Bacillus subtilis JLYCSK-54 and application thereof

Abstract

The invention discloses bacillus subtilis JLYCSK-54 and application thereof, and aims to solve the technical problems that the content of starch in baked sun-cured tobacco is high, burnt smell and irritant harmful gas are generated during burning and smoking, the smoking quality and safety of cigarettes are affected, and tobacco leaves lack a microbiological method for improving the quality of the tobacco leaves. Various metabolic enzyme products produced by the bacillus subtilis JLYCSK-54 after enzyme activity regulation and control can reduce the content of starch in the tobacco leaves and comprehensively improve the quality of the tobacco leaves (for example, the hay fragrance and the costustoot are improved, the nicotine content is reduced, and the content of neutral aroma substances is increased); the process flow for treating the tobacco leaves by using the metabolic enzyme product of the bacillus subtilis JLYCSK-54 is simple and convenient, the treatment period is relatively short, and the tobacco leaf treatment cost is relatively low.

Description

Technical Field

[0001] This application relates to the technical field of biological fermentation, and particularly relates to a Bacillus subtilis JLYCSK-54 and its application. Background Art

[0002] Some flue-cured tobacco leaves in Northeast China have problems such as insufficient aroma quantity, low smoke concentration, and relatively bland smoking quality, which affect their usability in cigarette formulations. Adding an appropriate amount of sun-cured tobacco to flue-cured cigarettes can effectively reduce tar, increase smoke concentration, and improve aroma quantity. Its internal chemical composition and smoking style will change greatly, mainly manifested in rich aroma, large smoke concentration, sufficient aroma quantity, higher irritation and strength, and having a blended style of flue-cured tobacco, oriental tobacco, cigar tobacco, and sun-cured tobacco. It is a new type of tobacco leaf raw material with unique characteristics and strong plasticity.

[0003] However, the style characteristics of this raw material are too typical, and there are still some obstacles in its application in traditional flue-cured cigarette formulations, mainly manifested in the relatively high starch content, which produces burnt and pungent harmful gases during combustion, affecting the smoking quality and safety of cigarettes. Therefore, choosing to use a suitable method to degrade the starch in sun-cured tobacco and convert it into substances such as reducing sugars is one of the effective ways to improve its compatibility characteristics and style quality with flue-cured tobacco.

[0004] Microbial fermentation methods have advantages such as simple operation, short fermentation cycle, and low investment in aspects such as material transformation, macromolecule degradation, and quality style improvement of tobacco leaves. Currently, it has become a research hotspot for tobacco science and technology workers. However, there are still problems such as low enzyme activity of strains and unstable effects in the application of using microorganisms to improve tobacco leaf quality, and there is relatively little research on the changes in volatile components of tobacco leaves due to enzyme activity regulation. Based on this, screening specific microorganisms for application in tobacco leaf fermentation has very important practical significance for effectively degrading macromolecular substances in tobacco leaves, rapidly improving tobacco leaf quality, expanding the use range of raw materials for new product research and development in the industry, and improving the utilization rate of tobacco raw materials. Summary of the Invention

[0005] The inventors found through research that: the Bacillus subtilis JLYCSK-54 strain isolated and screened from flue-cured tobacco in the early stage of the laboratory, its metabolic enzyme products can reduce the starch content in tobacco leaves and comprehensively improve the quality of sun-cured tobacco (such as enhancing hay aroma, reducing bad odors such as burnt and earthy odors, and improving permeability, etc.).

[0006] According to one aspect of the present application, a Bacillus subtilis JLYCSK-54 was screened and obtained. Its taxonomic name is Bacillus subtilis, and it was deposited at the China Center for Type Culture Collection (Address: Inside Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postcode: 430072) on February 28, 2025, with the deposit number CCTCC NO: M 2025335.

[0007] According to another aspect of the present application, there is provided a microbial preparation containing the Bacillus subtilis JLYCSK-54 and / or its metabolic enzyme products.

[0008] According to still another aspect of the present application, the application of the Bacillus subtilis JLYCSK-54 or the microbial preparation in any one of the following (1) to (7):

[0009] (1) Improving the quality of tobacco leaves or preparing a preparation for improving the quality of tobacco leaves;

[0010] (2) Degrading the starch in tobacco leaves or preparing a preparation for degrading the starch in tobacco leaves;

[0011] (3) Increasing the total sugar content and / or the reducing sugar content of tobacco leaves;

[0012] (4) Increasing the content of neutral aroma components in tobacco leaves, where the neutral aroma components include at least one of megastigmatrienone, geranyl acetone, β-ionone, and solanesol;

[0013] (5) Preparing at least one of amylase, pectinase, xylanase, and cellulase;

[0014] (6) Reducing at least one of the total nitrogen and protein in tobacco leaves;

[0015] (7) Increasing at least one of the hay aroma, woody aroma, aroma volatilization, and soft and delicate degree of tobacco leaves.

[0016] In some embodiments, the tobacco leaves are flue-cured and sun-cured tobacco.

[0017] According to still another aspect of the present application, the preparation method of the microbial preparation includes the following steps:

[0018] (1) Activating the strain: Inoculating the Bacillus subtilis JLYCSK-54 on an NA solid medium and culturing it at 28 - 32 °C for 24 h - 72 h;

[0019] (2) Preparing the seed liquid: Scraping the cultured bacterial cells in step (1) and inoculating them into an NA liquid medium, and culturing them on a shaker at 28 - 32 °C for 14 - 16 h;

[0020] (3) Subculture: Inoculate the seed liquid obtained in step (2) into the subculture medium at an inoculation amount of 2% - 4%, and culture it on a shaker at a rotation speed of 180 - 220 rpm and a temperature of 28 - 32°C for 24 - 48 h;

[0021] (4) Preparation of crude enzyme preparation: Centrifuge the fermentation broth obtained in step (3), and take the supernatant to obtain it.

[0022] In some embodiments, the composition of the NA solid medium is as follows: calculated in g / L, beef extract 3.0, peptone 10.0, NaCl 5.0, agar 20, sterilized at 121°C.

[0023] In some embodiments, the composition of the NA liquid medium is as follows: calculated in g / L, beef extract 3.0, peptone 10.0, NaCl 5.0, sterilized at 121°C.

[0024] In some embodiments, the composition of the subculture medium is as follows: calculated in g / L, wheat bran 3.50, soybean meal 20.41, KCl 4.64, sterilized at 121°C.

[0025] In some embodiments, in step (3), the inoculation amount is 2%, the shaker rotation speed is 200 rpm, and the culture time is 36 h.

[0026] According to another aspect of the present application, there is provided a method for treating tobacco leaves, including the following steps:

[0027] (1) Adjust the moisture content of the tobacco leaves to be treated to 18% - 22%;

[0028] (2) According to 20% - 30% of the mass ratio of the tobacco leaves, evenly spray the metabolic enzyme product of Bacillus subtilis JLYCSK - 54 described in claim 1 or the microbial preparation described in claim 2 on the surface of the tobacco leaves, and then perform fermentation treatment under the conditions of a temperature of 40 - 50°C and a humidity of 75% - 85%;

[0029] (3) After the fermentation is completed, perform inactivation treatment at 80°C for 10 - 15 min to obtain the finished product.

[0030] One or more technical solutions provided in the embodiments of the present application have at least any one of the following technical effects or advantages:

[0031] 1. The screened Bacillus subtilis JLYCSK - 54, whose metabolic products can effectively reduce the starch in tobacco leaves, improve the smoking quality or taste of tobacco leaves, such as increasing the hay aroma and wood aroma, the aroma is more intense, the softness and fineness are improved, the miscellaneous odor is reduced, and the aftertaste is improved.

[0032] 2. The enzyme activity fingerprint of Bacillus subtilis JLYCSK-54 was detected. Its metabolites mainly contain amylase, neutral protease, alkaline protease, cellulase, xylanase and pectinase. Under the synergistic action of various enzymes, the quality of tobacco leaves was effectively improved.

[0033] 3. A relatively simple technical scheme for treating tobacco leaves with enzyme agents was designed. Its technological process is relatively simple, the cost of treating tobacco leaves is relatively low, and the treatment cycle is relatively short. It has technical advantages such as significantly reducing the starch content in tobacco leaves and improving the quality of tobacco leaves. Therefore, it has good popularization and application value in the field of tobacco leaf fermentation technology. Description of the Drawings

[0034] Figure 1 This is the phylogenetic tree constructed based on the 16S rDNA gene sequence of strain JLYCSK-54 in the embodiment of this application.

[0035] Figure 2 This is the effect of different culture medium components on the amylase activity of strain JLYCSK-54 in the embodiment of this application.

[0036] Figure 3 This is the effect of the interaction of different culture medium components on the amylase activity of strain JLYCSK-54 in the embodiment of this application.

[0037] Figure 4 This is the effect of different culture conditions on the enzyme activity and growth of strain JLYCSK-54 in the embodiment of this application.

[0038] Figure 5 This is the enzyme activity fingerprint of the crude enzyme solution of strain JLYCSK-54 in the embodiment of this application.

[0039] Figure 6 This is the effect of the crude enzyme solution of strain JLYCSK-54 on the volatile substances of tobacco leaves in the embodiment of this application. Detailed Embodiments

[0040] In the following embodiments, the instrument and equipment involved are all conventional instrument and equipment unless otherwise specified; the reagents and culture media involved are all commercially available conventional reagents and culture media unless otherwise specified; the test and detection methods involved are all conventional methods unless otherwise specified.

[0041] In order to better understand the technical solution of this application, the above technical solution will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0042] Example 1. Obtaining and Identification of Strains

[0043] The strain was isolated and screened from the B2F primary flue-cured tobacco leaves in Xiang County, Xuchang, Henan by Fu Bo et al. in 2024 at the Tobacco Quality Improvement and Aroma Enhancement Laboratory of the Tobacco College, Henan Agricultural University.

[0044] Using the 16S rDNA gene fragment as a universal primer, PCR and clone sequencing were performed on this strain, and a 1082bp sequence was obtained. Phylogenetic analysis showed that the strain JLYCSK-54 had a homology of over 99% with Bacillus subtilis, that is, it had the closest genetic relationship with Bacillus subtilis (as Figure 1 shown).

[0045] Example 2: Smoking evaluation screening and identification of Bacillus subtilis JLYCSK-54 strain

[0046] The screening method was as follows: Various strains in the laboratory were used to ferment cut tobacco according to the treatment method in Example 7. After fermentation, the cut tobacco was placed in a thermostatic and humidified box to balance the moisture content at (22±1)°C and relative humidity (60±2)% so that the moisture content of the cut tobacco was 12.5%±0.5% to meet the rolling requirements. The cut tobacco with qualified moisture content was fully mixed evenly, rolled on the same cigarette machine using the same cigarette materials, and the rolling weight and other indicators were controlled. The rolled cigarette samples were balanced for moisture content according to the method in GB / T16447-2004. A smoking evaluation group was composed of 7-9 smoking evaluation experts from the Technology R & D Center of Jilin Tobacco Industry Co., Ltd., and a team leader was set. The sensory deviation reference method was used for sensory evaluation. As shown in Table 1, compared with the sterile water treatment (CK), the treatment with the JLYCSK-54 strain increased the hay aroma and wood aroma, the aroma was more prominent, the softness and fineness were improved, the miscellaneous odor was reduced, and the aftertaste was improved.

[0047] Table 1 Smoking quality evaluation results of JLYCSK-54

[0048]

[0049] Example 3: Preparation of enzyme solution of Bacillus subtilis JLYCSK-54 strain

[0050] First, the JLYCSK-54 strain was prepared into an enzyme solution for easy use:

[0051] (1) Activation of the strain: The JLYCSK-54 strain was placed on an NA plate (calculated in g / L, containing 3.0 of beef extract, 10.0 of peptone, 5.0 of NaCl, 20g of agar, natural pH, sterilized at 121°C for 20 min), and cultured in a constant temperature incubator at 30°C for 1-2 days;

[0052] (2) Preparation of seed liquid: Scrape the bacteria cultured in step (1) in a laminar flow hood, and inoculate them into a conical flask containing 20 mL of liquid NA medium (calculated in g / L, containing 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of NaCl, pH 6.5 - 7.0, sterilized at 121 °C for 20 min). Culture at 30 °C and 180 rpm for 15 h;

[0053] (3) Subculture: Transfer the seed liquid in step (2) to a sterilized 30 mL conical flask containing 20 mL of liquid NA medium (calculated in g / L, containing 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of NaCl, natural pH, sterilized at 121 °C for 20 min) at a ratio of 0.4 mL of bacterial suspension / 20 mL of NA medium (2% volume ratio). Culture at 30 °C and 180 rpm for 24 h;

[0054] (4) Preparation of microbial agent: Centrifuge the bacterial liquid in step (3) at 8000 rpm for 10 min, and take the supernatant, which is the enzyme solution used.

[0055] (5) Determine the amylase in the metabolites of Bacillus subtilis strain JLYCSK - 54. Using the DNS method, use maltose as the standard solution, and use starch solution as the substrate. After reacting precisely in a 50 °C water bath for 30 min, add DNS and react in a boiling water bath for 10 min for color development, and then measure its absorbance at 540 nm. Calculate its enzyme activity according to the formula.

[0056] Example 4. Optimization of the enzyme - producing medium of Bacillus subtilis strain JLYCSK - 54

[0057] Optimize the amylase - producing medium of the strain:

[0058] Carbon source, nitrogen source, inorganic salt: On the basis of single - factor experiments (as Figure 2 shown), using carbon source, nitrogen source, and inorganic salt as influencing factors respectively, a 3 - factor 3 - level response surface optimization was carried out. The specific design is shown in Table 2.

[0059] Table 2 Response surface optimization method

[0060]

[0061] Performing regression analysis, the multiple regression equation of each factor on the enzyme activity of the strain can be obtained as:

[0062] Enzyme activity = 57.19 + 2.18A + 0.9112B - 0.6816C - 1.62AB - 0.6013AC - 0.0688BC - 4.31A² - 3.16B² - 2.35C².

[0063] The response surface optimization design and results are shown in Table 3

[0064] Table 3 Response surface optimization design and results

[0065]

[0066]

[0067] Through response surface analysis (as Figure 3 shown), the optimal medium formula for strain JLYCSK-54 was predicted to be wheat bran 3.50 g / L, soybean meal 20.41 g / L, and KCl 4.64 g / L, and the predicted amylase activity was 57.56 U / ml. After experiments, the enzyme activity measured under this medium formula was 57.01 U / mL, which was basically similar to the predicted value, indicating that the model was relatively reliable. The optimal medium formula for enzyme production was determined.

[0068] Example 5: Optimization of enzyme production conditions for Bacillus subtilis strain JLYCSK-54

[0069] On the basis of the optimal medium for enzyme production, the culture conditions of the strain were further optimized:[[]]

[0070] Inoculum size, shaker speed, and culture time: On the basis of single-factor experiments (as Figure 4 shown), taking the inoculum size, shaker speed, and culture time as influencing factors respectively, a three-factor and three-level orthogonal experiment was carried out for optimization, and the specific design is shown in Table 4.

[0071] Table 4 Orthogonal experiment design

[0072]

[0073] The results of the orthogonal experiment are shown in Table 5.

[0074] Table 5 Results of orthogonal experiment

[0075]

[0076]

[0077] Through orthogonal experiment analysis, the shaker speed had the greatest influence on the amylase activity of strain JLYCSK-54, followed by the culture time and inoculum size. The optimal combination of enzyme production conditions was determined as D1 E2 F2 through the k value, that is, the inoculum size was 2%, the shaker speed was 200 rpm, and the culture time was 36 h. After three experiments for verification, the average enzyme activity was obtained as (72.32 ± 2.01) U / ml. The optimal culture conditions for enzyme production were determined.

[0078] Example 6: Determination of multiple enzyme activities and construction of enzyme activity fingerprints of the enzyme solution of strain JLYCSK-54

[0079] The strain JLYCSK-54 was cultured according to the optimal enzyme-producing medium formula and the optimal enzyme-producing culture conditions. The enzyme solution produced by it was measured for various enzyme activities, and an enzyme activity fingerprint was constructed:

[0080] For the determination of cellulase, pectinase, and xylanase, the DNS method was used. Glucose, galacturonic acid, and xylose were used as standard solutions respectively, and sodium carboxymethylcellulose solution, pectin solution, and xylan solution were used as substrates. After reacting precisely in a water bath at 50 °C for 30 min, DNS was added and then reacted in a boiling water bath for 10 min for color development. Then, the absorbance was measured at 540 nm, and the enzyme activity was calculated according to the formula.

[0081] For the determination of protease, the Folin-Ciocalteu method was used. Neutral protease and alkaline protease were determined respectively. A tyrosine solution was used to make a standard curve, casein was used as the substrate, and the absorbance value was measured at 680 nm, and the enzyme activity was calculated according to the formula.

[0082] For the construction of the enzyme activity fingerprint, see Figure 5 . After optimizing the enzyme-producing medium and enzyme-producing conditions, the amylase, pectinase, xylanase, and cellulase activities of the strain JLYCSK-54 increased, while the neutral protease and alkaline protease decreased.

[0083] Example 7. Application of the enzyme solution of strain JLYCSK-54 in the treatment of flue-cured sun-cured tobacco

[0084] The middle leaves of the second flue-cured sun-cured tobacco variety Piaohe in Jilin in 2023 were treated by the method of the enzyme produced in Example 5. The specific steps are as follows:

[0085] When treating the tobacco leaves, the moisture content of the tobacco leaves was adjusted to 20%; according to the tobacco leaf mass ratio of 25%, the optimized microbial agent (diluted 10 times with sterile water) was evenly sprayed on the surface of the tobacco leaves (T2) with a sprayer. Spraying an equal amount of sterile water (CK) and the microbial agent before optimization (T1) were used as controls respectively. Under the conditions of a temperature of 45 °C and a humidity of 80%, the fermentation treatment was carried out for 24 d. After the fermentation was completed, it was treated at 80 °C for 10 min to inactivate the bacteria and the protease they produced.

[0086] 1. Comparison of the conventional chemical component contents of flue-cured sun-cured tobacco

[0087] The determination of total water-soluble sugar, reducing sugar, total alkaloid, total nitrogen, protein, potassium, chlorine, and starch was carried out with reference to the tobacco industry standard (YC / T) of the People's Republic of China. The test results are shown in Table 6.

[0088] Table 6 Conventional chemical component contents of the tobacco leaves after treatment

[0089]

[0090] As can be seen from Table 6, compared with CK, the total sugar content in the T2 treatment increased by 16.45%, the reducing sugar content increased by 20.76%, and the starch degradation rate was 17.21%. Compared with the T1 treatment, the total sugar and reducing sugar contents in the T2 treatment increased, while the other chemical components decreased. This indicates that after the tobacco leaves were fermented with the enzyme solution of the optimized strain JLYCSK-54, the macromolecular substances such as starch in the tobacco leaves were effectively degraded to produce soluble sugars, which is helpful for improving the quality of tobacco leaves.

[0091] 2. Comparison of Differential Volatile Components in Sun-Cured Tobacco

[0092] Referring to the simultaneous distillation extraction-gas chromatography-mass spectrometry method, the differential volatile components of CK, T1, and T2 treatments were compared and analyzed. The test results are shown in Figure 6 . Compared with CK, the substances such as 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, solanesol, and linalool were more prominent in the T1 treatment. Among the volatile components that were more prominent in the T2 treatment, substances such as megastigmatrienone, geranyl acetone, β-ionone, and solanesol are all important neutral aroma components of tobacco leaves.

[0093] Although some preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0094] Based on the above embodiments of the present application, without explicit negation or conflict, the technical features of one embodiment can be beneficially combined with one or more other embodiments.

[0095] Although some specific embodiments of the present application have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration purposes and not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or some technical features can be equivalently replaced without departing from the scope and spirit of the present application. The scope of the present application is defined by the appended claims.

Claims

1. A Bacillus subtilis JLYCSK-54, with the preservation number of CCTCC NO: M2025335.

2. A microbial preparation, characterized in that, Containing the Bacillus subtilis JLYCSK-54 described in claim 1 and / or its metabolic enzyme products.

3. Use of the Bacillus subtilis JLYCSK-54 described in claim 1 or the microbial preparation described in claim 2 in any one of the following (1) to (7): (1) Improving the quality of tobacco leaves or preparing a preparation for improving the quality of tobacco leaves; (2) Degrading the starch in tobacco leaves or preparing a preparation for degrading the starch in tobacco leaves; (3) Increasing the total sugar content and / or the reducing sugar content of tobacco leaves; (4) Increasing the content of neutral aroma components in tobacco leaves, where the neutral aroma components include at least one of megastigmatrienone, geranylacetone, β-ionone, and solanesol; (5) Preparing at least one of amylase, pectinase, xylanase, and cellulase; (6) Reducing at least one of the total nitrogen and protein in tobacco leaves; (7) Improving at least one of the hay aroma, woody aroma, aroma volatilization, and soft and delicate degree of tobacco leaves.

4. The application according to claim 3, wherein The tobacco leaves are flue-cured and sun-cured tobacco.

5. Preparation method of the microbial preparation described in claim 2, including the following steps: (1) Activating the strain: Inoculating the Bacillus subtilis JLYCSK-54 described in claim 1 on an NA solid medium and culturing at 28 - 32 °C for 24 h - 72 h; (2) Preparing a seed solution: Scraping the cultured bacterial cells in step (1) and inoculating them into an NA liquid medium, and culturing on a shaker at 28 - 32 °C for 14 - 16 h; (3) Scale-up culture: Transferring the seed solution obtained in step (2) to an enlarged medium at an inoculation amount of 2% - 4%, with a shaker speed of 180 - 220 rpm, and culturing at 28 - 32 °C for 24 - 48 h; (4) Preparing a crude enzyme preparation: Centrifuging the fermentation broth obtained in step (3) and taking the supernatant.

6. The preparation method according to claim 5, characterized in that, The composition of the NA solid medium is: in g / L, beef extract 3.0, peptone 10.0, NaCl 5.0, agar 20, sterilized at 121 °C.

7. The preparation method according to claim 5, wherein The composition of the NA liquid medium is: in g / L, beef extract 3.0, peptone 10.0, NaCl 5.0, sterilized at 121 °C.

8. The preparation method according to claim 5, characterized in that, The composition of the enlarged medium is: in g / L, wheat bran 3.50, soybean meal 20.41, KCl 4.64, sterilized at 121 °C.

9. The preparation method according to claim 5, wherein In step (3), the inoculation amount is 2%, the shaker speed is 200 rpm, and the culture time is 36 h.

10. A method for treating tobacco leaves, including the following steps: (1) Adjusting the moisture content of the tobacco leaves to be treated to 18 - 22%; (2) Uniformly spraying the metabolic enzyme products of the Bacillus subtilis JLYCSK-54 described in claim 1 or the microbial preparation described in claim 2 on the surface of the tobacco leaves according to 20 - 30% of the mass ratio of the tobacco leaves, and then performing fermentation treatment under the conditions of a temperature of 40 - 50 °C and a humidity of 75 - 85%; (3) After the fermentation is completed, performing inactivation treatment at 80 °C for 10 - 15 min.

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