CN113475745B - Composite biological preparation for tobacco leaf treatment and fresh tobacco leaf treatment method - Google Patents

Composite biological preparation for tobacco leaf treatment and fresh tobacco leaf treatment method Download PDF

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CN113475745B
CN113475745B CN202110687891.2A CN202110687891A CN113475745B CN 113475745 B CN113475745 B CN 113475745B CN 202110687891 A CN202110687891 A CN 202110687891A CN 113475745 B CN113475745 B CN 113475745B
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micrococcus
biological agent
culture medium
tobacco
tobacco leaves
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CN113475745A (en
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冯颖杰
杨宗灿
杨永锋
张婷婷
张展
刘文召
彭玉富
刘向真
赵森森
牛洋洋
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China Tobacco Henan Industrial Co Ltd
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China Tobacco Henan Industrial Co Ltd
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    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/24—Treatment of tobacco products or tobacco substitutes by extraction; Tobacco extracts
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/20—Biochemical treatment
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/24—Treatment of tobacco products or tobacco substitutes by extraction; Tobacco extracts
    • A24B15/26—Use of organic solvents for extraction
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B3/00—Preparing tobacco in the factory
    • A24B3/10—Roasting or cooling tobacco
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B3/00—Preparing tobacco in the factory
    • A24B3/12—Steaming, curing, or flavouring tobacco

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Manufacture Of Tobacco Products (AREA)

Abstract

The invention discloses a tobacco leaf treatment composite biological agent and a fresh tobacco leaf treatment method, wherein the tobacco leaf treatment composite biological agent comprises micrococcus ZY02 with the preservation number of CGMCC NO.21313 and acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO. 21544. The tobacco leaf treatment composite biological agent disclosed by the invention can enable fresh tobacco leaves to generate fragrance components under a high-temperature baking condition, and effectively improves the quality of the fresh tobacco leaves.

Description

Composite biological preparation for tobacco leaf treatment and fresh tobacco leaf treatment method
Technical Field
The invention relates to the field of tobacco leaf treatment, in particular to a composite biological agent for tobacco leaf treatment and a method for treating fresh tobacco leaves.
Background
The tobacco leaves are required to be subjected to processes of primary baking, secondary baking, alcoholization and the like from the picking to the cigarette processing. The freshly picked tobacco leaves are called fresh tobacco leaves, and have high moisture content, inconsistent chemical components, high macromolecular content and few flavor components.
Through the processes of primary baking, secondary baking, alcoholization and the like, the moisture of the tobacco leaves is reduced to about 12 percent, macromolecular substances are gradually degraded, and the fragrance components are accumulated. However, some fresh tobacco leaves have poor quality, chemical components in the fresh tobacco leaves cannot be completely converted into coordination through processes of primary baking, secondary baking, alcoholization and the like, macromolecular substances are difficult to completely degrade, and the macromolecular conversion needs to be completed with the assistance of microorganisms and biological enzymes.
Therefore, how to provide a biological agent capable of treating fresh tobacco leaves becomes a technical problem which needs to be solved urgently in the field.
Disclosure of Invention
The invention aims to provide a novel technical scheme of a tobacco leaf treatment composite biological agent capable of treating fresh tobacco leaves.
According to a first aspect of the present invention, there is provided a composite biological agent for tobacco treatment.
The composite biological preparation for treating tobacco leaves comprises micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 and acetobacter gluconicum (Gluconacetobacter sp.) ZT-01 with the preservation number of CGMCC NO. 21544.
According to a second aspect of the invention, the invention provides an application of the composite biological preparation for tobacco leaf treatment in the field of fresh tobacco leaf treatment.
According to a third aspect of the invention, a method of processing fresh tobacco leaves is provided.
The processing method of the fresh tobacco leaves comprises the following steps:
step (1): inoculating micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 into a liquid culture medium, culturing for 6-36h in a shaker at 25-40 ℃ and 100-200r/min to obtain micrococcus seed liquid, centrifuging the micrococcus seed liquid at the low temperature of 4000r/min and 4 ℃, removing the supernatant, adding sterile water for redissolving, adjusting OD (optical density) to obtain a new micrococcus seed liquid 600 2.0, obtaining micrococcus seeds;
step (2): preparing an aqueous solution containing glucose, peptone, yeast powder and citric acid monohydrate, adjusting pH, sterilizing, inoculating Gluconacetobacter sp ZT-01 with the preservation number of CGMCC NO.21544, and performing shake culture at constant temperature for a period of time to obtain a seed solution of Gluconacetobacter gluconicum;
and (3): mixing tobacco powder and deionized water according to the mass ratio of 1 (2-20), extracting at 30-100 ℃ for 0.5-5 h, filtering, adding soluble starch and soluble protein into the filtrate, mixing uniformly and sterilizing to obtain a tobacco fermentation induction culture medium;
and (4): mixing micrococcus seeds and a tobacco fermentation induction culture medium according to a volume ratio of 1 (50-1000), stirring and fermenting for 1-5 days at a temperature of 25-40 ℃ at a speed of 50-300r/min, centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic crushing in an ice water bath, centrifuging at a low temperature, and taking supernatant to obtain a ZY02 biological agent;
and (5): mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1 (10-1000), firstly stirring and fermenting for 12-48h at the temperature of 25-40 ℃ and 50-300r/min, then standing for 12-72h, centrifuging the fermented tobacco fermentation induction culture medium, then ultrasonically crushing in an ice water bath, then centrifuging at low temperature, and taking the supernatant to obtain the ZT-01 biological agent;
and (6): mixing the ZY02 biological agent and the ZT-01 biological agent according to the volume ratio of (1-10) to obtain a composite biological agent;
and (7): before baking fresh tobacco leaves, adding the composite biological agent into the fresh tobacco leaves in a spraying manner according to the volume-mass ratio of the composite biological agent to the fresh tobacco leaves being 1 (50-10000).
Optionally, the step (1) is specifically as follows:
inoculating micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 into a liquid culture medium, culturing for 12h in a shaking table at 30 ℃ and 150r/min to obtain micrococcus seed liquid, centrifuging the micrococcus seed liquid at 4000r/min and 4 ℃ for 20min at low temperature, removing supernatant, adding sterile water for redissolving, and adjusting OD (optical density) to obtain the micrococcus seed liquid 600 The yield was 2.0, and micrococcus seeds were obtained.
Optionally, the step (2) is specifically as follows:
preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating the gluconacetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 25-40 ℃ for 24-48h under the condition of 100-200r/min to prepare the seed solution of the gluconacetobacter gluconicum.
Optionally, the step (3) is specifically as follows:
sieving tobacco powder, mixing the sieved tobacco powder with deionized water according to the mass ratio of 1 (2-20), extracting at 30-100 ℃ for 0.5-5 h, filtering, adding soluble starch and soluble protein into filtrate according to the addition of 10g/L of soluble starch and 10g/L of soluble protein, uniformly mixing and sterilizing to obtain the tobacco fermentation induction culture medium.
Optionally, the step (4) is specifically as follows:
step (4-1): mixing micrococcus seeds with a tobacco fermentation induction culture medium according to a volume ratio of 1 (50-1000), and stirring and fermenting for 1-5d at 25-40 deg.C and 50-300 r/min;
step (4-2): centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath with power of 50-300W for 2-30min, performing low-temperature centrifugation after ultrasonic disruption, and collecting supernatant to obtain ZY02 biological agent.
Optionally, the step (5) is specifically as follows:
step (5-1): mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1 (10-1000), stirring and fermenting for 12-48h at the temperature of 25-40 ℃ and 50-300r/min, and standing for 12-72 h;
step (5-2): centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath with power of 50-300W for 2-30min, performing low-temperature centrifugation after ultrasonic disruption, and collecting supernatant to obtain ZT-01 biological preparation.
Optionally, the step (6) is specifically as follows:
mixing the ZY02 biological agent and ZT-01 biological agent according to the volume ratio of 2:1 to obtain the compound biological agent.
Optionally, the step (7) is specifically as follows:
before the baking of the fresh tobacco leaves, the composite biological agent is added into the fresh tobacco leaves in a spraying mode according to the volume mass ratio of the composite biological agent to the fresh tobacco leaves being 1 (50-10000), the first-stage baking is carried out at the temperature of 40 ℃, and the second-stage baking is carried out at the temperature of 70 ℃.
The tobacco leaf treatment composite biological agent disclosed by the invention can enable fresh tobacco leaves to generate fragrant components under a high-temperature baking condition, and effectively improves the quality of the fresh tobacco leaves.
Other features of the present invention and advantages thereof will become apparent from the following detailed description of exemplary embodiments thereof, which proceeds with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 is a statistical chart of the flavor components of tobacco leaves after the primary flue-curing of the present disclosure.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.
Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.
In all examples shown and discussed herein, any particular value should be construed as exemplary only and not as limiting. Thus, other examples of the exemplary embodiments may have different values.
The present disclosure provides a composite biological preparation for tobacco leaf treatment, which comprises micrococcus (Micrococcaceae Pribram) ZY02 with a preservation number of CGMCC NO.21313 and Acetobacter gluconicum (Gluconaceobacter sp.) ZT-01 with a preservation number of CGMCC NO. 21544.
The Gluconacetobacter sp ZT-01 is deposited in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC NO.21544, the strain name is the Gluconacetobacter gluconicum, the strain number is ZT-01, the strain is classified and named as the Gluconacetobacter gluconicum, the Gluconacetobacter sp, and the preservation time is 2020, 12 and 23 days.
The micrococcus (Microccceae Pribram) ZY02 is deposited in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.21313, the strain name is Micrococcus, the strain number is ZY02, the classification name is Micrococcus, Microccceae Pribram, and the preservation time is 12 months and 07 days in 2020.
The disclosure also provides application of the tobacco treatment composite biological agent in the field of fresh tobacco treatment.
The processing method of the fresh tobacco leaves provided by the disclosure comprises the following steps:
step (1): inoculating a colony of Micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 into a liquid culture medium, culturing for 6-36h in a shaker at 25-40 ℃ and 200r/min at 100 ℃ to obtain Micrococcus seed liquid, centrifuging the Micrococcus seed liquid at the low temperature of 4000r/min and 4 ℃, removing the supernatant, adding sterile water for redissolving, adjusting OD (optical density) 600 The yield was 2.0, and micrococcus seeds were obtained.
The step (1) may be specifically as follows:
inoculating a colony of Micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 into a liquid culture medium, culturing for 12h in a shaking table at 30 ℃ and 150r/min to obtain Micrococcus seed liquid, centrifuging the Micrococcus seed liquid at the temperature of 4000r/min and 4 ℃ for 20min, removing supernatant, adding sterile water for redissolving, and adjusting OD (optical density) 600 The yield was 2.0, and micrococcus seeds were obtained.
Step (2): preparing an aqueous solution containing glucose, peptone, yeast powder and citric acid monohydrate, adjusting pH, sterilizing, inoculating acetobacter gluconicum (Gluconacetobacter sp.) ZT-01 with the preservation number of CGMCC NO.21544, and performing shake culture at constant temperature for a period of time to obtain an acetobacter gluconicum seed solution.
The step (2) may be specifically as follows:
preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating the gluconacetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 25-40 ℃ for 12-48h under the condition of 100-200r/min to prepare the seed solution of the gluconacetobacter gluconicum.
And (3): mixing tobacco powder and deionized water according to the mass ratio of 1 (2-20), extracting at 30-100 ℃ for 0.5-5 h, filtering, adding soluble starch and soluble protein into the filtrate, mixing uniformly and sterilizing to obtain the tobacco fermentation induction culture medium. The tobacco powder can be high-quality tobacco powder.
The step (3) may be embodied as follows:
sieving tobacco powder, mixing the sieved tobacco powder with deionized water according to the mass ratio of 1 (2-20), extracting at 30-100 ℃ for 0.5-5 h, filtering, adding soluble starch and soluble protein into filtrate according to the addition of 10g/L of soluble starch and 10g/L of soluble protein, uniformly mixing and sterilizing to obtain the tobacco fermentation induction culture medium.
And (4): mixing micrococcus seeds and a tobacco fermentation induction culture medium according to the volume ratio of 1 (50-1000), stirring and fermenting for 1-5 days at the temperature of 25-40 ℃ at the speed of 50-300r/min, centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic crushing in an ice water bath, then centrifuging at low temperature, and taking supernatant to obtain the ZY02 biological agent.
The step (4) may be embodied as follows:
step (4-1): mixing micrococcus seeds with a tobacco fermentation induction culture medium according to a volume ratio of 1 (50-1000), and stirring and fermenting for 1-5 days at a temperature of 25-40 ℃ and at a speed of 50-300 r/min.
Step (4-2): centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath with power of 50-300W for 2-30min, performing low-temperature centrifugation after ultrasonic disruption, and collecting supernatant to obtain ZY02 biological agent.
And (5): mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1 (10-1000), firstly stirring and fermenting for 12-48h at the temperature of 25-40 ℃ and 50-300r/min, then standing for 12-72h, centrifuging the fermented tobacco fermentation induction culture medium, then ultrasonically crushing in an ice water bath, then centrifuging at low temperature, and taking the supernatant to obtain the ZT-01 biological agent.
The step (5) may be embodied as follows:
step (5-1): mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1 (10-1000), stirring and fermenting for 12-48h at the temperature of 25-40 ℃ and 50-300r/min, and standing for 12-72 h.
Step (5-2): centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath with power of 50-300W for 2-30min, performing low-temperature centrifugation after ultrasonic disruption, and collecting supernatant to obtain ZT-01 biological preparation.
And (6): mixing the ZY02 biological agent and ZT-01 biological agent according to the volume ratio of (1-10) to obtain the compound biological agent.
The step (6) may be embodied as follows:
mixing the ZY02 biological preparation and the ZT-01 biological preparation according to the volume ratio of 2:1 to obtain the compound biological preparation.
And (7): before baking fresh tobacco leaves, adding the composite biological agent into the fresh tobacco leaves in a spraying manner according to the volume-mass ratio of the composite biological agent to the fresh tobacco leaves being 1 (50-10000).
The step (7) may be embodied as follows:
before the baking of the fresh tobacco leaves, the composite biological agent is added into the fresh tobacco leaves in a spraying mode according to the volume mass ratio of the composite biological agent to the fresh tobacco leaves being 1 (50-10000), the first-stage baking is carried out at the temperature of 40 ℃, and the second-stage baking is carried out at the temperature of 70 ℃.
The action of microorganisms and biological enzymes needs to satisfy sufficient moisture and appropriate temperature conditions. The moisture content in fresh tobacco leaves is high, about 85 percent. When the fresh tobacco leaves are primarily baked, a three-stage baking process is generally adopted, and the first-stage baking temperature is controlled to be about 40 ℃ and is suitable for the temperature of most biological enzymes.
The experimental procedures used in the following examples are conventional unless otherwise specified, the materials and reagents used are commercially available unless otherwise specified, and the equipment used in the experiments are well known to those skilled in the art.
Example 1
Inoculating the colony of Micrococcus ZY02 with the preservation number of CGMCC NO.21313 into LB liquid culture medium with an inoculating loop, culturing in a shaking table at 30 ℃ and 150r/min for 12h to obtain Micrococcus seed liquid, centrifuging the Micrococcus seed liquid at 4000r/min and 4 ℃ for 20min, removing supernatant, adding sterile water for redissolving, and adjusting OD 600 The yield was 2.0, and micrococcus seeds were obtained.
Preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 30 ℃ and 150r/min for 24h to prepare an acetobacter gluconicum seed solution.
Sieving tobacco powder with a 50-mesh sieve, mixing the sieved tobacco powder with deionized water according to the mass ratio of 1:10, extracting at 70 ℃ for 1.5h, filtering with a 200-mesh sieve, taking filtrate, adding soluble starch and soluble protein according to the addition of 10g/L of soluble starch and 10g/L of soluble protein, uniformly mixing, and sterilizing at 121 ℃ for 10min to obtain the tobacco fermentation induction culture medium.
Mixing micrococcus seeds with a tobacco fermentation induction culture medium according to a volume ratio of 1:200, and stirring and fermenting for 2d at the temperature of 30 ℃ and at the speed of 150 r/min. Centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath for 10min at power of 150W for 5s and 5s of intermittent operation, performing low-temperature centrifugation after ultrasonic disruption, and taking supernatant to obtain ZY02 biological agent.
Mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1:250, stirring and fermenting for 36 hours at the temperature of 30 ℃ and at the speed of 150r/min, and standing for 48 hours. Centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic crushing in ice water bath for 10min at power of 150W for 5s at interval of 5s, performing low-temperature centrifugation after ultrasonic crushing, and taking supernatant to obtain the ZT-01 biological agent.
Mixing the ZY02 biological agent and ZT-01 biological agent according to the volume ratio of 2:1 to obtain the compound biological agent.
Before baking fresh tobacco leaves (picking fresh tobacco leaves at the middle position of a production area in Henan Xiang county as a sample), adding a composite biological agent into the fresh tobacco leaves in a spraying mode according to the volume-to-mass ratio of the composite biological agent to the fresh tobacco leaves being 1:500, carrying out first-stage baking at the temperature of 40 ℃, and carrying out second-stage baking at the temperature of 70 ℃ to obtain primary-baked tobacco leaves 1 #. The composite biological agent is inactivated at the high temperature of the second stage baking.
Comparative example 1
Before baking fresh tobacco leaves (picking fresh tobacco leaves at the middle position of a production area in Henan Xiang county as a sample), adding sterile water into the fresh tobacco leaves in a spraying mode according to the volume-to-mass ratio of the sterile water to the fresh tobacco leaves being 1:500, carrying out first-stage baking at the temperature of 40 ℃, and carrying out second-stage baking at the temperature of 70 ℃ to obtain primary-baked tobacco leaves 2 #.
The conventional chemical components and macromolecular substances in the flue-cured tobacco leaves 1# and 2# are detected by a flow analysis method. Meanwhile, the flue-cured tobacco leaves 1# and 2# are extracted by dichloromethane, and the flavor components are detected by GC-MS.
TABLE 1 analysis of the conventional chemical composition of flue-cured tobacco leaves (unit:%)
Figure BDA0003125439120000081
The conventional chemical components and macromolecular substances of the flue-cured tobacco leaves are shown in the table 1. As can be seen from Table 1, the starch and protein contents of the flue-cured tobacco leaf No. 1 treated by the composite biological agent are obviously lower than those of the flue-cured tobacco leaf No.2, and reducing sugar and total sugar are generated in a part of degraded macromolecules, so that the reducing sugar and total sugar contents of the flue-cured tobacco leaf No. 1 are also obviously increased.
The statistics of the fragrance components of the primary flue-cured tobacco leaves are shown in figure 1 and table 2. As can be seen from FIG. 1 and Table 2, the contents of alcohol, ketone and Maillard product flavor components in flue-cured tobacco leaf No. 1 are significantly increased, and the Maillard product is significantly increased. The protein and the starch are degraded to generate micromolecular sugar and amino acid, and Maillard reaction is easy to occur under the high-temperature baking condition to generate flavor components, so that the quality of the fresh tobacco leaves is improved.
TABLE 2 statistics of primary flue-cured tobacco leaf flavor components (unit: ug/g)
Figure BDA0003125439120000091
Although some specific embodiments of the present invention have been described in detail by way of example, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the invention. It will be appreciated by those skilled in the art that modifications can be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims (9)

1. A method for processing fresh tobacco leaves is characterized by comprising the following steps:
step (1): inoculating a colony of Micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 into a liquid culture medium, culturing for 6-36h in a shaker at 25-40 ℃ and 200r/min at 100 ℃ to obtain Micrococcus seed liquid, centrifuging the Micrococcus seed liquid at the low temperature of 4000r/min and 4 ℃, removing the supernatant, adding sterile water for redissolving, adjusting OD (optical density) 600 2.0, obtaining micrococcus seeds;
step (2): preparing an aqueous solution containing glucose, peptone, yeast powder and citric acid monohydrate, adjusting pH, sterilizing, inoculating acetobacter gluconicum (Gluconacetobacter sp.) ZT-01 with the preservation number of CGMCC NO.21544, and performing constant-temperature shaking culture for a period of time to obtain an acetobacter gluconicum seed solution;
and (3): mixing tobacco powder and deionized water according to the mass ratio of 1 (2-20), extracting at 30-100 ℃ for 0.5-5 h, filtering, adding soluble starch and soluble protein into the filtrate, mixing uniformly and sterilizing to obtain a tobacco fermentation induction culture medium;
and (4): mixing micrococcus seeds and a tobacco fermentation induction culture medium according to a volume ratio of 1 (50-1000), stirring and fermenting for 1-5 days at a temperature of 25-40 ℃ at a speed of 50-300r/min, centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic crushing in an ice water bath, centrifuging at a low temperature, and taking supernatant to obtain a ZY02 biological agent;
and (5): mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1 (10-1000), firstly stirring and fermenting for 12-48h at the temperature of 25-40 ℃ and 50-300r/min, then standing for 12-72h, centrifuging the fermented tobacco fermentation induction culture medium, then ultrasonically crushing in an ice water bath, then centrifuging at low temperature, and taking the supernatant to obtain the ZT-01 biological agent;
and (6): mixing the ZY02 biological agent and the ZT-01 biological agent according to the volume ratio of (1-10) to obtain a composite biological agent;
and (7): before baking fresh tobacco leaves, adding the composite biological agent into the fresh tobacco leaves in a spraying manner according to the volume-mass ratio of the composite biological agent to the fresh tobacco leaves being 1 (50-10000).
2. The method for treating fresh tobacco leaves according to claim 1, wherein the step (1) is as follows:
inoculating a colony of Micrococcus (Micrococcus Pribram) ZY02 with the preservation number of CGMCC NO.21313 into a liquid culture medium, culturing for 12h in a shaking table at 30 ℃ and 150r/min to obtain Micrococcus seed liquid, centrifuging the Micrococcus seed liquid at the temperature of 4000r/min and 4 ℃ for 20min, removing supernatant, adding sterile water for redissolving, and adjusting OD (optical density) 600 The yield was 2.0, and micrococcus seeds were obtained.
3. The fresh tobacco leaf processing method according to claim 1, wherein the step (2) is specifically as follows:
preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating the gluconacetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 25-40 ℃ for 12-48h under the condition of 100-200r/min to prepare the seed solution of the gluconacetobacter gluconicum.
4. The method for treating fresh tobacco leaves according to claim 1, wherein the step (3) is specifically as follows:
sieving tobacco powder, mixing the sieved tobacco powder with deionized water according to the mass ratio of 1 (2-20), extracting at 30-100 ℃ for 0.5-5 h, filtering, adding soluble starch and soluble protein into filtrate according to the addition of 10g/L of soluble starch and 10g/L of soluble protein, uniformly mixing and sterilizing to obtain the tobacco fermentation induction culture medium.
5. The method for treating fresh tobacco leaves according to claim 1, wherein the step (4) is as follows:
step (4-1): mixing micrococcus seeds with a tobacco fermentation induction culture medium according to a volume ratio of 1 (50-1000), and stirring and fermenting for 1-5d at 25-40 deg.C and 50-300 r/min;
step (4-2): centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath with power of 50-300W for 2-30min, performing low-temperature centrifugation after ultrasonic disruption, and collecting supernatant to obtain ZY02 biological agent.
6. The method for treating fresh tobacco leaves according to claim 1, wherein the step (5) is as follows:
step (5-1): mixing the acetobacter gluconicum seed liquid and the tobacco fermentation induction culture medium according to the volume ratio of 1 (10-1000), stirring and fermenting for 12-48h at the temperature of 25-40 ℃ and 50-300r/min, and standing for 12-72 h;
step (5-2): centrifuging the fermented tobacco fermentation induction culture medium, performing ultrasonic disruption in ice water bath with power of 50-300W for 2-30min, performing low-temperature centrifugation after ultrasonic disruption, and collecting supernatant to obtain ZT-01 biological preparation.
7. The method for treating fresh tobacco leaves according to claim 1, wherein the step (6) is as follows:
mixing the ZY02 biological agent and ZT-01 biological agent according to the volume ratio of 2:1 to obtain the compound biological agent.
8. The method for treating fresh tobacco leaves according to claim 1, wherein the step (7) is as follows:
before the baking of the fresh tobacco leaves, the composite biological agent is added into the fresh tobacco leaves in a spraying mode according to the volume mass ratio of the composite biological agent to the fresh tobacco leaves being 1 (50-10000), the first-stage baking is carried out at the temperature of 40 ℃, and the second-stage baking is carried out at the temperature of 70 ℃.
9. The complex biological preparation for tobacco treatment based on the method for treating fresh tobacco leaves according to any one of claims 1 to 8, characterized by comprising micrococcus (Micrococcus Pribram) ZY02 with collection number CGMCC NO.21313 and Acetobacter gluconicum (Gluconaceobacter sp.ZT) -01 with collection number CGMCC NO. 21544.
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DE2816427C2 (en) * 1977-05-06 1982-09-16 Fabriques de Tabac Réunies S.A., 2003 Neuchâtel Process for refining tobacco
US4392501A (en) * 1981-07-06 1983-07-12 Brown & Williamson Tobacco Corporation Method of processing fresh tobacco leaves
CN101449854B (en) * 2008-12-30 2011-12-21 无锡华馨香精香料有限公司 Fermentation optimization method of tobacco essence
CN102078027B (en) * 2010-11-30 2014-12-03 昆明贵勋科技有限公司 Method for separating microorganisms on surface of tobacco leaf at high temperature by utilizing pure tobacco leaf leach liquor solid plate
CN103535849A (en) * 2012-07-10 2014-01-29 朱大恒 New flue-cured tobacco leaf concocting method, tobacco products and application
US11033050B2 (en) * 2017-10-13 2021-06-15 Kombucha Biomaterials Llc Cigarette rolling papers formed from kombucha biofilms
CN110693075B (en) * 2019-10-12 2021-10-08 中国科学院青岛生物能源与过程研究所 Four-stage fermentation method of cigar core tobacco leaves
CN110786534B (en) * 2019-11-26 2023-06-02 上海龙殷生物科技有限公司 Method for improving aroma of tobacco extract by using carotenoid-producing microorganism

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