Bacteria-enzyme synergistic microbial preparation as well as preparation method and application thereof

Abstract

The invention discloses a bacterium-enzyme synergistic microbial preparation and a preparation method and application thereof, and the bacterium-enzyme synergistic microbial preparation comprises a mixed bacterium suspension containing bacillus velezensis and bacillus altitudinis; the thallus concentration ratio of the bacillus velezensis to the bacillus altitudinis is (3: 1)-(1: 3); the mixed bacterial suspension also contains cellulase with the mass fraction of 0.5%-3%. After bacteria-enzyme synergistic fermentation treatment, the coordination of conventional chemical components of the tobacco leaves is improved. Moreover, after the bacteria-enzyme synergistic fermentation treatment, the volatile aroma components of the tobacco leaves are remarkably increased, and the content of the volatile aroma components such as megastigmatrienone, benzyl alcohol, phenethyl alcohol and damascenone is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of tobacco biotechnology, specifically to a microbial preparation with synergistic effects of bacteria and enzymes, its preparation method, and its application. Background Technology

[0002] Tobacco is an important economic crop in my country, playing a vital role in the country's economic development. The tobacco plant is divided into upper, middle, and lower leaves, which account for approximately 35% of the total yield. Upper leaves generally suffer from poor internal chemical composition, low sugar-alkali ratio, poor overall sensory quality, insufficient aroma, and noticeable irritation and off-flavors. Therefore, improving the industrial usability of upper leaves is a current research hotspot. Studies have found that microorganisms play a crucial role in the decomposition and transformation of macromolecular organic matter in tobacco leaves. Applying microbial agents before harvesting can improve tobacco quality and thus enhance its industrial applications.

[0003] During microbial fermentation, these microorganisms can effectively convert some large molecules in tobacco leaves into smaller aroma components, thereby improving tobacco quality. For example, by uniformly spraying a compound microbial preparation onto the surface of tobacco leaves, the chemical composition of the tobacco leaves becomes more harmonious after fermentation, the aroma components are significantly enhanced, and the quality of the tobacco leaves is noticeably improved. Alternatively, Aspergillus flavus and its application in the fermentation of unsuitable tobacco leaves can be used to prepare microbial preparations for fermenting tobacco leaves, which can increase the sweet aroma and mellowness of the tobacco leaves, improve the quality of unsuitable tobacco leaves, and increase the usability of the tobacco leaves. However, current technologies lack microbial agents specifically designed to improve the quality of upper leaves. Summary of the Invention

[0004] To address the lack of specific microbial agents for improving the quality of upper tobacco leaves in existing technologies, this invention proposes a synergistic microbial preparation of bacteria and enzymes, its preparation method, and its application. This method provides a reference for improving the quality of upper tobacco leaves and lays the foundation for further industrial application. The technical solution adopted in this invention is as follows: A microbial preparation with synergistic effects of bacteria and enzymes, comprising: A mixed bacterial suspension containing Bacillus belyssus and Bacillus hygroscopicus; the ratio of the bacterial concentrations of Bacillus belyssus and Bacillus hygroscopicus is 3:1 to 1:3; The mixed bacterial suspension also contains 0.5%-3% by mass of cellulase.

[0005] Optionally, the total bacterial concentration of *Bacillus belyssioides* and *Bacillus hygroscopicus* in the mixed bacterial suspension is 10. 7 ~10 9 cfu / mL.

[0006] Optionally, the ratio of the bacterial concentrations of *Bacillus belyssus* and *Bacillus hygroscopicus* is 1:2 to 2:1; Preferably, the ratio of the bacterial concentrations of Bacillus belyssus and Bacillus hygroscopicus is 1:1.

[0007] Optionally, the mixed bacterial suspension also contains 1.5% to 2.5% by mass of cellulase; The mixed bacterial suspension also contains 2% by mass of cellulase.

[0008] The preparation method of the microbial enzyme synergistic preparation includes the following steps: Step 1: Resuspend the cultured Bacillus belye and Bacillus hygroscopicus in water to obtain a mixed bacterial suspension; Step 2: Add cellulase to the mixed bacterial suspension.

[0009] Optionally, the cultured Bacillus belye and the cultured Bacillus glomeratus are obtained by culturing in a culture medium comprising 2-20 parts by weight of peptone, 2-20 parts by weight of yeast powder, 2-20 parts by weight of NaCl, and 500-2000 parts by weight of water; after dissolution, the mixture is autoclaved at 100-121°C for 10-50 minutes.

[0010] In step one, the obtained culture medium is centrifuged to remove the supernatant, and the wet bacterial cells are collected and resuspended in sterile deionized water to make the bacterial concentrations of the two strains consistent. They are then mixed in a certain proportion to obtain a mixed bacterial suspension.

[0011] This invention proposes the application of the above-mentioned microbial preparation with synergistic microbial activity in improving the quality of tobacco leaves.

[0012] This invention proposes a method for improving the quality of tobacco leaves, comprising the following steps: Spray the above-mentioned microbial preparation containing bacteria and enzymes onto the tobacco leaves to be initially cured; then ferment the tobacco leaves at a temperature of 30-35℃ for 24-48 hours; after fermentation, perform the initial curing of the tobacco leaves.

[0013] Optionally, the tobacco leaves are upper leaves, and the moisture content of the tobacco leaves is adjusted to 25-35% during the fermentation process; Preferably, the weight ratio of the tobacco leaves to be initially cured to the microbial preparation containing bacteria and enzymes is 20:1 to 5:1.

[0014] Optionally, the weight ratio of the tobacco leaves to be initially cured to the microbial preparation containing bacteria and enzymes is 10:1; the tobacco leaves to be initially cured are fermented at 32°C for 36 hours.

[0015] The present invention has the following beneficial effects: The co-fermentation treatment with bacteria and enzymes proposed in this invention improves the balance of conventional chemical components in tobacco leaves. Furthermore, the co-fermentation treatment significantly increases the content of volatile aroma components in tobacco leaves, particularly megastigmatrienone, benzyl alcohol, phenethyl alcohol, and damascene.

[0016] The microbial preparation for tobacco fermentation provided by this invention is simple to prepare, easy to store, and convenient to use without requiring special treatment. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0018] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] The experimental materials used in the examples were: mature Yunyan 87 upper leaves awaiting initial curing, provided by Guangxi China Tobacco Co., Ltd.

[0022] Experimental equipment and detection methods used in the embodiments: (1) Determination of conventional chemical components of tobacco leaves Nicotine, potassium, and chlorine in tobacco were determined using a SANPLUS 8505 continuous flow analyzer (SKALAR, Netherlands) according to the standards YCT160-2002 Determination of Total Alkaloids in Tobacco and Tobacco Products by Continuous Flow Method, YCT162-2011 Determination of Chlorine in Tobacco and Tobacco Products by Continuous Flow Method, and YCT 217-2007 Determination of Potassium in Tobacco and Tobacco Products by Continuous Flow Method. Total sugar and reducing sugar in tobacco were determined using the standard YC / T 159-2019 Determination of Water-Soluble Sugars in Tobacco and Tobacco Products by Continuous Flow Method.

[0023] (2) Determination of volatile aroma components in tobacco leaves The volatile aroma components of tobacco leaves were determined using an Agilent 6890A-5975C GC-MS system (Agilent Instruments, Inc., USA). Chromatographic conditions: column: HP-5MS (60 m * 0.25 mm * 0.25 μm); carrier gas: high-purity helium; injection volume: 1 uL; injection port temperature: 230℃; flow rate: 1.0 mL / min; split ratio: 10:1; solvent delay: 10 min; temperature program: initial temperature 50℃, increased to 280℃ at 4℃ / min, held for 10 min.

[0024] Mass spectrometry conditions / interface temperature: 270℃; ion source temperature: 230℃; quadrupole temperature: 150℃; ionization mode: E1; electron energy: 70eV; mass scan range: 35~550m / z.

[0025] (3) Sensory quality evaluation of tobacco leaves The tobacco leaf samples from each treatment group were scored using the "Sensory Evaluation Method for Tobacco and Tobacco Products" (YC / T138-1998) based on six indicators: gloss, aroma, harmony, irritation, off-flavors, and aftertaste.

[0026] Example 1: Synergistic Biological Agent of Bacteria and Enzymes Bacillus belye with accession number CCTCC AB 2018290 and Bacillus glaciformis with accession number CCTCC AB 2018316 were purchased from the China Center for Type Culture Collection (CCTCC AB 2018290 was deposited on September 25, 2018, and CCTCC AB 2018316 was deposited on October 12, 2018). Cellulase was purchased from Shanghai Yuanye Technology Co., Ltd.

[0027] Two loops of each strain were inoculated from solid culture medium into 100 mL LB liquid medium (10 g peptone, 10 g yeast extract, 10 g NaCl, 1000 mL water, dissolved and autoclaved at 110 °C for 40 min). The medium was then cultured on a shaker at 32 °C for 36 h. The resulting seed culture was centrifuged at 6000 rpm for 7 min, the supernatant was discarded, and the wet cells were resuspended in sterile deionized water. The dilution factor was determined using a spectrophotometer, and the cell concentration was determined using the OD600 value. The cell concentration was stabilized at 10⁻⁶. 8 The cfu / mL concentration was used as a bacterial suspension for later use. Bacillus belye and Bacillus glaberiae bacterial suspensions were prepared into mixed bacterial solutions at a 1:1 ratio. Then, cellulase was added to the bacterial suspension at a concentration of 2% to prepare a synergistic bacterial-enzyme biological agent.

[0028] Example 2 The concentration of Example 1 was 10. 8 Bacillus belye and Bacillus hygroscopicus suspensions at CFU / mL were prepared into mixed bacterial solutions at ratios of 3:1, 2:1, 1:1, 0:1, 1:0, 1:2, and 1:3, respectively. Cellulase at a mass ratio of 2.0% was added to prepare a synergistic biological agent. 30g of tobacco leaves were taken, and 3g of the microbial agent was evenly sprayed onto the surface of the tobacco leaves. The moisture content of the tobacco leaves was adjusted to 30% with sterile water. Separately, an equal amount of cellulase used to prepare the synergistic biological agent was dissolved in sterile water and evenly sprayed onto the surface of the tobacco leaves. The moisture content of the tobacco leaves was adjusted to 30% with sterile water. An equal amount of tobacco leaves sprayed with sterile water served as a blank control (CK). The experimental tobacco leaves were placed in sealed bags and fermented at 32℃ for 36 hours. Sensory evaluation was performed on both the original and fermented tobacco leaves after rolling. Among the tested samples, unfermented tobacco leaves exhibited low aroma content, poor aroma quality, high irritation, and an unpleasant aftertaste. When the ratio of *Bacillus vesalis* to *Bacillus albopictus* was 3:1 or 0:1, the tobacco leaves showed low aroma content, poor aroma quality, and a heavy off-flavor. Tobacco leaves with a ratio of 1:3 or 1:0, as well as those with added cellulase, showed high irritation and a strong spiciness. When the ratio of *Bacillus vesalis* to *Bacillus albopictus* was 1:2 to 2:1, the aroma content and quality of the tobacco leaves were significantly improved, while off-flavors and irritation were significantly reduced, and overall harmony was significantly enhanced. The 1:1 ratio of *Bacillus vesalis* to *Bacillus albopictus* showed the best results.

[0029] Example 3 The concentration of Example 1 was 10. 8A synergistic biological agent was prepared by adding cellulase at mass ratios of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0% to a 1:1 CFU / mL suspension of *Bacillus belye* and *Bacillus glaber*. 30g of tobacco leaves were used as a control, and 3g of the microbial agent was evenly sprayed onto the surface of the leaves. The moisture content of the tobacco leaves was then adjusted to 30% with sterile water. An equal volume of tobacco leaves sprayed with sterile water served as a blank control (CK). The inoculated tobacco leaves were placed in sealed bags and fermented at 32℃ for 48 hours. Sensory evaluation was performed on both the unprocessed and fermented tobacco leaves after rolling. Among the various types of tobacco, unfermented tobacco leaves exhibited lower aroma intensity, poor aroma quality, higher irritation, and an unpleasant aftertaste. When the enzyme content was less than 2.0%, the tobacco leaves had low aroma intensity, poor aroma quality, and a strong off-flavor. When the enzyme content was greater than 2.0%, the tobacco leaves were highly irritating and had a strong spiciness. When the enzyme content was between 1.5% and 2.5%, the aroma intensity and quality of the tobacco leaves were significantly improved, while the off-flavors and irritation were significantly reduced, and the overall harmony was significantly improved. Therefore, an enzyme content of 2.0% yielded the best results.

[0030] Example 4: Optimization of Biological Agent Spraying Dosage The concentration of Example 1 was 10. 8 A synergistic biological agent was prepared by adding 1.0% cellulase to a 1:1 CFU / mL suspension of *Bacillus belye* and *Bacillus glaber*. 30g of tobacco leaves were uniformly sprayed onto the surface of the leaves at tobacco-to-microbial agent ratios of 20:1, 15:1, 10:1, and 5:1. The moisture content of the tobacco leaves was then adjusted to 30% with sterile water. An equal volume of tobacco leaves sprayed with sterile water served as a blank control (CK). The inoculated tobacco leaves were placed in sealed bags and fermented at 32℃ for 36 hours. Sensory evaluation was performed on both the uninoculated and fermented tobacco leaves after rolling. Among them, unfermented tobacco leaves had a low aroma content, poor aroma quality, high irritation, and unpleasant aftertaste; when the mass ratio of microbial preparation to tobacco leaves was 20:1 and 15:1, the tobacco leaves had a low aroma content, poor aroma quality, and heavy off-flavors; when the mass ratio of microbial preparation to tobacco leaves was 5:1, the tobacco leaves were highly irritating, had a strong spiciness, and the smoke was accompanied by a strong burnt smell; when the mass ratio of microbial preparation to tobacco leaves was 10:1, the aroma content and quality of the tobacco leaves were significantly improved, the off-flavors and irritation were significantly reduced, and the overall harmony was significantly improved.

[0031] Example 5: Optimization of Fermentation Time for Biopharmaceuticals The concentration of Example 1 was 10. 8A synergistic biological agent was prepared by adding 2.0% cellulase to a 1:1 CFU / mL suspension of *Bacillus belyssima* and *Bacillus glaber*. 30g of tobacco leaves were uniformly sprayed onto the surface of the leaves at a tobacco-to-microbial agent mass ratio of 10:1. The moisture content of the tobacco leaves was then adjusted to 30% with sterile water. An equal volume of sterile water-sprayed tobacco leaves served as a blank control (CK). The inoculated tobacco leaves were placed in sealed bags and fermented at 32℃ for 12h, 24h, 36h, 48h, and 60h. Sensory evaluation was performed on both the unprocessed and fermented tobacco leaves after rolling. Among them, the blank control (CK) tobacco leaves had a lower aroma content, poorer aroma quality, higher irritation, and an unpleasant aftertaste. When the fermentation time was 12 hours, the tobacco leaves had less aroma content, poorer aroma quality, and more off-flavors. When the fermentation time was 60 hours, the tobacco leaves were highly irritating, had a strong spiciness, and the smoke was accompanied by a strong burnt smell. When the mass ratio of microbial preparation to tobacco leaves was 24-48 hours, the aroma content and quality of the tobacco leaves were significantly improved, the off-flavors and irritation were significantly reduced, and the overall harmony was significantly improved. Among them, the fermentation time of 36 hours had the best effect.

[0032] Example 6: The effect of optimal fermentation conditions on the aroma enhancement of tobacco leaves The concentration of Example 1 was 10. 8 A synergistic biological agent was prepared by adding 1.0% cellulase to a 1:1 CFU / mL suspension of *Bacillus belyssima* and *Bacillus glaber*. 30g of tobacco leaves were uniformly sprayed onto the surface of the leaves at a tobacco-to-microbial agent mass ratio of 10:1. The moisture content of the tobacco leaves was then adjusted to 30% with sterile water. An equal volume of tobacco leaves sprayed with sterile water served as the control group (CK). The inoculated tobacco leaves were then placed in sealed bags and fermented at 32℃ for 36 hours.

[0033] (1) Determination of conventional chemical components of tobacco leaves As shown in Table 1, compared with the control group, the fermented tobacco leaves showed significant changes in total potassium, nicotine, reducing sugar, total sugar, reducing sugar, sugar-to-alkali ratio, and potassium-to-chloride ratio (P < 0.05). Among these, total potassium, nicotine, total sugar, reducing sugar, and potassium-to-chloride ratio were significantly decreased, while the sugar-to-alkali ratio was significantly increased. Higher total sugar and reducing sugar content in flue-cured tobacco resulted in better aroma quality and quantity, lighter off-flavors, and improved tobacco leaf quality. Generally, a sugar-to-alkali ratio of 6-10 is considered suitable for flue-cured tobacco; this ratio is often used to evaluate the strength and comfort of the tobacco. The sugar-to-alkali ratio of the fermented tobacco leaves was significantly higher than that of the control group.

[0034] Table 1. Effects of synergistic effects of bacteria and enzymes on conventional chemical components of tobacco leaves.

[0035] (2) Detection of aroma components in tobacco leaves 25g of tobacco leaf samples from both the fermentation group and the control group were weighed, pulverized, and passed through a 40-mesh sieve. Simultaneously, they were distilled and extracted for 2.5 hours using CH2Cl2 as the extractant. After co-distillation, the extract was cooled to room temperature, and 50μL of 0.871mg / mL phenethyl acetate internal standard was added. Then, anhydrous sodium sulfate was added, and the mixture was allowed to stand overnight before being concentrated in a 50℃ water bath. The concentrated extract was filtered through an organic filter membrane and then transferred to a chromatographic bottle for GC / MS analysis. As shown in Table 2, the results indicated that the FJ treatment group showed significant increases in the contents of alcohols, esters, hydrocarbons, organic acids, furans, nitrogen-containing heterocycles, and phenols. Among the alcohols, the contents of phenylethanol and benzyl alcohol were significantly higher than those in the CK treatment group. Benzyl alcohol and phenylethanol are aromatic amino acid cleavage products that can enhance the floral aroma and flavor of the tobacco leaves. Among ketones, the contents of 4-cyclopenten-1,3-dione, damascene, β-ionone, and megastigmatrienone were significantly higher than those in the CK treatment group. β - Ionone, damascene, and megastigmatrienone are carotenoid degradation products. β - Ionone and damascene contribute floral aromas to cigarettes, while megastigmatrienone has a hay-like sweet aroma that softens the smoke. 4-Cyclopenten-1,3-dione and methylcyclopentenolone contribute sweet and roasted sweet aromas. Among aldehydes, only phenylacetaldehyde showed a slight increase in content, a metabolite of phenylalanine and lignin, contributing floral aromas and adding a rose-like fragrance to the tobacco. Esters in tobacco leaves contribute significantly to aroma components, typically exhibiting characteristic fruity aromas. The content of dihydroactinolone was significantly increased compared to the control group. Dihydroactinolone is an important carotenoid degradation product that plays a crucial role in eliminating smoke irritation, providing a mellow woody and fruity aroma. Among hydrocarbons, the content of neophydeadienes increased significantly. Neophydeadienes are 20-carbon isoprene-like aroma compounds and important aroma-producing substances in tobacco leaves.

[0036] Table 2. GC-MS analysis results of volatile aroma components of tobacco leaves before and after fermentation.

[0037] (3) Sensory quality evaluation results Table 3 Sensory evaluation results of tobacco leaves before and after fermentation

[0038] Sensory evaluations were conducted on the tobacco leaves from the fermentation group and the control group after rolling. As shown in Table 3, the tobacco leaves without microbial treatment had lower aroma content, poor aroma quality, higher irritation, and an unpleasant aftertaste; after treatment with microbial agents, the aroma content and quality were significantly improved, the off-flavors and irritation were significantly reduced, and the overall harmony was significantly improved.

[0039] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A microbial preparation with synergistic effects of bacteria and enzymes, characterized in that, include: A mixed bacterial suspension containing Bacillus belyssus and Bacillus hygroscopicus; the ratio of the bacterial concentrations of Bacillus belyssus and Bacillus hygroscopicus is 3:1 to 1:3; The mixed bacterial suspension also contains 0.5%-3% by mass of cellulase.

2. The microbial preparation with synergistic effects of bacteria and enzymes according to claim 1, characterized in that, The total bacterial concentration of *Bacillus belysinus* and *Bacillus hygroscopicus* in the mixed bacterial suspension was 10. 7 ~10 9 cfu / mL.

3. The microbial preparation with synergistic effects of bacteria and enzymes according to claim 1, characterized in that, The ratio of the bacterial concentrations of *Bacillus belyssus* and *Bacillus hygroscopicus* is 1:2 to 2:

1. Preferably, the ratio of the bacterial concentrations of Bacillus belyssus and Bacillus hygroscopicus is 1:

1.

4. The microbial preparation with synergistic effects of bacteria and enzymes according to claim 1, characterized in that, The mixed bacterial suspension contains 1.5% to 2.5% by mass of cellulase; The mixed bacterial suspension contains 2% by mass of cellulase.

5. The method for preparing the microbial preparation with synergistic effects of bacteria and enzymes according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Resuspend the cultured Bacillus belye and Bacillus hygroscopicus in water to obtain a mixed bacterial suspension; Step 2: Add cellulase to the mixed bacterial suspension.

6. The preparation method according to claim 5, characterized in that, The cultured Bacillus belye and the cultured Bacillus glomeratus were obtained by culturing in a culture medium comprising 2-20 parts by weight of peptone, 2-20 parts by weight of yeast powder, 2-20 parts by weight of NaCl, and 500-2000 parts by weight of water.

7. The application of the microbial preparation with synergistic microbial activity according to any one of claims 1 to 4 in improving the quality of tobacco leaves.

8. A method for improving the quality of tobacco leaves, characterized in that, Includes the following steps: Spray the tobacco leaves to be initially cured with the microbial preparation of any one of claims 1 to 4; then ferment the tobacco leaves to be initially cured at a temperature of 30 to 35°C for 24 to 48 hours; after fermentation, perform initial curing of the tobacco leaves.

9. The method for improving tobacco leaf quality according to claim 8, characterized in that, The tobacco leaves are upper leaves, and the moisture content of the tobacco leaves is adjusted to 25-35% during the fermentation process. Preferably, the weight ratio of the tobacco leaves to be initially cured to the microbial preparation containing bacteria and enzymes is 20:1 to 5:

1.

10. The method for improving tobacco leaf quality according to claim 8, characterized in that, The weight ratio of the tobacco leaves to be initially cured to the microbial preparation containing bacteria and enzymes is 10:1; the tobacco leaves to be initially cured are fermented at 32°C for 36 hours.

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