Klebsiella pneumoniae for enhancing the aroma of tobacco concentrate and its application

Abstract

This invention discloses a Klebsiella strain for enhancing the aroma of tobacco concentrate and its application. The Klebsiella strain for enhancing the aroma of tobacco concentrate is Klebsiella sp. HNYJ-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327. This strain can not only effectively degrade macromolecules in tobacco, but also utilize glycoside hydrolases and oxidoreductases produced by itself to generate key aroma compounds from some chemical components in tobacco, thereby improving the quality and aroma of the prepared reconstituted tobacco leaves.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Klebsiella pneumoniae strain for enhancing the aroma of tobacco concentrate and its applications. Background Technology

[0002] Klebsiella pneumoniae is a facultative anaerobic bacterium widely found in tobacco. Various types of Klebsiella have been reported to be isolated from samples including the interior of tobacco plants, the surface of tobacco leaves, and aged tobacco flakes. Klebsiella pneumoniae has been shown to produce macromolecular degrading enzymes such as amylase, pectinase, and polysaccharide-degrading enzymes. Furthermore, some literature reports its ability to produce aroma compounds such as succinic acid, butanediol, 3-hydroxypropionic acid, and lactic acid.

[0003] Reconstituted tobacco using the papermaking method utilizes tobacco materials (stems, dust, and broken tobacco leaves) as the main raw materials. After water extraction and concentration, the solid portion is processed into a sheet base, and the liquid portion forms a concentrated liquid. This is further formulated into a coating solution and applied to the sheet base to form a tobacco product. During the formation of the concentrated liquid, some large molecules dissolve in water and are retained, increasing the viscosity of the concentrated liquid and affecting the coating rate. These substances also act as precursors to off-flavors. Furthermore, the aroma components in the concentrated liquid, due to their low boiling point, undergo significant volatilization, reducing the smoking quality of the reconstituted tobacco. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Klebsiella pneumoniae for improving the aroma of tobacco concentrate and its application, which can achieve the effect of enhancing aroma and quality.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0006] A Klebsiella strain for enhancing the aroma of tobacco concentrate, wherein the Klebsiella sp. HNYJ-1 is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 24327, and the 16S rDNA sequence of the Klebsiella is shown in SEQ ID NO. 1.

[0007] A method for preparing flavor-enhancing tobacco concentrate using Klebsiella pneumoniae includes the following steps:

[0008] (1) Inoculate a single colony of Klebsiella HNYJ-1 into LB liquid medium with an inoculation loop and culture it in a shaker at 25-40℃ and 100-200r / min for 6-36h to obtain seed culture;

[0009] (2) The seed liquid obtained in step (1) is mixed with the reconstituted tobacco concentrate at a volume ratio of 1:0.5%-1:10%, and allowed to stand for 1-24 hours at a temperature of 40℃-50℃. During this period, the mixture is slowly stirred once every 0.5 hours to allow it to react fully. After the reaction is complete, the aroma-enhancing tobacco concentrate is obtained.

[0010] Preferably, in the above technical solution, in step (1), the culture conditions of the culture medium are 30°C, the shaking speed is 150 r / min, and the culture time is 12 h.

[0011] Preferably, in the above technical solution, in step (2), the ratio of seed liquid to reconstituted tobacco concentrate is 1:2%, and the settling time is 12h.

[0012] A concentrated tobacco liquid with strong aroma.

[0013] Application of a flavor-enhancing tobacco concentrate in tobacco leaf processing.

[0014] Preferably, in the above technical solution, the protein and pectin content in the flavor-enhancing tobacco concentrate is reduced, and the viscosity is also reduced; the total amount of flavor components is increased, with a significant increase in alcohols, aldehydes, ketones, esters, and lactones.

[0015] The above-described technical solution of the present invention has the following beneficial effects:

[0016] This patent discloses a type of Klebsiella pneumoniae that can not only effectively degrade macromolecules in tobacco, but also utilize some chemical components in tobacco to generate key aroma substances through some glycoside hydrolases and oxidoreductases produced by itself, thereby improving the quality and aroma of the prepared reconstituted tobacco leaves. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0018] Figure 1 Image of Klebsiella pneumoniae HNYJ-1 colony.

[0019] Figure 2 This is the phylogenetic tree of Klebsiella pneumoniae HNYJ-1. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. 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.

[0021] A Klebsiella sp. HNYJ-1 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327 on January 17, 2022. The 16S rDNA sequence of the Klebsiella sp. is shown in SEQ ID NO. 1.

[0022] Example 1: Strain Characteristics

[0023] Microscopic images: Colony images of Klebsiella pneumoniae HNYJ-1, as shown below. Figure 1 As shown, the colonies are white, raised, round, with a smooth, wrinkle-free surface and regular edges.

[0024] Evolutionary tree: The evolutionary tree of HNYJ-1 is as follows Figure 2 As shown in the phylogenetic tree, this species is closely related to Klebsiella variegata and belongs to the same genus, Klebsiella.

[0025] Whole-genome sequencing: Statistical analysis of the whole-genome next-generation sequencing data of HNYJ-1 was performed, and the results are shown in Table 1. As shown in the table, the total number of reads was 8,087,512, with only 0.0038% being fuzzy bases, 55.81% being GC bases, 98.21% having a base identification accuracy of over 99%, and 94.36% having a base identification accuracy of over 99.9%.

[0026] Table 1 Sequencing data statistics

[0027]

[0028] Functions: Klebsiella pneumoniae HNYJ-1 can effectively degrade some macromolecular substances in reconstituted tobacco concentrate; it can transform some tobacco-specific glycosides and other aroma precursors to form key aroma components of tobacco.

[0029] Example 2: Cultivation of Klebsiella pneumoniae HNYJ-1

[0030] A single colony of Klebsiella pneumoniae HNYJ-1 was inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200 r / min (optimal 150 r / min) for 6-36 h (optimal 12 h) to obtain the seed culture.

[0031] Example 3: Application in reconstituted tobacco concentrate

[0032] The seed liquor and the reconstituted tobacco concentrate are mixed at a volume ratio of 1:0.5% to 1:10% (optimal 1:2%) and allowed to stand for 1-24 hours (optimal 12 hours) at any temperature between 40℃ and 50℃, with slow stirring every 0.5 hours to ensure complete reaction. After the reaction is complete, the flavor-enhancing concentrate is obtained.

[0033] Application Example 1

[0034] The TS-005 (light aroma) reconstituted tobacco concentrate from Henan Tobacco Industry Co., Ltd. was selected as the test sample. The untreated reconstituted tobacco concentrate was designated as CK, and the reconstituted tobacco concentrate with added Klebsiella pneumoniae HNYJ-1 was designated as T, with the addition amount being 2% of the concentrate volume ratio. Fermentation was carried out at 40℃ for 12 hours.

[0035] The content of macromolecular substances in the concentrate was detected by flow analysis. Simultaneous distillation extraction was performed using dichloromethane as solvent. The aroma components of the reconstituted tobacco concentrate were detected by GC-MS. The changes in macromolecular substances are shown in Table 2, and the results of detectable aroma substances are shown in Table 3.

[0036] Table 2. Changes in macromolecular substances and viscosity of reconstituted tobacco concentrate before and after enzyme treatment.

[0037]

[0038] Table 3. Changes in aroma components of reconstituted tobacco concentrate before and after enzyme treatment (μg / mL)

[0039]

[0040] As can be seen from the results in Tables 2 and 3, after treatment with Klebsiella pneumoniae HNYJ-1, the protein and pectin content in the concentrate decreased significantly, and the viscosity improved; the total amount of aroma components increased, with a significant increase in alcohols, aldehydes, ketones, esters and lactones, and the overall quality of the concentrate was improved.

[0041] 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 Klebsiella bacteria for improving the aroma of tobacco concentrate, characterized in that, The Klebsiella is Klebsiella sp. HNYJ-1, which is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 24327, and the 16S rDNA sequence of the Klebsiella is shown as SEQ ID NO.

1.

2. A method for preparing a flavor-enhanced tobacco concentrate using the Klebsiella of claim 1, characterized by, The method comprises the following steps: (1) a single colony of Klebsiella HNYJ-1 is inoculated into LB liquid medium by an inoculating loop, and is cultured in a shaker at a temperature of 25-40°C and a rotating speed of 100-200 r / min for 6-36 h to obtain a seed liquid; (2) the seed liquid obtained in step (1) is mixed with a reconstituted tobacco concentrate according to a volume ratio of 1:0.5%-1:10%, and is allowed to stand at a temperature of 40-50°C for 1-24 h, during which the mixture is slowly stirred once every 0.5 h to allow a full reaction, and after the reaction is completed, a flavored tobacco concentrate is obtained.

3. The method of claim 2, wherein, In step (1), the culture condition of the medium is 30°C, the rotating speed of the shaker is 150 r / min, and the culture time is 12 h.

4. The method of claim 2, wherein, In step (2), the ratio of the seed liquid to the reconstituted tobacco concentrate is 1:2%, and the standing time is 12 h.

5. The flavored tobacco concentrate prepared by the method according to any one of claims 2-4.

6. The flavored tobacco concentrate according to claim 5 in the application of tobacco processing.

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