Application of Klebsiella extracellular enzyme Eyj-1 in reconstituted tobacco concentrate

Abstract

This invention discloses the application of Klebsiella extracellular enzyme Eyj-1 in reconstituted tobacco concentrate. The Klebsiella extracellular enzyme Eyj-1 is prepared by the following method: (1) Seed liquid preparation: Klebsiella is inoculated and cultured to obtain a seed liquid; (2) Microbial enzyme fermentation: The seed liquid is mixed with reconstituted tobacco concentrate and cultured to obtain a seed; tobacco dust is added and the mixture is shaken to obtain a fermentation broth; (3) Preparation of extracellular enzyme: The fermentation broth is centrifuged to remove the precipitate, yielding a crude extracellular enzyme; the crude extracellular enzyme is mixed with anhydrous ammonium sulfate, and the precipitate is removed to obtain supernatant A; A is mixed with anhydrous ammonium sulfate, and the precipitate is removed, leaving supernatant B; B is separated to obtain the extracellular enzyme, i.e., Eyj-1. The aroma-enhancing concentrate of this application shows a significant overall increase in aroma content, a significant decrease in protein and pectin content, and a decrease in viscosity; the content of alcohols, aldehydes, ketones, and phenols is increased, and the total aroma components are increased by 18.15%, effectively improving the smoking quality of tobacco leaves.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of Klebsiella extracellular enzyme Eyj-1 in reconstituted tobacco concentrate. Background Technology

[0002] Reconstituted tobacco using the papermaking method is a tobacco product made from tobacco materials (tobacco stems, tobacco dust, tobacco flakes, etc.) as the main raw materials. Through processes such as extraction, pulping, concentration, papermaking, coating, and drying, it plays a vital role in the development of cigarette brands and the stable improvement of cigarette quality.

[0003] Reconstituted tobacco concentrate is made by extracting tobacco leaves with water and then concentrating it, providing the main aroma source for reconstituted tobacco. However, reconstituted tobacco often has problems such as excessively high content of macromolecules and low aroma content, resulting in excessively high viscosity during coating and insufficient aroma in the reconstituted tobacco prepared after coating.

[0004] Some microorganisms produce enzymes that can effectively degrade macromolecules in concentrates and transform them into aroma components, making them an important means of improving concentrate quality. Microorganisms can secrete numerous enzymes, divided into intracellular and extracellular enzymes. Extracellular enzymes mainly include some macromolecular degrading enzymes and a small number of enzymes with aroma-enhancing functions, but also include some enzymes that have no beneficial effect on concentrate quality or even have adverse effects. Therefore, it is necessary to purify the enzymes produced by microorganisms that have a positive effect on concentrate quality, thereby improving concentrate quality more efficiently. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of Klebsiella extracellular enzyme Eyj-1 in reconstituted tobacco concentrate. Klebsiella HNYJ-1 produces extracellular enzymes, which are prepared by a simple separation and purification method and used to treat reconstituted tobacco concentrate to improve its quality.

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

[0007] A method for preparing the Klebsiella extracellular enzyme Eyj-1 includes the following steps:

[0008] (1) Preparation of seed solution:

[0009] A single colony of Klebsiella sp. HNYJ-1, deposited at the China General Microbiological Culture Collection Center (CGMCC No. 24327), was inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ and 100-200 rpm for 6-36 hours to obtain the seed culture.

[0010] (2) Microbial enzyme-producing fermentation:

[0011] The seed liquid was mixed with 10% concentrated reconstituted tobacco leaf concentrate at a volume ratio of 0.5%-10%:1, and cultured at 25-40℃ and 100-300r / min for 12-72h to obtain seeds;

[0012] Add pre-sterilized tobacco powder under sterile conditions, adding 0.1g of tobacco powder per milliliter of seed liquid, and continue to place it at 25-40℃ and shake at 100-300r / min for 12-72h to obtain fermentation liquid;

[0013] (3) Preparation of extracellular enzymes:

[0014] The fermentation broth was centrifuged at 4000 r / min and 4℃ for 20 min to remove the precipitated bacterial cells, yielding crude extracellular enzyme. The crude extracellular enzyme was mixed with anhydrous ammonium sulfate at a mass ratio of 1:0.2–0.4 at 4℃, and the precipitate was removed to obtain supernatant A.

[0015] Supernatant A is mixed with anhydrous ammonium sulfate at a mass ratio of 1:0.4 to 0.8 at 4°C. The precipitate is removed, and the supernatant is retained to obtain supernatant B.

[0016] Supernatant B was separated using a DEAE-FF anion exchange column with a equilibration buffer of 20 mmol / L Tris-HCl (pH 8.5). After elution began, the fraction that did not adhere to the column was collected and dialyzed to obtain the extracellular enzyme HNYJ-1, which was named Eyj-1.

[0017] Preferably, in the above technical solution, the culture conditions of the original seed liquid in step (1) are: 30°C and 150 r / min in a shaker for 12 h.

[0018] Preferably, in the above technical solution, in step (2), 0.1g of tobacco powder is added to each milliliter of seed liquid, and the liquid is placed at 30°C and shaken at 220r / min for 24 hours to obtain fermentation liquid.

[0019] Preferably, in the above technical solution, in step (3), the extracellular enzyme crude enzyme and anhydrous ammonium sulfate are mixed at a mass ratio of 1:0.25.

[0020] Preferably, in the above technical solution, in step (3), the supernatant A and anhydrous ammonium sulfate are mixed at a mass ratio of 1:0.5.

[0021] Application of Klebsiella extracellular enzyme Eyj-1 in reconstituted tobacco concentrate.

[0022] A flavoring concentrate was prepared using the Klebsiella extracellular enzyme Eyj-1. The preparation method is as follows:

[0023] The reconstituted tobacco leaf concentrate was mixed with the extracellular enzyme Eyj-1 at a mass ratio of 1:0.5% to 1:10%, and allowed to stand for 1-12 hours at any temperature between 40℃ and 50℃, with slow stirring every 0.5 hours during the period to allow it to react fully and obtain the aroma-enhancing concentrate.

[0024] Preferably, in the above technical solution, the concentrate and the extracellular enzyme Eyj-1 are mixed at a mass ratio of 1:1% and allowed to stand for 3 hours.

[0025] Preferably, in the above technical solution, the content of protein and pectin in the flavor-enhancing concentrate is significantly reduced, and the viscosity is decreased. The content of alcohols, aldehydes, ketones, and phenols is increased, thereby improving the smoking quality of the tobacco leaves.

[0026] The above-described technical solution of the present invention has the following beneficial effects:

[0027] The processed concentrate of this application shows a significant overall improvement in aroma content, a significant reduction in protein and pectin content, and a 16.19% decrease in viscosity. The content of alcohols, aldehydes, ketones, and phenols is increased, resulting in an 18.15% increase in total aroma components, effectively enhancing the smoking quality of tobacco leaves. Attached Figure Description

[0028] Figure 1 This is a chromatogram of a DEAE-FF anion exchange column. Detailed Implementation

[0029] 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.

[0030] Application of an extracellular enzyme of Klebsiella sp. HNYJ-1 in reconstituted tobacco concentrate. Klebsiella sp. HNYJ-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24327 on January 17, 2022.

[0031] Example 1: Seed Liquid Preparation

[0032] The preparation conditions for the extracellular enzyme are as follows: Klebsiella pneumoniae HNYJ-1 single colony is inoculated into LB liquid medium with an inoculation loop and cultured in a shaker at 25-40℃ (optimal 30℃) and 100-200r / min (optimal 150r / min) for 6-36h (optimal 12h) to obtain the seed culture.

[0033] Example 2 Microbial enzyme production fermentation

[0034] The seed culture and the 10% concentrated solution were mixed at a volume ratio of 0.5%-10%:1 (optimal 2%:1), and cultured at 25-40℃ (optimal 30℃) and 100-300r / min (optimal 220r / min) for 12-72h (optimal 24h) to obtain the seeds.

[0035] Add pre-sterilized tobacco powder under sterile conditions, at a rate of 0.1g of tobacco powder per milliliter of seed liquid, and continue to place it at 25-40℃ (optimal 30℃) and shake at 100-300r / min (optimal 220r / min) for 12-72h (optimal 24h) to obtain fermentation broth.

[0036] Example 3: Preparation of extracellular enzymes

[0037] The fermentation broth was centrifuged at 4000 r / min and 4℃ for 20 min to remove the precipitated bacterial cells, yielding crude extracellular enzyme. Crude extracellular enzyme and anhydrous ammonium sulfate were mixed at a mass ratio of 1:0.2–0.4 (optimal 1:0.25) at 4℃, and the precipitate was removed to obtain supernatant A. Supernatant A and anhydrous ammonium sulfate were mixed at a mass ratio of 1:0.4–0.8 (optimal 1:0.5) at 4℃, and the precipitate was removed, leaving supernatant B.

[0038] Supernatant B was separated using a DEAE-FF anion exchange column with a equilibration buffer of 20 mmol / L Tris-HCl (pH 8.5). After elution began, the fraction that did not adhere to the column was collected and dialyzed to obtain the extracellular enzyme HNYJ-1, which was named Eyj-1.

[0039] Example 5: Concentrate Treatment

[0040] Mix the concentrated solution with the extracellular enzyme Eyj-1 at a mass ratio of 1:0.5% to 1:10% (optimal 1:1%), and let it stand for 1-12 hours (optimal 3 hours) at any temperature between 40℃ and 50℃, stirring slowly every 0.5 hours to ensure complete reaction. After the reaction is complete, the flavor-enhancing concentrated solution is obtained.

[0041] Application Example 1

[0042] TS-005 (light aroma) reconstituted tobacco concentrate from Henan Tobacco Industry Co., Ltd. was selected as the test sample. Untreated reconstituted tobacco concentrate was designated CK, and reconstituted tobacco concentrate with added extracellular enzyme Eyj-1 was designated T, with an addition amount of 1% of the concentrate volume. The concentrate was treated at 40℃ for 3 hours. GC-MS was used to detect the content of macromolecules and aroma components in the concentrate. Changes in macromolecules are shown in Table 1, and the results of detectable aroma components are shown in Table 2. The results show that the overall aroma content of the treated T concentrate was significantly improved, while the content of protein and pectin decreased significantly, and the viscosity decreased by 16.19%. The content of alcohols, aldehydes, ketones, and phenols increased, and the total aroma components increased by 18.15%, effectively improving the smoking quality of the tobacco.

[0043] Table 1. Changes in macromolecular substances and viscosity of reconstituted tobacco concentrate before and after enzyme treatment.

[0044]

[0045] Table 2. Changes in aroma components of reconstituted tobacco concentrate before and after enzyme treatment (μg / mL)

[0046]

[0047] 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 for producing Klebsiella exoenzyme Eyj-1, characterized by, Comprise the following steps: (1) Preparation of seed liquid: The Klebsiella sp. HNYJ-1 single colony preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 24327 is inoculated into LB liquid medium with an inoculation loop, and cultured at 25-40℃ in a shaker at 100-200 r / min for 6-36 h to obtain a seed liquid; (2) Microbial enzyme production fermentation: The seed liquid is mixed with a 10% concentration of reconstituted tobacco concentrate at a volume ratio of 0.5%-10%:1, and first cultured at 25-40℃ at 100-300 r / min for 12-72 h to obtain a seed; In a sterile environment, add sterilized tobacco powder in advance, add 0.1 g of tobacco powder per milliliter of seed, and continue to place it at 25-40℃ at 100-300 r / min for 12-72 h to obtain a fermentation liquid; (3) Preparation of extracellular enzyme: The fermentation liquid is centrifuged at 4000 r / min at 4℃ for 20 min to remove the precipitated bacteria, and the extracellular enzyme crude enzyme is obtained, and the extracellular enzyme crude enzyme is mixed with anhydrous ammonium sulfate at a mass ratio of 1:0.2~0.4 in a 4℃ environment, the precipitate is removed, and the supernatant A is obtained; Supernatant A is mixed with anhydrous ammonium sulfate at a mass ratio of 1:0.4~0.8 in a 4℃ environment, the precipitate is removed, and the supernatant is obtained, and supernatant B is obtained; Supernatant B is separated by DEAE-FF anion exchange column, the equilibrium buffer is 20 mmol / L Tris-HCl with pH 8.5, and the penetrated components not attached to the column are collected after elution, and after dialysis, HNYJ-1 extracellular enzyme is obtained, named Eyj-1.

2. The process for the preparation of Klebsiella oxytoca extracellular enzyme Eyj-1 as claimed in claim 1 wherein, In step (1), the seed liquid is cultured at a temperature of 30℃ and a shaking speed of 150 r / min for 12 h.

3. The process for the preparation of Klebsiella extracellular enzyme Eyj-1 as claimed in claim 1 wherein, In step (2), 0.1 g of tobacco powder is added per milliliter of seed liquid, and the mixture is continuously placed at 30℃ and shaken at 220 r / min for 24 h to obtain a fermentation liquid.

4. The process for the preparation of Klebsiella extracellular enzyme Eyj-1 as claimed in claim 1 wherein, In step (3), the extracellular enzyme crude enzyme is mixed with anhydrous ammonium sulfate at a mass ratio of 1:0.

25.

5. The process for the preparation of Klebsiella extracellular enzyme Eyj-1 as claimed in claim 1 wherein, In step (3), supernatant A is mixed with anhydrous ammonium sulfate at a mass ratio of 1:0.

5.

6. The use of Klebsiella exoenzyme Eyj-1 prepared according to the method of any one of claims 1-5 in a reconstituted tobacco leaf concentrate, characterized in that, The application is to prepare a flavoring concentrate using Klebsiella extracellular enzyme Eyj-1, and the preparation method of the flavoring concentrate is as follows: Mix the reconstituted tobacco concentrate and the extracellular enzyme Eyj-1 at a mass ratio of 1:0.5%-1:10%, and place it at a temperature of 40℃-50℃ for 1-12 h, and stir it slowly every 0.5 h during the period to make it fully react, and obtain a flavoring concentrate.

7. Use according to claim 6, characterized in that, Mix the reconstituted tobacco concentrate and the extracellular enzyme Eyj-1 at a mass ratio of 1:1%, and place it for 3 h.

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