Preparation of a microbial intracellular glycoside hydrolase Eyj-2 and its application in tobacco leaves

Abstract

This invention discloses the preparation of a microbial intracellular glycoside hydrolase, Eyj-2, and its application in tobacco leaves. The microbial intracellular glycoside hydrolase is prepared by the following method: (1) Preparation of seed culture: Klebsiella pneumoniae HNYJ-1 single colony is inoculated and cultured to obtain seed culture; (2) Preparation of tobacco crude glycosides; (3) Microbial enzyme-producing fermentation: The seed culture is mixed with 10% concentrated reconstituted tobacco leaf extract and cultured to obtain enzyme-producing seeds; tobacco crude glycosides are added, and the mixture is shaken to obtain fermentation broth; (4) Preparation of intracellular enzyme glycosidase: The fermentation broth is centrifuged, the supernatant is removed, the bacterial precipitate is broken, centrifuged again, saturated ammonium sulfate solution is added to the supernatant, incubated overnight, the precipitate is collected by centrifugation, dialyzed, and the glycosidase is obtained, named Eyj-2, and stored at 4℃ for later use. The content of aroma substances in the treated tobacco leaves is significantly improved overall, and all of these substances have a positive effect on cigarette aroma, 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 preparation of a microbial intracellular glycoside hydrolase Eyj-2 and its application in tobacco leaves. Background Technology

[0002] Domestically produced tobacco leaves generally suffer from low levels of aroma compounds, resulting in insufficient aroma, a lack of prominent characteristic aromas, and particularly poor sweetness. Current methods involve adding flavorings and additives to improve this, but this cannot completely solve the problem for all tobacco leaves. It often results in unbalanced aromas, insufficient improvement in aroma, and less than ideal results, while also being costly. Tobacco leaves contain a certain amount of glycosides, mostly located within the leaves' cells. These substances themselves have limited impact on the smoking quality of tobacco, but the aroma components produced after degradation become the main aroma compounds, significantly improving the smoking quality and enhancing the sweetness of the smoke. Some glycosides undergo spontaneous conversion and degradation during tobacco curing and processing, but this is usually insufficient to meet the requirements of high-quality tobacco. After initial curing, the cell structure of the tobacco leaves is broken down, and some of the intracellular glycosides are released to the leaf surface. Selecting appropriate glycosidases can effectively degrade these glycosides, forming rich aroma components.

[0003] Microorganisms secrete numerous enzymes, divided into intracellular and extracellular enzymes. Extracellular enzymes are mostly macromolecular hydrolases such as starch and protein, while intracellular enzymes are mostly oxidoreductases and glycoside hydrolases. Extracting and purifying suitable glycoside hydrolases can effectively degrade glycosides in tobacco, producing key aroma components and significantly improving the smoking quality of tobacco leaves. Furthermore, bio-enzymatic treatment of tobacco leaves not only significantly improves quality but also has low cost, simple process, and is easy to promote. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation of microbial intracellular glycoside hydrolase Eyj-2 and its application in tobacco leaves. The intracellular glycoside hydrolase produced by Klebsiella pneumoniae HNYJ-1 catalyzes the regeneration of tobacco aroma precursors in tobacco leaf concentrate to achieve the purpose of enhancing aroma.

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

[0006] A method for preparing the microbial intracellular glycoside hydrolase Eyj-2 includes the following steps:

[0007] (1) Preparation of seed solution:

[0008] A single colony of Klebsiella sp. HNYJ-1, deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 24327, was inoculated into LB liquid medium using an inoculation loop and cultured in a shaker at 25-40℃ and 100-200 r / min for 6-36 h to obtain the seed culture.

[0009] (2) Preparation of tobacco crude glycosides:

[0010] (21) Grind the tobacco leaves into powder and pass them through a 200-mesh sieve;

[0011] (22) The tobacco powder and anhydrous ethanol were mixed in a mass ratio of 1:2 to 1:10, and sonicated for 2 hours. After sonication, the supernatant was filtered and concentrated under reduced pressure at 40°C until the ethanol evaporated to dryness. Deionized water of the same volume as the ethanol before concentration was added to obtain an aqueous suspension.

[0012] (23) Three rounds of extraction were carried out using petroleum ether, dichloromethane and n-butanol. Each round of extraction was repeated three times. The organic phase was separated and concentrated. The solvent was removed and deionized water of the same mass as the original tobacco powder was added to dissolve the organic phase to obtain tobacco crude glycosides.

[0013] (3) Microbial enzyme-producing fermentation:

[0014] The seed liquid and 10% concentrated reconstituted tobacco leaf concentrate were mixed at a volume ratio of 0.5%-10%:1 and cultured at 25-40℃ and 100-300r / min for 12-72h to obtain enzyme-producing seeds.

[0015] Add pre-sterilized tobacco crude glycosides under sterile conditions, at a rate of 0.1 mL crude glycosides per milliliter of seed liquid, and continue to place it at 25-40℃ and shake at 100-300 r / min for 12-72 h to obtain fermentation broth;

[0016] (4) Preparation of intracellular enzyme glycosidase:

[0017] The fermentation broth from step (3) was centrifuged at 10,000 r / min and 4℃ for 20 min to remove the supernatant. The cell pellet was disrupted 2-3 times at 1000 Pa using an ultra-high pressure cell disruptor, and then centrifuged at 10,000 r / min for 20 min at 4℃. Two volumes of saturated ammonium sulfate solution were added to the supernatant. After precipitation overnight, the pellet was collected by centrifugation at 10,000 rpm for 20 min. The pellet was dialyzed three times with 10 mm PBS (pH 7.3) buffer to obtain the glycosidase, named Eyj-2, which was stored at 4℃ for later use.

[0018] 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.

[0019] Preferably, in the above technical solution, in step (2), the mass ratio of tobacco dust to anhydrous ethanol is 1:5.

[0020] Preferably, in the above technical solution, in step (3),

[0021] The seed liquid was mixed with 10% concentrated reconstituted tobacco leaf concentrate at a volume ratio of 2%:1, and cultured at 30℃ and 220r / min for 24h to obtain enzyme-producing seeds.

[0022] Preferably, in the above technical solution, in step (3),

[0023] Add pre-sterilized tobacco crude glycosides under sterile conditions, at a rate of 0.1 mL crude glycosides per milliliter of seed liquid, and continue to place it at 30°C and shake at 220 r / min for 24 h to obtain fermentation broth.

[0024] A tobacco leaf for enhancing flavor and reducing viscosity, prepared using a flavor-enhancing and viscosity-reducing bacterial agent, is characterized by the following preparation method:

[0025] Tobacco leaves and glycosidase Eyj-2 are mixed at a mass ratio of 1:0.5% to 1:10%, and left to stand for 1-12 hours at any temperature between 30℃ and 45℃, turning the mixture every 0.5 hours to ensure full reaction, thus obtaining tobacco leaves for flavor enhancement.

[0026] Preferably, in the above technical solution, the mass ratio of tobacco leaves to glycosidase Eyj-2 is 1:1%, and the standing time is 6 hours.

[0027] The above-described technical solution of the present invention has the following beneficial effects:

[0028] The content of aroma substances in the tobacco leaves treated in this application is significantly improved overall. These substances all have a positive effect on the aroma of cigarettes and can effectively improve the smoking quality and sweetness of tobacco leaves. 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] A Klebsiella sp. HNYJ-1 strain has been 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 Preparation of tobacco crude glycosides

[0034] (1) Grind the tobacco leaves into powder and pass them through a 200-mesh sieve.

[0035] (2) Smoke dust: Anhydrous ethanol is mixed in a mass ratio of 1:2 to 1:10 (optimal 1:5), and sonicated for 2 hours. After sonication, the supernatant is filtered and concentrated under reduced pressure at 40°C until the ethanol evaporates to dryness. Deionized water of the same volume as the ethanol before concentration is added to obtain an aqueous suspension.

[0036] (3) Three rounds of extraction were carried out using organic substances such as petroleum ether, dichloromethane and n-butanol. Each round of extraction was repeated three times. The organic phase was separated and concentrated. The solvent was removed and deionized water of the same mass as the original tobacco powder was added to dissolve the organic phase to obtain tobacco crude glycosides.

[0037] Example 3 Microbial enzyme production fermentation

[0038] The seed liquid and 10% concentrated reconstituted tobacco leaf concentrate 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 enzyme-producing seeds.

[0039] Add pre-sterilized tobacco crude glycosides under aseptic conditions, at a rate of 0.1 mL of crude glycosides per mL of seed liquid. Continue to place it at 25-40℃ (optimal 30℃) and shake at 100-300 r / min (optimal 220 r / min) for 12-72 h (optimal 24 h) to obtain the fermentation broth.

[0040] Example 4: Preparation of intracellular enzyme glycosidase

[0041] The fermentation broth was centrifuged at 10,000 r / min and 4℃ for 20 min. The supernatant was removed, and the cell pellet was disrupted 2-3 times at 1000 Pa using an ultra-high pressure cell disruptor. Then, it was centrifuged at 10,000 r / min for 20 min at 4℃. Twice the volume of saturated ammonium sulfate solution was added to the supernatant, and the pellet was allowed to precipitate overnight. After centrifugation at 10,000 rpm for 20 min, the pellet was collected and dialyzed three times with 10 mm PBS (pH 7.3) buffer to obtain the glycosidase, named Eyj-2, which was stored at 4℃ for later use.

[0042] Example 5: Tobacco Leaf Processing

[0043] Tobacco leaves and glycosidase Eyj-2 were mixed at a mass ratio of 1:0.5% to 1:10% (optimal 1:1%) and allowed to stand for 1-12 hours (optimal 6 hours) at any temperature between 30℃ and 45℃, with the mixture being slowly turned every 0.5 hours to ensure complete reaction. After the reaction was complete, tobacco leaves with enhanced flavor were obtained.

[0044] Preferably, tobacco leaves are mixed with glycosidase Eyj-2, and then further mixed at a volume ratio of tobacco leaves to crude glycosides of 1:0.01% to 1:0.1%. The mixture is thoroughly combined and allowed to stand at any temperature between 30℃ and 45℃ for 1-12 hours (ideally 6 hours), turning the mixture every 0.5 hours to ensure complete reaction. After the reaction is complete, the enhanced tobacco leaves are obtained.

[0045] Application Example 1

[0046] The experimental tobacco leaves were selected from C3F grade tobacco leaves grown in Xiangxian County, Henan Province in 2020. TS-005 (light aroma) reconstituted tobacco concentrate from Henan Tobacco Industry Co., Ltd. was selected as the concentrate for enzyme fermentation. Untreated tobacco leaves were designated as CK, tobacco leaves with only extracellular enzyme Eyj-2 added as T1, tobacco leaves with only crude glycosides added as T2, and tobacco leaves with both extracellular enzyme Eyj-2 and crude glycosides added as T3, with a volume ratio of 1:1% for each. The mixture was allowed to stand at 40℃ for 6 hours, with the leaves turned over every 0.5 hours.

[0047] GC-MS analysis of aroma components revealed the following results, which are shown in Table 1. The results indicate a significant overall increase in aroma content in the treated T3 tobacco leaves. All of these substances positively contribute to cigarette aroma and effectively enhance the smoking quality of the tobacco.

[0048] It is worth noting that the improvement in the quality of treated tobacco leaves is related to both the catalysis of Eyj-2 and the addition of crude glycosides. Simply adding crude glycosides or glycosidases does not significantly increase the aroma compounds. The surface of tobacco leaves already contains a certain amount of tobacco glycosides, which is insufficient to meet the requirements, necessitating the addition of additional glycosides. However, simply adding glycosides cannot smoothly convert them into aroma compounds; they require catalysis by extracellular enzymes to achieve an overall improvement in quality.

[0049] Table 1. Changes in some aroma compounds of tobacco leaves before and after treatment (unit: ug / mL)

[0050]

[0051] 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 a microbial intracellular glycoside hydrolase Eyj-2, characterized by, The method comprises the following steps: (1) Preparation of seed liquid: A single colony of Klebsiella sp. HNYJ-1 preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 24327 is inoculated into LB liquid medium by using an inoculation loop, and then cultured at 25-40 °C in a shaker at a rotation speed of 100-200 r / min for 6-36 h to obtain a seed liquid; (2) Preparation of tobacco crude glycoside: (21) The tobacco leaves are ground into powder and passed through a 200-mesh sieve; (22) The tobacco powder is mixed with anhydrous ethanol at a mass ratio of 1:2-1:10, and then subjected to ultrasonic treatment for 2 h; after the ultrasonic treatment, the supernatant is obtained by filtration; the supernatant is concentrated at a temperature of 40 °C under reduced pressure until the ethanol is evaporated; an equal volume of deionized water to the ethanol before concentration is added to obtain a water suspension; (23) The water suspension is subjected to three rounds of extraction with petroleum ether, dichloromethane and n-butanol, respectively, and each round of extraction is repeated three times; the organic phase is separated and concentrated; the solvent is removed; an equal mass of deionized water to the original tobacco powder is added to dissolve the tobacco crude glycoside; (3) Microbial enzyme production fermentation: The seed liquid is mixed with a 10% concentration of reconstituted tobacco leaf concentrate at a volume ratio of 0.5%-10%:1; the mixture is first cultured at 25-40 °C at a rotation speed of 100-300 r / min for 12-72 h to obtain an enzyme-producing seed; The tobacco crude glycoside is added to the enzyme-producing seed in a sterile environment at an addition amount of 0.1 mL of crude glycoside per milliliter of seed; the mixture is further placed at 25-40 °C and subjected to continuous shaking at a rotation speed of 100-300 r / min for 12-72 h to obtain a fermentation liquid; (4) Preparation of intracellular enzyme glycosidase: The fermentation liquid obtained in step (3) is subjected to low-temperature centrifugation at 10,000 r / min and 4 °C for 20 min; the supernatant is removed; the bacterial cell precipitate is broken by using an ultrahigh-pressure cell disrupter at 1000 Pa for 2-3 times; then the precipitate is subjected to centrifugation at 10,000 r / min and 4 °C for 20 min; 2 volumes of saturated ammonium sulfate solution are added to the supernatant; the precipitate is collected after overnight precipitation and centrifugation at 10,000 rpm for 20 min; the precipitate is dialyzed three times in a 10 mm PBS buffer with a pH of 7.3 to obtain microbial intracellular glycoside hydrolase, which is named Eyj-2 and stored at 4 °C for standby use.

2. The method for preparing the microbial intracellular glycoside hydrolase Eyj-2 according to claim 1, characterized by, In step (1), the seed liquid is cultured at a temperature of 30 °C and a rotation speed of 150 r / min in a shaker for 12 h.

3. The method of claim 1, wherein the microorganism is a strain of Escherichia coli. In step (2), the mass ratio of the tobacco powder to anhydrous ethanol is 1:

5.

4. The method for preparing the microbial intracellular glycoside hydrolase Eyj-2 according to claim 1, characterized by, In step (3), the seed liquid is mixed with a 10% concentration of reconstituted tobacco leaf concentrate at a volume ratio of 2%:1; the mixture is first cultured at 30 °C at a rotation speed of 220 r / min for 24 h to obtain an enzyme-producing seed.

5. The method for preparing the microbial intracellular glycoside hydrolase Eyj-2 according to claim 1, characterized by, In step (3), the tobacco crude glycoside is added to the enzyme-producing seed in a sterile environment at an addition amount of 0.1 mL of crude glycoside per milliliter of seed; the mixture is further placed at 30 °C and subjected to continuous shaking at a rotation speed of 220 r / min for 24 h to obtain a fermentation liquid.

6. A flavoured tobacco leaf, characterised in that, The preparation method is as follows: The tobacco leaves and the microbial intracellular glycosidase Eyj-2 are mixed according to a mass ratio of 1:0.5%-1:10%, and are allowed to stand at a temperature of 30-45 DEG C for 1-12 h, during which the mixture is stirred every 0.5 h to allow a full reaction, thereby obtaining the flavored tobacco leaves; wherein the microbial intracellular glycosidase Eyj-2 is prepared according to the preparation method of any one of claims 1-5.

7. The aromatic tobacco leaf of claim 6, wherein, The mass ratio of the tobacco leaves to the glycosidase Eyj-2 is 1:1%, and the standing time is 6 h.

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