature resistant active composite biological agent is dissolved in water and then sprayed onto the surface of tobacco leaves to obtain tobacco leaves treated with the high temperature resistant active composite biological agent; The tobacco leaves treated with the high temperature resistant active composite biological agent are placed at 25° C. in a dark, ventilated and dry place for 20 to 35 days to obtain aged tobacco leaves.

6. The method for applying the high temperature resistant active composite biological agent according to claim 5, characterized in that: The time for spraying the high temperature resistant active composite biological agent on the surface of the tobacco leaves is before the tobacco leaves are re-roasted.

7. The method for applying the high temperature resistant active composite biological agent according to claim 5, characterized in that: The tobaccos include Yunyan 87, Honghua Dajinyuan and Yibin K326.

8. The method for applying the high temperature resistant active composite biological agent according to claim 5, characterized in that: The mass ratio of the composite biological agent to the tobacco leaf sample is 1: (800-1200) under laboratory conditions, while the ratio is 5: (800-1200) in industrial production.

9. The method for applying the high temperature resistant active composite biological agent according to claim 5, characterized in that: The tobacco quality includes aroma quality, aroma quantity, concentration, pungency, aftertaste and sweetness.

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