A biological agent for treating fresh tobacco leaves and a method for treating fresh tobacco leaves.

Abstract

This invention discloses a biological agent for treating fresh tobacco leaves and a method for treating fresh tobacco leaves. The biological agent for treating fresh tobacco leaves is prepared by the following method: S1. A single colony of *Pseudomonas paraxanthum* ZY-03 (CGMCC NO.24422) is selected to prepare a seed culture; S2. Field-discarded fresh tobacco leaves are washed, ground at low temperature, and then the fresh tobacco pulp is ultrasonically crushed and filtered at low temperature. The filtrate is filtered through a membrane to obtain fresh tobacco pulp; S3. The seed culture is added to the fresh tobacco pulp, and fermentation is carried out; S4. The fermented pulp is ultrasonically crushed at low temperature, centrifuged, and the supernatant is collected, which is the biological agent for treating fresh tobacco leaves. When the biological agent of this application is sprayed onto fresh tobacco leaves and subjected to initial curing, the aroma components generated from the chemical transformation of the tobacco leaves, and the Maillard reaction that occurs under high-temperature curing conditions, further improve the quality of the fresh tobacco leaves.

Description

Technical Field

[0001] This invention relates to the field of tobacco leaf processing technology, and in particular to a biological agent for processing fresh tobacco leaves and a method for processing fresh tobacco leaves. Background Technology

[0002] Freshly picked tobacco leaves are called fresh tobacco leaves. A huge amount of fresh tobacco leaves in the field are discarded or abandoned due to climate, pests and diseases, or improper management. Even qualified fresh tobacco leaves have problems such as high moisture content, unbalanced chemical composition, high content of macromolecules, and low aroma components. Therefore, tobacco leaves need to go through processes such as initial curing, re-curing, and aging from picking to cigarette processing.

[0003] After fresh tobacco leaves are harvested from the field, they are placed in a curing barn. The drying process is influenced by artificially controlling the temperature and using natural or forced ventilation to achieve the goal of curing the tobacco leaves until they turn yellow, become fragrant, and are dry, thus forming primary cured tobacco leaves. Currently, primary curing uses air as the heat transfer medium, resulting in excessively long yellowing and color-fixing periods. This leads to the consumption of a large amount of original aroma substances, resulting in fewer aroma compounds being formed. During the color-fixing stage, the high temperature and large air volume cause excessive water loss, leading to the decomposition, transformation, and volatilization of aroma substances, which greatly reduces the amount of aroma. In addition, some fresh tobacco leaves themselves have relatively few types and amounts of endogenous enzymes, so it is impossible to completely transform their internal chemical components into a harmonious state through primary curing, re-curing, and aging processes. Therefore, it is necessary to rely on external microorganisms and biological enzymes to complete the process.

[0004] Therefore, how to provide a biological agent for treating fresh tobacco leaves to promote the coordinated transformation of chemical components in the treatment process and improve the quality of fresh tobacco leaves has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a biological agent for treating fresh tobacco leaves and a method for treating fresh tobacco leaves. After the biological agent is sprayed onto the fresh tobacco leaves and subjected to initial curing, the aroma components generated by the transformation of the chemical components of the tobacco leaves, as well as the Maillard reaction that occurs under high-temperature curing conditions, further improve the quality of the fresh tobacco leaves.

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

[0007] A biological agent for treating fresh tobacco leaves, characterized in that the biological agent for treating fresh tobacco leaves is prepared by the following method:

[0008] S1: Select a single colony of Pseudomonas paraxanthii ZY-03 with preservation number CGMCC NO.24422 and inoculate it into YPD liquid medium. Incubate for 12-72 h at a shaking speed of 100-250 r / min to obtain Pseudomonas paraxanthii seed liquid.

[0009] S2: Wash the waste fresh tobacco leaves from the field, grind them at low temperature, and then further crush the fresh tobacco pulp under low temperature conditions at 300-600W. Crush for 30 seconds, pause for 30 seconds, and circulate for 20-40 minutes. Filter the crushed fresh tobacco pulp and sterilize the filtrate through a 0.22um filter membrane to obtain fresh tobacco pulp.

[0010] S3: Add 2%-8% of Pseudomonas paraxanthizobium seed liquid to fresh tobacco leaf slurry, stir and ferment in a fermenter at 150-200 r / min and 30-40℃ for 48-96 h, and introduce sterile air at 0.2-0.8 V / V·min.

[0011] S4: Under low temperature conditions, the fermented slurry is ultrasonically crushed at 200-400W for 30 seconds, followed by a 30-second interval, and then circulated for 20-40 minutes. It is then centrifuged at 8000-12000 r / min and 4℃ for 10-20 minutes. The supernatant is then collected, which is the biological agent for treating fresh tobacco leaves.

[0012] Preferably, in the above technical solution, the YPD liquid culture medium is composed of 10 g / L yeast powder, 20 g / L peptone and 20 g / L glucose, and is prepared by autoclaving at 121°C for 15 min.

[0013] Preferably, in the above technical solution, the low temperature in S2 is 0-4℃, the ultrasonic crushing is 450W, the cycle is 30min, and the crushed material is filtered through gauze.

[0014] Preferably, in the above technical solution, S3 specifically involves: adding 5% *Pseudomonas paraxanthum* to fresh tobacco slurry, stirring and fermenting in a fermenter at 180 r / min and 30°C for 72 h, and introducing sterile air at 0.3 V / V·min.

[0015] Preferably, in the above technical solution, S4 specifically involves: ultrasonically crushing the fermented slurry at a low temperature of 4°C using 300W for 30 seconds, followed by a 30-second interval, and then circulating for 30 minutes. The slurry is then centrifuged at 12000 r / min and 4°C for 15 minutes, and the supernatant is taken as the biological agent for fresh tobacco leaf treatment.

[0016] A method for processing fresh tobacco leaves, wherein the method involves adding the biological agent to the fresh tobacco leaves by spraying at a volume-to-mass ratio of 1:1000, followed by initial curing.

[0017] Preferably, in the above technical solution, the initial roasting of the fresh tobacco leaves adopts a three-stage roasting method, wherein the temperature of the first stage yellowing period is 32-42℃, the temperature of the second stage color fixing period is 45-55℃, and the temperature of the third stage drying period is 60-68℃.

[0018] Application of a biological agent for treating fresh tobacco leaves in the initial curing process of tobacco leaves.

[0019] Preferably, in the above technical solution, before the fresh tobacco leaves are picked and cured, the biological agent is added to the fresh tobacco leaves by spraying according to the volume-to-mass ratio of biological agent to fresh tobacco leaves of 1:1000, and then the initial curing is carried out. The temperature of the first yellowing stage of the initial curing is 32-42℃, the temperature of the second color fixing stage is 45-55℃, and the temperature of the third drying stage is 60-68℃.

[0020] Preferably, in the above technical solution, the reducing sugar and total sugar of the fresh tobacco leaves after initial curing with biological agents increase, while the total nitrogen decreases, resulting in an increased sugar-nitrogen ratio and improved quality of the fresh tobacco leaves.

[0021] The above-described technical solution of the present invention has the following beneficial effects:

[0022] The *Pseudomonas paraxanthum* ZY-03 of this application can tolerate the nicotine environment in waste fresh tobacco leaves. It utilizes the sugars, amino acids, and other nutrients in the waste fresh tobacco leaves to ferment and produce aroma substances and abundant extracellular enzymes. In addition, waste fresh tobacco leaves themselves contain endogenous enzymes that promote the transformation of chemical substances. By using membrane filtration for sterilization, rather than high-pressure high-temperature sterilization, the endogenous enzymes in the fresh tobacco leaves are also retained in the inoculant. When the ZY-03 inoculant is applied to fresh tobacco leaves, the abundant enzymes in the inoculant in the low-temperature baking stage can promote the coordinated transformation of chemical substances in the fresh tobacco leaves and produce aroma substances. The high-temperature baking stage can promote the Maillard reaction and further increase the aroma substances in the tobacco leaves. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0024] The tobacco materials used in this application are obtained as follows: the tobacco leaves are obtained from a cigarette factory under Henan China Tobacco Industry Co., Ltd., and are provided by Henan China Tobacco Co., Ltd.

[0025] The *Pseudomonas parafulva* ZY-03 of this application is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 24422, on February 23, 2022.

[0026] This strain was isolated from the surface of tobacco leaf fragments. It can tolerate the complex environment of tobacco itself, efficiently degrade lignin macromolecules, and has a certain aroma-enhancing effect.

[0027] Example 1

[0028] (1) Prepare YPD medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose) and autoclave at 121℃ for 15min.

[0029] (2) Select a single colony of Pseudomonas paraxanthizobium ZY-03 with preservation number CGMCC NO.24422 and inoculate it into YPD liquid medium. Incubate at 30℃ and shaker speed of 120r / min for 48h.

[0030] (3) Wash the waste fresh tobacco leaves from the field, grind them at 4℃, and then further crush the fresh tobacco pulp at 4℃ and 450W for 30s, 30s interval, and 30min circulation. Filter the crushed fresh tobacco pulp with gauze, and sterilize the filtrate through a 0.22um filter membrane to obtain fresh tobacco pulp.

[0031] (4) Add 5% Pseudomonas paraxanthizobium to fresh tobacco slurry, stir and ferment in a fermenter at 180 r / min and 30 ℃ for 72 h, and introduce sterile air at 0.3 V / V·min.

[0032] (5) The fermented slurry was ultrasonically crushed at 300W at a low temperature of 4℃ for 30s, intermittent for 30s, and circulated for 30min. It was then centrifuged at 12000r / min and 4℃ for 15min. The supernatant was taken as the ZY-03 preparation.

[0033] (6) Before the fresh tobacco leaves are picked and cured, the biological agent is added to the fresh tobacco leaves by spraying according to the volume-to-mass ratio of biological agent to fresh tobacco leaves of 1:1000. The initial curing is carried out according to the normal production process. The initial curing of fresh tobacco leaves is generally a three-stage curing process: the first stage yellowing stage temperature is 32-42℃, the second stage color fixing stage temperature is 45-55℃, and the third stage dry rib stage temperature is 60-68℃.

[0034] Comparative Example 1

[0035] Fresh tobacco leaves from the same production area and part of the plant used in Example 1 were initially cured according to conventional processes. The temperature for the first yellowing stage was 32-42℃, the temperature for the second color fixing stage was 45-55℃, and the temperature for the third drying stage was 60-68℃.

[0036] The conventional chemical components of the pre-cured tobacco leaves in Comparative Example 1 and Example 1 were determined by flow analysis. Lignin was determined according to the industry standard YC / T 347-2010 "Determination of Neutral Detergent Fibers, Acid Detergent Fibers and Acid-Treated Lignin in Tobacco and Tobacco Products - Detergent Method". Aroma components were determined by GC-MS. The conventional chemical components and macromolecules of the pre-cured tobacco leaves are shown in Table 1.

[0037] Table 1. Analysis of conventional chemical components and lignin content in freshly cured tobacco leaves (unit: %)

[0038]

[0039] As shown in Table 1, the lignin content of Example 1 treated with ZY03 biological agent was significantly lower than that of Comparative Example 1, while the reducing sugar and total sugar content were significantly increased, the total nitrogen content was decreased, and the sugar-nitrogen ratio was increased. The abundant bacterial extracellular enzymes and tobacco endogenous enzymes in the inoculant promoted the coordinated transformation of chemical components.

[0040] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A method of treating fresh tobacco leaves, characterized by, The processing method is that the biological agent is added to fresh tobacco leaves in a spraying manner according to a volume-mass ratio of 1:1000, and primary curing is performed; the primary curing of the fresh tobacco leaves adopts a three-stage curing method, wherein the first-stage yellowing period temperature is 32-42 DEG C, the second-stage color fixing period temperature is 45-55 DEG C, and the third-stage dry stem period temperature is 60-68 DEG C; The biological agent is prepared by the following method: S1: a single colony of the Pseudomonas paraficusiens ZY-03 with a preservation number of CGMCC NO. 24422 is inoculated into a YPD liquid culture medium, and seed liquid of the Pseudomonas paraficusiens is obtained by culturing under the condition of a shaking bed speed of 100-250 r / min for 12-72 h; S2: field waste fresh tobacco leaves are washed, and then are ground into pulp at 0-4 DEG C; then the fresh tobacco leaf pulp is further subjected to ultrasonic crushing under the condition of 300-600 W at 0-4 DEG C, the crushing is performed for 30 s, the interval is 30 s, and the cycle is 20-40 min; the crushed fresh tobacco leaf pulp is filtered, the filtrate is sterilized through a 0.22 um filter membrane, and fresh tobacco leaf slurry is obtained; S3: 2%-8% seed liquid of the Pseudomonas paraficusiens is added into the fresh tobacco leaf slurry, the fermentation tank is stirred and fermented under the condition of 150-200 r / min and 30-40 DEG C, sterile air is introduced at 0.2-0.8 V / V·min for 48-96 h; S4: the fermented slurry is subjected to ultrasonic crushing under the condition of 200-400 W at 4 DEG C, the crushing is performed for 30 s, the interval is 30 s, the cycle is 20-40 min, and the supernatant is obtained by centrifugation at 8000-12000 r / min and 4 DEG C for 10-20 min, and the supernatant is the biological agent.

2. The method of treating fresh tobacco leaves according to claim 1, characterized in that, The YPD liquid culture medium is composed of 10 g / L of yeast powder, 20 g / L of peptone and 20 g / L of glucose, and is prepared after high-pressure sterilization at 121 DEG C for 15 min.

3. The method of treating fresh tobacco leaves according to claim 1, characterized in that, The ultrasonic crushing in S2 is 450 W, the cycle is 30 min, and the crushed product is filtered through gauze.

4. The method of treating fresh tobacco leaves according to claim 1, characterized in that, S3 specifically is that 5% Pseudomonas paraficusiens is added into the fresh tobacco leaf slurry, the fermentation tank is stirred and fermented under the condition of 180 r / min and 30 DEG C, sterile air is introduced at 0.3 V / V·min for 72 h.

5. The method of treating fresh tobacco leaves according to claim 1, wherein S4 specifically is that the fermented slurry is subjected to ultrasonic crushing under the condition of 300 W, the crushing is performed for 30 s, the interval is 30 s, the cycle is 30 min, the supernatant is obtained by centrifugation at 12000 r / min and 4 DEG C for 15 min, and the supernatant is the fresh tobacco leaf processing biological agent.

6. Use of a fresh tobacco leaf treatment biologic in the curing process of tobacco leaf, characterized in that, Before the fresh tobacco leaves are picked and cured, the biological agent is added to the fresh tobacco leaves in a spraying manner according to a volume-mass ratio of 1:1000, and primary curing is performed, wherein the first-stage yellowing period temperature is 32-42 DEG C, the second-stage color fixing period temperature is 45-55 DEG C, and the third-stage dry stem period temperature is 60-68 DEG C; The biological agent is prepared by the following method: S1: a single colony of the Pseudomonas paraficusiens ZY-03 with a preservation number of CGMCC NO. 24422 is inoculated into a YPD liquid culture medium, and seed liquid of the Pseudomonas paraficusiens is obtained by culturing under the condition of a shaking bed speed of 100-250 r / min for 12-72 h; S2: washing the field abandoned fresh tobacco leaves, 0-4 DEG C grinding pulp, and then further fresh tobacco leaf pulp at 0-4 DEG C, 300-600W ultrasonic crushing, crushing 30s, intermittent 30s, cycle 20-40min, the crushed fresh tobacco leaf pulp filtration, filter 0.22um filter membrane sterilization, to obtain fresh tobacco leaf slurry; S3: adding 2%-8% of the Pseudomonas paraflavescens seed liquid to the fresh tobacco leaf slurry, 150-200r / min, 30-40 DEG C, fermentation tank stirring fermentation, 48-96h, the sterile air 0.2-0.8V / V·min is imported; S4: the fermented slurry 200-400W ultrasonic crushing under the condition of 4 DEG C, crushing 30s, intermittent 30s, cycle 20-40min, 8000-12000r / min, 4 DEG C centrifugation 10-20min, take the supernatant, that is, biological agent.

7. Use according to claim 6, characterized in that, The fresh tobacco leaf after primary curing with the biological agent has increased reducing sugar and total sugar, reduced total nitrogen, increased sugar-nitrogen ratio, and improved fresh tobacco leaf quality.

8. Use according to claim 6, characterized in that The YPD liquid culture medium is composed of yeast powder 10g / L, peptone 20g / L and glucose 20g / L, prepared after high pressure sterilization at 121 DEG C for 15min; the ultrasonic crushing in S2 is 450W, cycle 30min, and the crushed pulp is filtered with gauze.

9. Use according to claim 6, characterized in that, S3 specifically is: adding 5% of the Pseudomonas paraflavescens to the fresh tobacco leaf slurry, 180r / min, 30 DEG C, fermentation tank stirring fermentation, 72h, the sterile air 0.3V / V·min is imported.

10. Use according to claim 6, characterized in that, S4 specifically is: the fermented slurry 300W ultrasonic crushing, crushing 30s, intermittent 30s, cycle 30min, 12000r / min, 4 DEG C centrifugation 15min, take the supernatant, that is, fresh tobacco leaf processing biological agent.

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