Bacillus for increasing ambergris content in cigars and application

Abstract

The invention belongs to the field of bioengineering, and discloses bacillus for increasing the content of ambergris in cigars and application. When the bacillus provided by the invention is used for fermenting the cigar tobacco leaves, rapid conversion of labdan compounds in the cigar tobacco leaves can be efficiently realized, and the content of ambrox in the cigar tobacco leaves is increased, so that the tobacco leaf quality is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to an engineered bacterial strain, specifically a Bacillus that increases the ambroxol content in cigar smoke and its application. Background Technology

[0002] Lipatanoid compounds in tobacco, mainly including salinomyol and cis-fiberol, are the main components of tobacco glandular secretions and are primarily found in cigars and flavored tobacco. During the curing process, they can degrade and transform into ambroxol. Ambroxol has a strong ambergris aroma, which harmonizes with the characteristic aroma of tobacco. In cigarettes, it can mask off-flavors, improve the smoking quality of tobacco, and enhance the aroma and taste of cigarettes.

[0003] During the aging process of cigar tobacco leaves, lypatanoids in the leaves degrade to produce ambroxol and other ambergris-like aroma compounds, thereby enhancing the flavor of the tobacco. However, the aging time for cigar tobacco leaves typically ranges from several months to several years, and the amount of ambroxol produced during this process is relatively small. Therefore, how to rapidly degrade lypatanoids in cigar tobacco leaves to produce ambroxol, thereby improving the quality of cigar tobacco leaves, has become a critical issue that urgently needs to be addressed in the current cigar manufacturing industry.

[0004] This invention provides a Bacillus strain that increases the ambroxol content in cigar tobacco and its application. When used in the fermentation of cigar tobacco leaves, it can greatly increase the rate at which lysine compounds in cigar tobacco leaves are converted into ambroxol, thereby increasing the ambroxol content in cigar tobacco leaves and improving the quality of cigar tobacco leaves. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus strain that can increase the ambroxol content in cigars and its application. The aim is to achieve rapid and efficient conversion of ambroxol in cigars through bio-fermentation, thereby improving the quality of cigars and providing a new approach for the preparation of ambroxol.

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

[0007] A method for applying a Bacillus species that increases the ambroxol content in cigar smoke includes the following steps:

[0008] S1. Inoculate the preserved Bacillus ST-4 strain into LB liquid medium at an inoculation rate of 1%, and culture at a temperature of 28-35℃ and a rotation speed of 120-170 rpm for 24-48 h to obtain the bacterial culture.

[0009] S2. Apply the bacterial solution to the surface of the cigar tobacco leaves at a ratio of 5-8% by weight of the tobacco leaves, adjust the moisture content of the tobacco leaves to 25%-30%, and ferment for 90 days at a temperature of 35℃ and a humidity of 70%.

[0010] Furthermore, Bacillus sp. ST-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30674 and deposit date of May 17, 2024.

[0011] Furthermore, the formulation of LB liquid culture medium is as follows: tryptone: yeast extract: NaCl: distilled water in a mass ratio of 2:1:2:200, with a pH of 7.0.

[0012] Furthermore, it also includes the preparation of neutral component samples, comprising the following steps:

[0013] Take 20 g of tobacco leaves fermented with Bacillus ST-4 and 20 g of unfermented tobacco leaves respectively, remove the midrib, dry at 75-80℃ for 2-3 h, pulverize and pass through a 10-mesh sieve, weigh 10 g of tobacco powder, and extract with 15-20 times the mass of saturated saline and 6-8 times the mass of dichloromethane as solvents using simultaneous distillation extraction technology for 2-3 h to obtain the extract. The organic phase is heated in a water bath at 55-60℃, and the aqueous phase is heated in an oil bath at 160-180℃. The obtained extract is first washed with a 5-8% hydrochloric acid aqueous solution 3-5 times, and the hydrochloric acid washings are combined and then back-extracted with 20 mL of dichloromethane 3-5 times. The organic phases are combined, and then washed with a 5-8% sodium hydroxide aqueous solution 3-5 times. The sodium hydroxide washings are combined and then back-extracted with 20 mL of dichloromethane 3-5 times. The organic phases are combined, anhydrous sodium sulfate is added and dried overnight, and then concentrated to 1 mL to obtain the neutral component concentrate.

[0014] Furthermore, the method also includes the detection of ambroxol content. The neutral component concentrate is separated by semi-preparative high performance liquid chromatography, and each fraction is concentrated and analyzed by GC-MS to obtain the fraction containing ambroxol. The content change is calculated using the internal standard method.

[0015] Furthermore, the semi-preparative high-performance liquid chromatography (HPLC) conditions were as follows: semi-preparative silica gel column, column temperature 20℃; UV-Vis detector, detection wavelength 254 nm; mobile phase A: n-hexane, B: isopropyl ether, C: isopropanol; gradient elution program: 0–10 min, 100% A, flow rate 2 mL / min; 10–12 min, 100% A, flow rate 2 mL / min–1 mL / min; 12–50 min, 100% A–(62% A + 38% B), flow rate 1 mL / min; 50–110 min, (62% A + 38% B), flow rate 1 mL / min; 110–120 min, (62% A + 38% B)–100% C, flow rate 1 mL / min; 120–122 min, 100% C, flow rate 1 mL / min–2 mL / min; 122–160 min, 100% C, flow rate 2 mL / min. mL / min.

[0016] Furthermore, sensory quality evaluation is also included. Cigar tobacco leaves fermented with Bacillus ST-4 and naturally fermented are rolled into single-material tobacco and equilibrated at 22°C and 60% humidity for 48 hours before sensory quality evaluation is conducted.

[0017] The beneficial effects of this invention are:

[0018] Fermentation of cigar tobacco leaves using the Bacillus provided in this invention can achieve rapid conversion of lysine compounds in cigar tobacco leaves, increase the content of ambroxol in cigar tobacco leaves, and thus effectively improve the quality of tobacco leaves. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the specific implementation of the present invention.

[0020] Figure 2 This is a plate diagram of purified Bacillus ST-4.

[0021] Figure 3 This is the phylogenetic tree of Bacillus ST-4.

[0022] Figure 4 Total ion chromatogram of neutral fraction 3 after 90 days of natural fermentation of cigar tobacco leaves.

[0023] Figure 5 Total ion chromatogram of neutral fraction 3 after 90 days of fermentation with Bacillus ST-4 in cigar tobacco leaves. Detailed Implementation

[0024] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, and should not be construed as limiting the technical solution of the present invention.

[0025] Please see Figures 1 to 3 As shown, this application provides a Bacillus strain for increasing the ambroxol content in cigars and its application. The specific application method includes the following steps:

[0026] 1. Preparation of bacterial culture:

[0027] Bacterial LB liquid medium was prepared according to the mass ratio of tryptone: yeast extract: NaCl: distilled water of 2:1:2:200. The pH was adjusted to 7.0. The preserved Bacillus ST-4 strain was inoculated into the LB liquid medium at an inoculum of 1% and cultured at 32℃ and 150 rpm for 48 h to obtain the bacterial solution.

[0028] Bacillus sp. ST-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30674.

[0029] 2. Cigar tobacco fermentation:

[0030] The aforementioned bacterial solution was applied to the surface of cigar tobacco leaves at a ratio of 5% by weight of the tobacco leaves. The moisture content of the tobacco leaves was adjusted to 30%. Fermentation was carried out for 90 days at a temperature of 35°C and a humidity of 70%. A natural fermentation control group was also set up and fermented for 90 days.

[0031] 3. Preparation of neutral component samples:

[0032] 20 g of tobacco leaves fermented with Bacillus ST-4 and 20 g of unfermented tobacco leaves were taken separately. After removing the midrib, the leaves were dried at 75℃ for 2 h, pulverized, and passed through a 10-mesh sieve. 10 g of tobacco powder was weighed out and extracted for 2 h using 15 times the mass of saturated saline solution and 6 times the mass of dichloromethane as solvents. The organic phase was heated in a 55℃ water bath, and the aqueous phase was heated in a 160℃ oil bath. The resulting extract was washed 5 times with a 5% hydrochloric acid aqueous solution. The combined hydrochloric acid washes were then back-extracted 3 times with 20 mL of dichloromethane. The combined organic phases were washed 5 times with a 5% sodium hydroxide aqueous solution. The combined sodium hydroxide washes were then back-extracted 3 times with 20 mL of dichloromethane. The combined organic phases were dried overnight with anhydrous sodium sulfate and concentrated to 1 mL to obtain a neutral component concentrate.

[0033] 4. Ambroxol content detection:

[0034] 0.2 mL of the neutral component concentrate obtained in Example 3 was subjected to semi-preparative high-performance liquid chromatography (HPLC) separation, yielding 5 fractions. Each fraction was concentrated and analyzed by GC-MS. Ambroxol was found in fraction 3 (e.g., Figure 4 , Figure 5 As shown in the figure, the content of ambroxol was calculated using the internal standard method. After fermentation with Bacillus ST-4, the content of ambroxol in tobacco leaves increased from 9.6 ug / mL to 81.6 ug / mL, an increase of 8.5 times. This indicates that Bacillus ST-4 can rapidly and efficiently increase the content of ambroxol in cigar tobacco leaves.

[0035] Table 1. Ambroxol content in fraction 3

[0036]

[0037] Semi-preparative high-performance liquid chromatography (HPLC) conditions: semi-preparative silica gel column (10 mm × 250 mm, 10 μm), column temperature 20℃; UV-Vis detector, detection wavelength 254 nm; mobile phase A: n-hexane, B: isopropyl ether, C: isopropanol; gradient elution program (Table 1): 0–10 min, 100% A, flow rate 2 mL / min; 10–12 min, 100% A, flow rate 2 mL / min–1 mL / min; 12–50 min, 100% A–(62% A + 38% B), flow rate 1 mL / min; 50–110 min, (62% A + 38% B), flow rate 1 mL / min; 110–120 min, (62% A + 38% B)–100% C, flow rate 1 mL / min; 120–122 min, 100% C, flow rate 1 mL / min–2 mL / min; 122–160 min, 100% C, flow rate 2 mL / min.

[0038] GC-MS analysis conditions: Column: HP-5MS (30 m × 0.25 mm × 0.25 μm); Carrier gas: He; Flow rate: 1.0 mL / min; Injector temperature: 280℃; Injection volume: 1 μL; Split ratio: 2:1; Solvent delay: 8 min; Temperature program: Initial column temperature 50℃, hold for 4 min, increase to 70℃ at 3℃ / min, hold for 5 min, increase to 100℃ at 2℃ / min, hold for 10 min, increase to 140℃ at 2℃ / min, hold for 4 min, increase to 240℃ at 4℃ / min, hold for 5 min; Ion source: Electron impact (EI); Ion source temperature: 230℃; Quadrupole temperature: 150℃; Ionization energy: 70 eV; Scan mode: Full scan and Selected ion monitoring (SIM); Mass scan range: 35~550 amu.

[0039] 5. Sensory quality evaluation:

[0040] Cigar tobacco leaves fermented with Bacillus ST-4 and naturally fermented were rolled into single-origin tobacco and equilibrated at 22℃ and 60% humidity for 48 hours before sensory quality evaluation. The evaluation results are shown in Table 2. Compared with naturally fermented tobacco, the cigar tobacco leaves fermented with Bacillus ST-4 showed a significant improvement in woody aroma, harmonious smoke, better aroma quality, increased aroma intensity, reduced off-flavors, lower irritation, and a cleaner aftertaste, indicating a significant improvement in tobacco quality.

[0041] Table 2 Sensory quality evaluation results

[0042]

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Bacillus species for increasing the ambroxol content in cigar smoke, and its application method, characterized in that, Includes the following steps: S1. Inoculate the preserved Bacillus ST-4 strain into LB liquid medium at an inoculation rate of 1%, and culture at a temperature of 28-35℃ and a rotation speed of 120-170 rpm for 24-48 h to obtain the bacterial culture. S2. Apply the bacterial solution to the surface of the cigar tobacco leaves at a ratio of 5-8% by weight of the tobacco leaves, adjust the moisture content of the tobacco leaves to 25%-30%, and ferment for 90 days at a temperature of 35℃ and a humidity of 70%.

2. The method of applying the Bacillus subtilis for increasing the ambroxol content in cigars according to claim 1, characterized in that, Bacillus sp. ST-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 30674.

3. The method of applying the Bacillus subtilis for increasing the ambroxol content in cigars according to claim 1, characterized in that, The LB liquid medium formula is: tryptone: yeast extract: NaCl: distilled water in a mass ratio of 2:1:2:200, with a pH of 7.

0.

4. The method of applying the Bacillus subtilis for increasing the ambroxol content in cigars according to claim 1, characterized in that, It also includes the preparation of neutral component samples, comprising the following steps: Take 20 g of tobacco leaves fermented with Bacillus ST-4 and 20 g of unfermented tobacco leaves respectively, remove the midrib, dry at 75-80℃ for 2-3 h, pulverize and pass through a 10-mesh sieve, weigh 10 g of tobacco powder, and extract with 15-20 times the mass of saturated saline and 6-8 times the mass of dichloromethane as solvents using simultaneous distillation extraction technology for 2-3 h to obtain the extract. The organic phase is heated in a water bath at 55-60℃, and the aqueous phase is heated in an oil bath at 160-180℃. The obtained extract is first washed with a 5-8% hydrochloric acid aqueous solution 3-5 times, and the hydrochloric acid washings are combined and then back-extracted with 20 mL of dichloromethane 3-5 times. The organic phases are combined, and then washed with a 5-8% sodium hydroxide aqueous solution 3-5 times. The sodium hydroxide washings are combined and then back-extracted with 20 mL of dichloromethane 3-5 times. The organic phases are combined, anhydrous sodium sulfate is added and dried overnight, and then concentrated to 1 mL to obtain the neutral component concentrate.

5. The method of applying the Bacillus subtilis for increasing the ambroxol content in cigars according to claim 1, characterized in that, It also includes the detection of ambroxol content. The neutral component concentrate is separated by semi-preparative high performance liquid chromatography. Each fraction is concentrated and then analyzed by GC-MS to obtain the fraction containing ambroxol. The content change is calculated using the internal standard method.

6. The method of applying the Bacillus subtilis according to claim 5, which increases the ambroxol content in cigars, is characterized in that... The semi-preparative high-performance liquid chromatography (HPLC) conditions were as follows: semi-preparative silica gel column, column temperature 20℃; UV-Vis detector, detection wavelength 254 nm; mobile phase A: n-hexane, B: isopropyl ether, C: isopropanol; gradient elution program: 0–10 min, 100% A, flow rate 2 mL / min; 10–12 min, 100% A, flow rate 2 mL / min–1 mL / min; 12–50 min, 100% A–(62% A + 38% B), flow rate 1 mL / min; 50–110 min, (62% A + 38% B), flow rate 1 mL / min; 110–120 min, (62% A + 38% B)–100% C, flow rate 1 mL / min; 120–122 min, 100% C, flow rate 1 mL / min–2 mL / min; 122–160 min, 100% C, flow rate 2 mL / min. mL / min.

7. The method of applying the Bacillus subtilis for increasing the ambroxol content in cigars according to claim 1, characterized in that, It also includes sensory quality evaluation, in which cigar tobacco leaves fermented with Bacillus ST-4 and naturally fermented are rolled into single-material tobacco, and then equilibrated at 22°C and 60% humidity for 48 hours before sensory quality evaluation is conducted.

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