Biological agent for accelerating tobacco mellowing and improving tobacco quality and application thereof
A technology of biological preparations and tobacco leaves, which is applied in the fields of application, tobacco, and tobacco treatment. It can solve the problems of long time, poor treatment effect of tobacco leaves, and low alcoholization efficiency, and achieves fast growth, less infection with bacteria, and volatile components. score increase effect
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Embodiment 1
[0033] Example 1: Isolation and identification of strains
[0034] Step 1: Isolation of microorganisms from alcoholized tobacco leaves:
[0035] Tobacco leaves used for screening strains were from Liangshan, Sichuan, and were provided by Sichuan China Tobacco Industry Co., Ltd.
[0036] (1) Collection of microorganisms on the surface of tobacco leaves:
[0037] Select 10 g of non-mildew, moth-eaten, and non-destructive tobacco leaf samples from the alcoholization box, immerse them in a 250-mL conical flask containing 200 mL of 0.1 M, pH 7.2 sterile phosphate buffer, and shake at 220 rpm and 30 °C for 2 hours. Filter through two layers of sterile gauze to collect the filtrate containing microorganisms on the surface of the tobacco leaves.
[0038](2) Separation of microorganisms: After diluting the above bacterial liquid, take 100 μL of it and evenly spread it on the LB solid medium containing tobacco leaf extract, and cultivate at a constant temperature of 37°C for 1 day. Th...
Embodiment 2
[0054] Example 2: Preparation of Biologics
[0055] The strains screened in Example 1 were inoculated into the LB liquid containing tobacco leaf extract (10 g of tobacco leaf powder of 40 meshes was added to 100 mL, pH7.2 phosphate buffer solution, leached at room temperature for 1 h, filtered with gauze and stored at low temperature for later use). In the medium, shake at 37°C for 24h until the bacterial concentration is 1.0×10 7 ~1.0×10 10 cfu / mL, the fermentation broth was centrifuged at 10,000 g at 4°C for 10 min, the supernatant was discarded to collect the bacterial slurry, and the bacterial slurry was washed 2-3 times with sterile 0.85% normal saline. Then, trehalose with a final concentration of 3-7% (w / w) and sucrose solution with a final concentration of 4-8% (w / w) were added as protective agents, and vacuum freezing and concentration were carried out to prepare dry powder of inoculum, which was stored at 4°C for use.
Embodiment 3
[0056] Example 3: Application of biological preparations to unalcoholated fragrant tobacco leaves
[0057] 1. Alcoholized tobacco leaves with biological preparations
[0058] Dilute the biological preparation prepared in Example 2 with sterile water to a bacterial concentration of 1.0 × 10 7 ~1.0×10 10 After cfu / mL, spray evenly on the surface of the tobacco leaves according to the addition amount of 10% (w / w), and mix evenly. After culturing in a temperature-controlled humidity-controlled incubator for 24 hours at 37°C and a humidity of 70%, take out the tobacco leaves and place them on the surface. In an oven, the tobacco leaves are dried at a temperature of 80-90° C. to a moisture content of 13-14% (w / w), and the bacterial strain is terminated to continue metabolism.
[0059] The same amount of sterile water was sprayed on the tobacco leaves and fermented under the same culture conditions to obtain an experimental control sample.
[0060] 2. Tobacco smoking quality evalu...
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