Bacillus safensis and application thereof

Abstract

The invention provides bacillus safensis and application of the bacillus safensis. The bacillus safensis disclosed by the invention is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.30643. The rhodiola rosea flavor for the cigarettes is prepared through fermentation of the strain, the content of effective active ingredients including gallic acid, salidroside, rhodiola crenulata, rhodioloside and tyrosol in rhodiola rosea is increased, the medicinal value of the active ingredients of the rhodiola rosea is greatly protected, and the health-care effect of the rhodiola rosea is guaranteed and improved; moreover, the contents of aromatic substances such as n-caprylic alcohol, linalool, phenethyl alcohol, citronellol and geraniol are increased. The cigarette flavoring sensory evaluation shows that the cigarette flavor is harmonious and unique in fragrance, has a strong effect on fragrance such as faint scent and green fragrance in smoke style characteristics, makes a certain contribution to flower fragrance, radix aucklandiae and sweet fragrance, and has the effects of improving the cigarette fragrance amount, softening and smoothing smoke and reducing throat irritation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a Bacillus safensis and application thereof. BACKGROUND

[0002] Tobacco flavor is an additive specially used for flavoring and taste-correcting of various tobacco products, which can produce good smoke effect and is one of the core technologies in the tobacco industry. Through recent years of research, certain achievements have been made in the technology of tobacco flavor in China, which has contributed to the formation of the aroma and taste characteristics of Chinese-style cigarettes. Tobacco flavor is generally divided into natural tobacco flavor and artificially synthesized tobacco flavor.

[0003] Natural tobacco flavor has the advantages of natural composition, coordinated aroma, rich and full flavor, environmental protection, etc., and has great development prospects. Natural tobacco flavor can release aroma and flavor after being added to cigarettes and burned, and plays a very important role in cigarette production.

[0004] As a natural medicinal plant, Rhodiola crenulata can enhance the diffusion of oxygen in cells in the body, or the efficiency of oxygen use, and the ability of antioxidant, which helps to improve the adaptability of the body to hypoxic stimulation. The prepared Rhodiola crenulata tobacco flavor contains various active ingredients such as salidroside, rhodiolin, rhodioloside, tyrosol, and gallic acid, and has significant biological functions. Among them, the core component salidroside has multiple effects such as anti-tumor, anti-virus, anti-fatigue, anti-oxidation, anti-hypoxia, anti-aging, anti-radiation, improvement of cardiovascular function, and enhancement of immunity. However, due to limited raw materials and high extraction cost, it cannot meet the demand of the tobacco flavor market.

[0005] Natural tobacco flavor obtained by microbial fermentation not only ensures the naturalness of the source of the flavor, but also can obtain new types of tobacco flavor that cannot be produced by traditional extraction process, increase product flavor, and lay a foundation for in-depth research and application of tobacco flavor. In the prior art, there is no Rhodiola crenulata tobacco flavor fermented by Bacillus safensis, and there is no report on its application in cigarettes.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The first object of the present application is to provide a Bacillus safensis and a microbial inoculant thereof for preparing Rhodiola crenulata flavor.

[0008] The second object of the present application is to provide a preparation method of Rhodiola crenulata flavor, in which Bacillus safensis is used as a fermentation strain to participate in the preparation of Rhodiola crenulata flavor, the reaction condition is mild, and the content of characteristic components of Rhodiola crenulata is effectively improved.

[0009] The third object of the present application is to provide a tobacco Rhodiola herb flavor, which has multiple effective components, unique and coordinated fragrance, and high fragrance quality, thereby providing an important way for the development of tobacco flavors.

[0010] The fourth object of the present application is to provide an application of the tobacco Rhodiola herb flavor in the production of tobacco products.

[0011] To achieve the above objects, the technical scheme adopted by the present application is as follows:

[0012] In the first aspect of the present application, a Bacillus safensis is provided, and the strain is named NS18.

[0013] The Bacillus safensis of the present application is an endophytic strain screened from fresh tobacco plant samples, and is named Bacillus safensis NS18.

[0014] Further, the Bacillus safensis NS18 of the present application is a gram-positive bacterium, and the size of the strain is 0.7-1.5 μm x 2.2-3.8 μm, which is rod-shaped and has flagella.

[0015] Specifically, the nucleotide sequence of the 16S rDNA of the Bacillus safensis NS18 is shown in SEQ ID NO: 1.

[0016] Further, the culture method of the Bacillus safensis is as follows: the Bacillus safensis NS18 is inoculated into LB liquid medium, and cultured at 30℃ and 200 rpm for 12 hours to obtain a seed liquid.

[0017] Further, the culture conditions of the Bacillus safensis NS18 are as follows: the temperature is 30℃, and the rotation speed is 200 rpm.

[0018] Further, the formula of the LB liquid medium is as follows: tryptone 10 g / L, yeast powder 10 g / L, and sodium chloride 5 g / L.

[0019] The LB solid medium formula is: tryptone 10 g / L, yeast powder 10 g / L, sodium chloride 5 g / L, agar 20 g / L.

[0020] In a second aspect of the present invention, a microbial agent is provided, wherein the microbial agent contains the Bacillus saffron.

[0021] Preferably, the microbial agent uses Bacillus sabdariffa as the main microorganism.

[0022] Preferably, the microbial agent is a solid agent or a liquid agent.

[0023] Preferably, the microbial agent contains living cells of the Bacillus subtilis strain, dry cells of the Bacillus subtilis strain obtained by freeze-drying, immobilized cells of the Bacillus subtilis strain, or the Bacillus subtilis strain in any other form.

[0024] Preferably, the microbial agent is obtained by expanding the seed liquid of Bacillus sabdariffa.

[0025] The third aspect of the present invention provides the use of the Bacillus saffron or the microbial agent in the preparation of Rhodiola rosea spice.

[0026] Specifically, the efficacy of the Bacillus sabdariffa includes at least one of the following:

[0027] (I) increasing the content of gallic acid, salidroside, salidroside, salidroside, and tyrosol in Rhodiola rosea;

[0028] (II) Increase the content of octanol, linalool, phenylethyl alcohol, citronellol and geraniol.

[0029] In a fourth aspect of the present invention, a method for preparing Rhodiola rosea spice is provided, comprising the following steps:

[0030] (1) Pretreatment: crushing the Rhodiola rosea medicinal material to obtain Rhodiola rosea powder; preferably, the temperature during crushing is not higher than 40° C., and the Rhodiola rosea is treated under low temperature conditions to ensure the activity of the effective ingredients in the Rhodiola rosea and improve the extraction efficiency;

[0031] (2) Ultrasonic extraction: The Rhodiola rosea powder prepared in step (1) is uniformly mixed with water and glucose, and subjected to ultrasonic treatment to obtain a Rhodiola rosea solution; the Rhodiola rosea is crushed in a low temperature environment to obtain a powder and subjected to ultrasonic treatment, so that the effective active ingredients in the Rhodiola rosea are quickly and effectively leached, which is helpful for subsequent extraction and improves the extraction efficiency;

[0032] (3) Solution inactivation: inactivating the Rhodiola rosea solution obtained in step (2);

[0033] (4) Preparation of Bacillus sabdariffa NS18 seed solution: Activate Bacillus sabdariffa or its microbial inoculum on an LB plate, pick a single colony and inoculate it into LB liquid medium to prepare a seed solution;

[0034] (5) microbial fermentation treatment: inoculating the Bacillus NS18 seed solution obtained in step (4) into the Rhodiola rosea solution inactivated in step (3), and obtaining a Rhodiola rosea microbial fermentation liquid after fermentation;

[0035] (6) Purification: Using ethanol as an extractant, the Rhodiola rosea microbial fermentation liquid in step (5) is subjected to a first alcohol extraction, filtration, and a second alcohol extraction to obtain a Rhodiola rosea fermentation extract;

[0036] (7) filtering and concentrating the Rhodiola rosea fermentation extract obtained in step (6) to obtain the Rhodiola rosea tobacco flavoring.

[0037] Preferably, in step (2), the mass ratio of Rhodiola rosea powder to water and glucose is 1:(10-50):(3-5), and during ultrasonic treatment, the ultrasonic equipment is set to a temperature of 45-55° C., a power of 400-500 kW, and an extraction time of 30-60 min.

[0038] Preferably, the inactivation treatment conditions in step (3) are: sterilization at 121° C. for 15-30 min, and cooling to room temperature.

[0039] Preferably, the specific steps of preparing the Bacillus sabdariffa seed solution in step (4) are: inoculating Bacillus sabdariffa NS18 into LB liquid culture medium, and culturing at 30° C. and 200 rpm for 12 hours to obtain the seed solution.

[0040] Preferably, the fermentation conditions in step (5) are a fermentation temperature of 25-35° C., a stirring speed of 100-300 r / min, a culture time of 2-7 days, and an inoculation amount of the Bacillus sabdariffa seed solution of 1% to 5% by volume of the Rhodiola rosea solution. By fermenting the Rhodiola rosea solution with Bacillus sabdariffa, a Rhodiola rosea microbial fermentation liquid organically composed of multiple flavor components is obtained. The tobacco flavoring has a unique aroma, good coordination, and high flavor quality, providing an important approach for the development of tobacco flavorings.

[0041] Preferably, the conditions for the primary and secondary alcohol extractions in step (6) are: an ethanol concentration of 50-100% (V / V), and a volume ratio of the Rhodiola rosea microbial fermentation broth to the ethanol of 1:(10-15). The present invention improves the extraction efficiency of the active ingredients of Rhodiola rosea through ultrasonic treatment and multiple alcohol extractions.

[0042] Preferably, the filtration conditions in step (6) are: filtering with a filter cloth having a pore size of 60-80 mesh, and centrifuging, the centrifuge speed is 6000-10000 rpm, and the centrifugation time is 5-20 min.

[0043] Preferably, the filtration in step (7) is performed using a filter membrane with a pore size of 200-400 mesh, and the specific step of concentration is to use rotary distillation vacuum decompression concentration, the concentration temperature is 30-35°C, the vacuum degree is 30-35mbar, and the rotation speed is 60-75rpm, and the Rhodiola rosea fermentation extract is decompressed and concentrated to 1.20±0.005g / mL.

[0044] In a fifth aspect of the present invention, a tobacco-use Rhodiola rosea spice is provided, which is obtained by using the preparation method of the Rhodiola rosea spice.

[0045] In a sixth aspect of the present invention, a tobacco product is provided, wherein the tobacco product is added with the Rhodiola rosea flavoring.

[0046] Preferably, the tobacco-use Rhodiola rosea flavoring is added to tobacco products at a mass percentage of 0.01-0.05%.

[0047] Preferably, the tobacco products include: cigarettes, cigars, shredded tobacco, redried tobacco leaves, tobacco spices, tobacco extract, flue-cured tobacco leaves and cigar leaves.

[0048] The present invention has at least the following beneficial effects:

[0049] (1) The present invention utilizes microbial fermentation technology to prepare spices or transform spices. The prepared spices are natural-grade products. While improving the quality of spice products, it saves energy and reduces costs, thus realizing a revolution in natural spice processing technology.

[0050] (2) The microorganism used in the present invention is Bacillus saffron, which is used for fermentation treatment to increase the content of the effective active ingredients gallic acid, salidroside, salidroside, salidroside, and tyrosol in Rhodiola rosea, reduce the use of organic reagents, reduce the loss of active substances, greatly protect the medicinal value of the active ingredients of Rhodiola rosea, and ensure and improve the health care effect of Rhodiola rosea.

[0051] (3) The present invention utilizes Bacillus saffron to ferment and prepare biological Rhodiola rosea fragrance. Through the action of microbial fermentation, the content of flavor substances such as n-octanol, linalool, phenylethyl alcohol, citronellol, and geraniol is increased, thereby improving the aroma of the Rhodiola rosea fragrance. After sensory evaluation of cigarette flavoring, it was found that the tobacco fragrance has a harmonious and unique aroma, has a strong effect on the fragrance of the smoke style characteristics such as fresh fragrance and green fragrance, and also has a certain contribution to the fragrance of flowers, wood, and sweetness. It has the effect of increasing the aroma of cigarettes, softening and delicate smoke, and reducing throat irritation. Chemical analysis results show that the content of flavor substances such as n-octanol, linalool, phenylethyl alcohol, citronellol, and geraniol is increased.

[0052] (4) Compared with the traditional preparation of Rhodiola rosea extract, the microbial fermentation technology provided by the present invention has mild reaction conditions, easy product separation, and short production cycle for preparing Rhodiola rosea tobacco flavoring. It has the advantages of safety, environmental protection, simple operation, and easy regulation, which is conducive to saving process costs and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 The left picture is a growth picture on a plate, and the right picture is an observation picture under a microscope.

[0055] Figure 2 It is a phylogenetic tree analysis diagram of the Bacillus sabdariffa of the present invention.

[0056] Figure 3 This is a circle map of the whole genome of Bacillus saffron of the present invention.

[0057] Figure 4 The UPLC-MS / MS results of the effective active ingredients in the fermentation broth of the embodiment of the present invention are shown in FIG. 1 , wherein 1 is gallic acid, 2 is the internal standard, 3 is salidroside, 4 is salidroside, 5 is tyrosol, and 6 is salidroside.

[0058] Figure 5 GC-MS / MS test results of aroma components in the fermentation broth according to the embodiment of the present invention; wherein, 1 is the internal standard, 2 is n-octanol, 3 is linalool, 4 is phenylethyl alcohol, 5 is citronellol, and 6 is geraniol. DETAILED DESCRIPTION

[0059] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0060] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0061] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0062] The Bacillus safensis NS18 of the present invention is currently deposited in the General Biological Center of the China Microorganism Culture Collection Administration with the deposit number CGMCC No. 30643. The deposit date is May 16, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0063] The formula of LB liquid culture medium is: tryptone 10 g / L, yeast powder 10 g / L, sodium chloride 5 g / L.

[0064] The LB solid medium formula is: tryptone 10 g / L, yeast powder 10 g / L, sodium chloride 5 g / L, agar 20 g / L.

[0065] Example 1 Isolation and screening of Bacillus sabdariffa NS18

[0066] Under sterile conditions, roots, stems, and leaves of fresh tobacco plants from Baoji, Shaanxi (Qinyan 99) were washed with sterile water, then immersed in 1% sodium hypochlorite for 10 minutes and 75% ethanol for 2 minutes for disinfection. If no microbial growth was detected after the sterile water wash, the sample surface was successfully disinfected. A 20g sample was weighed and ground into a homogenate. Add 200mL of normal saline, shake at 200 rpm and 20°C for 30 minutes, and filter through two layers of gauze. The filtrate was spread on LB medium and incubated at 30°C for 28 hours. Single colonies of varying morphology were selected and stored for future use.

[0067] The above-mentioned preserved strain was inoculated into the Rhodiola solution after enrichment and activation in the LB medium, and was cultured at 30°C and 150 r / min for 36 h. After fermentation, preliminary sensory evaluation was performed, and the strain with obvious change in aroma was marked and numbered as NS18.

[0068] Example 2 Identification of Bacillus safensis NS18

[0069] NS18 was streak-inoculated into the LB solid medium, and was cultured at 30°C for 24 h. Morphological observation was performed after Gram staining, and the bacterial morphology was as shown in Figure 1 The Bacillus safensis NS18 of the present application is a gram-positive bacterium, and the size of the strain is 0.7-1.5 μm x 2.2-3.8 μm, which is rod-shaped and has flagella. The colony morphology on the LB plate is gray-white convex round, with neat edges and rough dry surface. The strain can grow well under aerobic and anaerobic conditions.

[0070] NS18 was inoculated into the LB liquid medium, and was cultured at 37°C and 200 rpm for overnight. Centrifugation was performed at 12000 r / min for 5 min, and the supernatant was discarded to obtain the bacterial body. The genomic DNA of the NS18 strain was extracted by using a bacterial genomic DNA extraction kit. PCR amplification was performed by using 16S universal primers 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID No. 2) and 1492R: TACGGYTACCTTGTTACGACTT (SEQ ID No. 3).

[0071] The PCR amplification system 25 μL: template DNA (20-50 ng / μL) 1 μL, forward and reverse primers (10 μmol / L) 0.5 μL each, dNTP mixture (2.5 mmol / L) 1 μL, Taq enzyme 0.2 μL, 10 x Buffer 2.5 μL, ultrapure water 19.8 μL.

[0072] The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; and 72°C for 10 min.

[0073] The amplified product was subjected to electrophoresis analysis by using 0.8% agarose gel, and the successfully amplified DNA fragment was detected. Sequence determination was performed by Beijing Chengke Biological Technology Co., Ltd., and the 16S rDNA sequence of the bacterium was obtained. The 16S rDNA sequence was compared and analyzed in the NCBI database by Blast, and the NS18 strain was identified as Bacillus safensis. The phylogenetic tree was constructed by using MEGA 11.0 software, and the phylogenetic tree was as shown in Figure 2 Figure 2 ​It can be seen that Bacillus safovici NS18 belongs to the Bacillus safensis evolutionary branch.

[0074]

[0075] The whole genome of strain NS18 was sequenced using the Illumina NovaSeq sequencing platform and the Pac Bio Sequel sequencing platform. Reads < 500 bp were filtered out. The whole genome of Bacillus saffron is shown in the figure below. Figure 3 shown.

[0076] Example 3 Preparation method of a biological Rhodiola rosea tobacco flavoring

[0077] The preparation method of a Rhodiola rosea fragrance for bio-cigarettes in this embodiment comprises the following steps:

[0078] (1) Pretreatment: crushing the Rhodiola rosea medicinal material to obtain Rhodiola rosea powder; the temperature during crushing is not higher than 40°C, and the Rhodiola rosea is treated under low temperature conditions to ensure the activity of the effective ingredients in the Rhodiola rosea and improve the extraction efficiency;

[0079] (2) Ultrasonic extraction: The Rhodiola rosea powder prepared in step (1) is uniformly mixed with water and glucose in a mass ratio of 1:30:5, and ultrasonically treated. The ultrasonic equipment is set to a temperature of 55°C, a power of 500KW, and an extraction time of 60 minutes to obtain a Rhodiola rosea solution; the Rhodiola rosea is crushed in a low temperature environment to obtain a powder and subjected to ultrasonic treatment. The effective active ingredients in the Rhodiola rosea will be quickly and effectively leached, which is helpful for subsequent extraction and improves the extraction efficiency;

[0080] (3) Solution inactivation: The Rhodiola rosea solution obtained in step (2) was inactivated by sterilization at 121° C. for 30 min, followed by cooling to room temperature;

[0081] (4) Preparation of Bacillus sabdariffa NS18 seed solution: Bacillus sabdariffa was activated on an LB plate, and a single colony was picked and inoculated into LB liquid medium to prepare a seed solution;

[0082] (5) Microbial fermentation treatment: inoculating the Bacillus NS18 seed solution obtained in step (4) into the Rhodiola solution inactivated in step (3), wherein the inoculation amount of the Bacillus NS18 seed solution is 1% by volume of the Rhodiola solution; performing fermentation treatment at a fermentation temperature of 28° C., a stirring speed of 210 r / min, and a culture time of 3 days to obtain a Rhodiola microbial fermentation liquid;

[0083] (6) Purification: Using 75% (V / V) ethanol as an extractant, the Rhodiola rosea microbial fermentation liquid and ethanol are mixed at a volume ratio of 1:10, and the Rhodiola rosea microbial fermentation liquid in step (5) is subjected to a first alcohol extraction; then, the mixture is filtered through a 60-mesh filter cloth and centrifuged at 10,000 rpm for 10 minutes to remove the Rhodiola rosea residue and the Bacillus sabdariffa cells; using 90% (V / V) ethanol as an extractant, the Rhodiola rosea microbial fermentation liquid and ethanol are mixed at a ratio of 1:10, and a second alcohol extraction is performed to obtain a Rhodiola rosea fermentation extract;

[0084] (7) The Rhodiola rosea fermentation extract obtained in step (6) is filtered using a filter membrane with a pore size of 200 mesh, and the filtrate is concentrated. The filtrate is concentrated under reduced pressure by rotary distillation at a concentration temperature of 30° C., a vacuum degree of 35 mbar, and a rotation speed of 75 rpm. The Rhodiola rosea fermentation extract is concentrated under reduced pressure to 1.20±0.005 g / mL to obtain a Rhodiola rosea tobacco flavoring.

[0085] Example 4 Analysis of characteristic components of Rhodiola rosea flavor for bio-cigarettes

[0086] UPLC-MS / MS was used to analyze the non-volatile active ingredients (gallic acid, salidroside, salidroside, tyrosol, and salidroside) in the Rhodiola rosea fragrance before and after NS18 fermentation in Example 3. To 1 g of the concentrated fragrance material, 0.1 mL of theophylline-d6 (1.5 μg / mL) internal standard solution was added. The solution was ultrasonically dissolved in 100 mL of a 50% methanol-water solution, and the organic phase was filtered through a 0.22 μm filter membrane before analysis.

[0087] GC-MS / MS was used to detect the volatile aroma components n-octanol, linalool, phenylethyl alcohol, citronellol, and geraniol in the Rhodiola rosea fragrance before and after NS18 fermentation in Example 3. 1 g of concentrated aroma raw material was added with 0.3 mL of acetophenone-d8 (0.12 mg / mL) internal standard solution, ultrasonically dissolved with 100 mL of methanol, and filtered through a 0.22 μm organic phase filter membrane before analysis.

[0088] Control sample: The control sample is 1 g of the fragrance raw material prepared under the same process conditions as in Example 3 without fermentation, treated under the same conditions, and filtered through a 0.22 μm organic phase filter membrane.

[0089] UPLC-MS / MS conditions: A ZORBAX Eclipse Plus C18 column (3.0 mm × 100 mm, 1.8 μm); mobile phase A: 0.02% formic acid in water, mobile phase B: acetonitrile, flow rate 0.3 mL / min, gradient elution program: 90% A from 0 to 1.5 min, 60% A from 2 min, 90% A from 4 min; injection volume 1 μL; column temperature 40°C; standards and samples were quantified using a 50% methanol-water solvent; internal standard method. Mass spectrometry conditions: electrospray ionization (ESI) source, drying gas temperature 250°C, drying gas flow rate 7.0 L / min, nebulizer gas pressure 30 psi, sheath gas temperature 325°C, sheath gas flow rate 11.0 L / min. MS parameters for each substance are shown in Table 1.

[0090] Table 1 Mass spectrometry parameter settings for compounds

[0091]

[0092] GC-MS / MS conditions: Column: DB-5MS UI flexible quartz capillary column (60 m × 0.25 mm × 0.25 μm); Injection temperature: 290°C; Splitless mode; Temperature program: Initial temperature: 40°C, hold for 3 min, then increase to 75°C at 5°C / min, then to 120°C at 1°C / min, and finally to 160°C at 8°C / min; Injection volume: 1 μL. Mass spectrometry conditions: Electron impact (EI) ionization mode; Filament current: 35 μA; Ionization energy: 70 eV; Transfer line temperature: 280°C; Ion source temperature: 280°C; Quadrupole temperature: 150°C; Carrier gas flow rate: 1.5 mL / min; Quench gas flow rate: 2.25 mL / min; Collision gas flow rate: 1.5 mL / min. MS parameters for each substance are shown in Table 2.

[0093] Table 2 Mass spectrometry parameter settings for compounds

[0094]

[0095]

[0096] The results of the comparison of the content of characteristic components of Rhodiola rosea spice before and after treatment with Bacillus saffron NS18 are shown in Table 3. The table shows the average of 6 determination results, and the RSD of the determination results of each substance is less than 10%.

[0097] Table 3 Detection results of characteristic components of Rhodiola rosea tobacco flavoring

[0098]

[0099] As shown in Table 3, the content of characteristic components of the Rhodiola rosea tobacco flavoring fermented with Bacillus saffron is increased, with salidroside, salidroside, linalool, and geraniol significantly increased (to 28.5%, 27.2%, 11.8%, and 13.9%, respectively). This shows that the Rhodiola rosea tobacco flavoring fermented with Bacillus saffron is of high quality.

[0100] Rhodiola rosea tobacco flavoring contains multiple active ingredients, including salidroside, salidroside, salidroside, tyrosol, and gallic acid, and exhibits significant biological functions. The core ingredient, salidroside, has multiple effects, including anti-tumor, anti-viral, anti-fatigue, antioxidant, anti-hypoxia, anti-aging, anti-radiation, cardiovascular function improvement, and immunity enhancement. The Rhodiola rosea flavoring prepared by the present invention, which combines cryogenic freezing, microbial fermentation, and distillation and reduced-pressure concentration, ensures the extraction efficiency and stability of the Rhodiola rosea active ingredients. The content of the active ingredient salidroside in the prepared Rhodiola rosea tobacco flavoring is significantly increased (by 28.5%) compared to the control sample, thereby enhancing its health benefits.

[0101] Among the flavor components of rhodiola rosea tobacco spices, geraniol has a warm and sweet floral aroma, which can be well transferred to the mainstream smoke during the smoking process of cigarettes, increasing the rose aroma and sweet fragrance of cigarettes, and harmonizing the soft and moist mouthfeel of cigarettes; n-octanol has a citrus, rose, waxy, sweet aroma, which can make the cigarette aroma mellow and rich in layers; linalool has a typical floral aroma and a citrus-like fruity aroma, which is soft and transparent, coordinates with the cigarette aroma, and can modify and correct the natural flavor; phenylethyl alcohol has a unique rose-like aroma, which can increase the roundness and mellowness of the cigarette aroma in cigarettes; citronellol has a rose-like honey-sweet aroma, a peaceful aroma, and a lot of green air, which coordinates with the cigarette aroma, enhances the richness, and improves the taste of the cigarette. The flavor components in the prepared rhodiola rosea tobacco spices are relatively stable, and the contents of linalool and geraniol are significantly increased (increased by 11.8% and 13.9% respectively), which can improve the smoking quality of cigarettes.

[0102] UPLC-MS / MS chart of characteristic components of Rhodiola rosea tobacco incense raw materials Figure 4 , GC-MS / MS is shown in Figure Figure 5 .

[0103] Example 5 Application of biological Rhodiola rosea tobacco flavoring in cigarettes and effect evaluation

[0104] In this example, the biological Rhodiola rosea tobacco flavoring prepared in Example 3 was sprayed onto the surface of tobacco shreds, processed into cigarettes, and then the sensory quality of the cigarettes was evaluated.

[0105] (1) Application of biological Rhodiola rosea spices in cigarettes:

[0106] Take 0.01% of the biological Rhodiola rosea fragrance by weight of cigarette tobacco, dilute it with ethanol and spray it on the surface of the leaf group tobacco of Gansu Tobacco Industry Co., Ltd., and balance it in a constant temperature and humidity chamber with a humidity of 70%±2.0 and a temperature of 22℃±2 for 48 hours. After the moisture balance is completed, the cigarettes are rolled and the sensory evaluation is carried out.

[0107] (2) Sensory evaluation:

[0108] The test cigarettes and the blank control group were subjected to sensory evaluation by nationally certified smokers. The efficacy of the cigarettes was analyzed, with a focus on their effects on aroma, taste, and flavor profiles. The results are shown in Table 4. The rhodiola rosea spice enhances the aroma quality of cigarettes, softens and refines the smoke, improves the taste of the smoke, and reduces throat irritation. It has a strong effect on the fresh and green notes of the smoke profile, and also has some effect on floral, woody, and sweet notes, resulting in a harmonious and unique overall aroma.

[0109] Table 4 Efficacy analysis of Rhodiola rosea scented raw material cigarettes

[0110]

[0111] The effects of microbial fermentation on the sensory quality of cigarettes before and after fermentation were examined, as shown in Table 5. Microbial fermentation significantly improved aroma, with the overall sensory quality score increasing by 0.92 points, with the aroma index being the most significant, increasing by 0.4 points. These results indicate that microbial fermentation significantly improves the sensory quality of cigarettes, manifested in an increase in aroma and a reduction in off-flavor and irritation.

[0112] Table 5 Impact of cigarette sensory quality

[0113]

[0114] In summary, the use of the Bacillus NS18 of the present invention to ferment and prepare biological Rhodiola rosea spices has increased the content of gallic acid, salidroside, salidroside, salidroside, and tyrosol in the effective active ingredients of Rhodiola rosea, greatly protecting the medicinal value of the active ingredients of Rhodiola rosea, and ensuring and improving the health benefits of Rhodiola rosea. Chemical analysis results show that the content of flavor substances such as n-octanol, linalool, phenylethyl alcohol, citronellol, and geraniol is increased. After sensory evaluation of cigarette flavoring, it was found that the aroma of the tobacco flavor is coordinated and unique, and has a strong effect on the fragrance of the fresh fragrance and green fragrance in the smoke style characteristics. It also contributes to the floral fragrance, woody fragrance, sweet fragrance, etc., and has the effect of increasing the aroma of cigarettes, softening and delicate smoke, and reducing throat irritation.

[0115] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A Bacillus safensis, characterized in that Its deposit number is CGMCC No.30643.

2. The Bacillus sabdariffa according to claim 1, wherein The nucleotide sequence of the Bacillus saffron 16S rDNA is shown in SEQ ID NO:

1.

3. A microbial agent, characterized in that: The microbial agent contains the Bacillus saffron according to any one of claims 1-2.

4. Use of the Bacillus saffron according to any one of claims 1 to 2 or the microbial agent according to claim 3 in the preparation of Rhodiola rosea spice.

5. The use according to claim 4, characterized in that The efficacy of the bacillus sabdariffa includes at least one of the following: (I) increasing the content of gallic acid, salidroside, salidroside, salidroside, and tyrosol in Rhodiola rosea; (II) Increase the content of octanol, linalool, phenylethyl alcohol, citronellol and geraniol.

6. A method for preparing Rhodiola rosea spice, characterized in that: The following steps are involved: (1) crushing Rhodiola rosea to obtain Rhodiola rosea powder; (2) uniformly mixing the Rhodiola rosea powder prepared in step (1) with water and glucose, and subjecting the mixture to ultrasonic treatment to obtain a Rhodiola rosea solution; (3) inactivating the Rhodiola rosea solution obtained in step (2); (4) activating the Bacillus sp. according to any one of claims 1 to 2 or the microbial agent according to claim 3 on an LB plate, picking a single colony and inoculating it into an LB liquid medium to prepare a seed solution; (5) inoculating the Bacillus saffron seed solution obtained in step (4) into the Rhodiola rosea solution inactivated in step (3), and obtaining a Rhodiola rosea microbial fermentation liquid after fermentation; (6) performing a primary alcohol extraction, filtering, and a secondary alcohol extraction on the Rhodiola rosea microbial fermentation broth in step (5) to obtain a Rhodiola rosea fermentation extract; (7) filtering and concentrating the Rhodiola rosea fermentation extract obtained in step (6) to obtain Rhodiola rosea spice.

7. A tobacco-use Rhodiola rosea spice, characterized in that: The product is prepared by the preparation method of Rhodiola rosea spice according to claim 6.

8. A tobacco product, characterized in that The tobacco product is added with the Rhodiola rosea flavoring according to claim 7.

9. The tobacco product according to claim 8, characterized in that The tobacco rhodiola rosea flavoring is added into tobacco products at a mass percentage of 0.01-0.05%.

10. The tobacco product according to claim 8, characterized in that The tobacco products include: cigarettes, cigars, shredded tobacco, redried tobacco leaves, tobacco spices, tobacco extract, flue-cured tobacco leaves and cigar leaves.

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