Bacillus velezensis and application thereof

Abstract

The invention provides bacillus velezensis and an application thereof. The bacillus velezensis disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30646. According to the seed melon spice for the tobacco prepared through fermentation of the strain, the characteristic component L-citrulline can be efficiently enriched, and the content of several key flavor substances including aldehydes (such as (E)-4-nonenal, (E)-2-nonenal and nonanal) and alcohols (such as Z-3-nonene-1-ol and 1-nonanol) is increased. Cigarette flavoring sensory evaluation shows that the spice can coordinate and fuse the original fragrance of tobacco, endows the cigarette with fresh and sweet note characteristics, effectively supplements the faint scent, the fruit fragrance and the sweet fragrance, remarkably improves the richness of the smoke fragrance, improves the texture of the cigarette smoke, softens and delicate the smoke, reduces the irritation and dryness of the throat, and integrally improves the smoking experience.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a Bacillus belye and its applications. Background Technology

[0002] Natural tobacco flavorings are green and healthy, with pure and natural aromas, which can improve the quality of cigarettes and meet personalized needs. They have become an important direction to replace traditional chemically synthesized flavorings. Their development needs to strengthen the screening of plant resources (focusing on safety, effectiveness, stability and sustainability) and improve extraction, preparation and separation detection technologies.

[0003] Seed melons are rich in nutrients and medicinal value. The *Compendium of Materia Medica* records that "seed melons are sweet in taste" and have the effects of moisturizing the lungs, promoting urination, reducing inflammation, warming the stomach, and strengthening the spleen. However, the pulp, which accounts for about 95% of its weight, is often discarded during processing, resulting in low economic value and waste. Developing tobacco flavorings from seed melons using microbial fermentation technology can ensure a natural source, create new flavorings, increase the added value of seed melons, and reduce pollution. Currently, there are no research reports on the fermentation of seed melons by *Bacillus belye* to prepare tobacco flavorings and their application in cigarettes.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a Bacillus vesiculosus and its microbial agent for preparing melon seed flavoring.

[0006] The second objective of this invention is to provide a method for preparing a melon seed flavoring, in which Bacillus bellis is used as a fermentation strain in the preparation of the melon seed flavoring. The reaction conditions are mild and effectively increase the content of characteristic components of melon seeds.

[0007] The third objective of this invention is to provide a tobacco seed flavoring that has a unique aroma, good harmony, and high flavor quality, thus providing an important pathway for the development of tobacco flavorings.

[0008] The fourth objective of this invention is to provide an application of a tobacco seed flavoring in the production of tobacco products. The main function of this flavoring in improving the sensory quality of tobacco products is to harmonize and blend the natural aroma of tobacco. This flavoring can harmonize and blend the natural aroma of tobacco, giving cigarettes a fresh and sweet aroma profile, effectively complementing the fresh, fruity, and sweet aromas, significantly enhancing the richness of the smoke aroma, improving the texture of the cigarette smoke, producing a smooth and delicate smoke, while reducing throat irritation and dryness, thus improving the overall smoking experience.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a Bacillus velezensis strain named NS42, which is taxonomically named Bacillus velezensis and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 30646.

[0011] The Bacillus velezensis strain of this invention is an endophytic strain screened from fresh tobacco plant samples, named Bacillus velezensis NS42, and was deposited on May 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30646, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] Furthermore, the *Bacillus belyssus* NS42 described in this invention is a Gram-positive bacterium with a size of 0.7-1.0 μm × 2.2-3.6 μm, rod-shaped, and flagellated; on LB agar plates, it appears as a milky-white, raised, round shape with neat edges and a smooth, moist surface. It can grow well under both aerobic and anaerobic conditions.

[0013] Specifically, the nucleotide sequence of the 16S rDNA of Bacillus belyssus NS42 is shown in SEQ ID NO:1.

[0014] Furthermore, the cultivation method of *Bacillus belyssus* is as follows: *Bacillus belyssus* NS42 is inoculated into LB liquid medium and cultured at 30°C and 210 rpm for 12 hours to obtain a seed culture. The *Bacillus belyssus* NS42 seed culture is then inoculated into LB liquid medium at an inoculation rate of 1% and cultured for 12 hours.

[0015] Furthermore, the culture conditions for the Bacillus belyssus NS42 are a temperature of 30°C and a rotation speed of 210 rpm.

[0016] Furthermore, the LB liquid culture medium formula is: 10 g / L tryptone, 10 g / L yeast extract, and 5 g / L sodium chloride.

[0017] The LB solid medium formula is: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 20 g / L agar.

[0018] A second aspect of this invention provides a microbial agent containing the aforementioned Bacillus belesii.

[0019] Preferably, the microbial agent uses Bacillus belyssus as the main microorganism.

[0020] Preferably, the microbial agent is a solid agent or a liquid agent.

[0021] Preferably, the microbial agent contains live cells of the *Bacillus berleis* strain, freeze-dried dried *Bacillus berleis* strain, immobilized cells of the *Bacillus berleis* strain, or the *Bacillus berleis* strain existing in any other form.

[0022] Preferably, the microbial agent is obtained by seed culture of Bacillus belye.

[0023] A third aspect of this invention provides the application of the aforementioned Bacillus berberis or the aforementioned microbial agent in the preparation of melon seed flavoring.

[0024] Specifically, the efficacy of the Bacillus belyssus includes at least one of the following:

[0025] (I) Increase the content of L-citrulline, a characteristic component in seed melon;

[0026] (II) Increase the content of volatile components such as (E)-4-nonenal, (E)-2-nonenal, nonanal, Z-3-nonen-1-ol, and 1-nonanol.

[0027] The fourth aspect of this invention provides a method for preparing a melon seed flavoring, comprising the following steps:

[0028] (1) Pretreatment: Select fresh, unrotten mature seed melons, wash and cut them, separate the seed melon pulp and seed melon peel, remove the seeds from the seed melon pulp, juice and filter to obtain seed melon juice; preferably, treat the seed melon under low temperature conditions to ensure the activity of the effective ingredients in the seed melon and improve the extraction efficiency.

[0029] (2) Ultrasonic extraction: The seed melon juice prepared in step (1) is mixed evenly with water and glucose, and ultrasonically treated to obtain seed melon solution.

[0030] (3) Solution inactivation: The seed melon solution obtained in step (2) is subjected to inactivation treatment;

[0031] (4) Preparation of seed culture of Bacillus vesiculus NS42: Bacillus vesiculus or its microbial agent was activated on LB plates, and a single colony was picked and inoculated into LB liquid medium to prepare seed culture;

[0032] (5) Microbial fermentation treatment: Inoculate the Bacillus NS42 seed liquid obtained in step (4) into the seed melon solution after inactivation in step (3), and ferment to obtain seed melon microbial fermentation liquid;

[0033] (6) Purification: Using ethanol as the extractant, the seed melon microbial fermentation broth in step (5) is subjected to one alcohol extraction, filtration, and two alcohol extractions to obtain seed melon fermentation extract.

[0034] (7) The fermented extract of seed melon obtained in step (6) is filtered and concentrated to obtain seed melon tobacco flavoring.

[0035] Preferably, in step (2), the mass ratio between the original juice of the seed melon, water, and glucose is 1:(10-50):(1-2), and the ultrasonic equipment is set to a temperature of 45-55℃, a power of 400-500KW, and an extraction time of 30-60min during ultrasonic treatment.

[0036] Preferably, the inactivation treatment conditions in step (3) are: sterilization at 121°C for 15-30 min, followed by cooling to room temperature.

[0037] Preferably, the specific steps for preparing the seed culture of Bacillus belysaeus in step (4) are as follows: Bacillus belysaeus NS42 is inoculated into LB liquid medium and cultured at 30°C and 210 rpm for 12 h to obtain the seed culture.

[0038] Preferably, the fermentation conditions in step (5) are a fermentation temperature of 25-35℃, a stirring speed of 100-300 r / min, a culture time of 2-7 days, and an inoculation amount of Bacillus vesiliflorus seed liquid of 1% to 5% of the volume percentage of the seed cucumber solution. Fermentation of the seed cucumber solution using Bacillus vesiliflorus yields a seed cucumber microbial fermentation broth composed of a variety of aroma components, resulting in a unique and well-coordinated aroma for tobacco flavorings, with high aroma quality, providing an important pathway for the development of tobacco flavorings.

[0039] Preferably, the conditions for the first and second alcohol extractions in step (6) are: an ethanol concentration of 50-100% (V / V) and a volume ratio of 1:(10-15) between the fermentation broth of the seed melon microorganisms and ethanol. This invention improves the extraction efficiency of the active ingredients from the seed melon through ultrasonic treatment and multiple alcohol extractions.

[0040] Preferably, the filtration conditions in step (6) are: using a filter cloth with a pore size of 60-80 mesh for filtration, followed by centrifugation, with a centrifuge speed of 6000-10000 rpm and a centrifugation time of 5-20 min.

[0041] Preferably, in step (7), filtration is performed using a 200-400 mesh membrane. The specific steps for concentration are as follows: vacuum concentration is performed by rotary distillation at a temperature of 30-35℃, a vacuum of 30-35mbar, and a rotation speed of 60-75rpm, to concentrate the fermented extract of the seed melon to 1.20±0.005g / mL.

[0042] The fifth aspect of this invention provides a seed melon flavoring for tobacco, obtained by the aforementioned method for preparing the seed melon flavoring.

[0043] A sixth aspect of this invention provides a tobacco product in which the aforementioned tobacco seed flavoring is added.

[0044] Preferably, the tobacco seed flavoring is added to the tobacco product at a weight percentage of 0.01-0.05%.

[0045] Preferably, the tobacco products include: cigarettes, cigars, shredded tobacco, re-dried tobacco leaves, tobacco flavorings, tobacco extracts, flue-cured tobacco leaves, and cigar tobacco leaves.

[0046] The present invention has at least the following beneficial effects:

[0047] (1) This invention utilizes microbial fermentation technology to prepare or transform fragrances. The resulting fragrances are natural products. While improving the quality of fragrance products, it saves energy and reduces costs, thus realizing a revolution in natural fragrance processing technology.

[0048] (2) The present invention utilizes Bacillus beryl to ferment and prepare biological seed melon flavoring, which can effectively increase the content of L-citrulline in seed melon, and greatly protect the medicinal value and health benefits of the active ingredients of seed melon.

[0049] (3) This invention utilizes Bacillus belye fermentation to prepare bio-seed melon flavoring. Through microbial fermentation, the content of aroma substances such as (E)-4-nonenal, (E)-2-nonenal, nonanal, Z-3-nonen-1-ol, and 1-nonanol is increased, thus improving the aroma of the seed melon flavoring. Sensory evaluation of cigarette flavoring revealed that this flavoring has a harmonious and unique aroma, effectively blending with the natural aroma of tobacco to give cigarettes a fresh and sweet flavor profile. It effectively complements the aromas of freshness, fruitiness, and sweetness, significantly enhancing the richness of the smoke aroma, improving the cigarette's texture, and producing a smooth and delicate smoke. At the same time, it reduces throat irritation and dryness, improving the overall smoking experience.

[0050] (4) Compared with the traditionally prepared melon seed extract, the microbial fermentation technology provided by the present invention has mild reaction conditions, low organic reagent usage, easy product separation, and short production cycle. It has the advantages of being safe and environmentally friendly, easy to operate and easy to control, which is conducive to saving process costs and is suitable for industrial production. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1This is a microscopic image of the Bacillus belesis of the present invention.

[0053] Figure 2 This is a phylogenetic tree diagram of Bacillus belesii of the present invention.

[0054] Figure 3 This is a complete genome diagram of Bacillus belyssus of the present invention.

[0055] Figure 4 The results of UPLC-MS / MS analysis of the non-volatile characteristic components of fermented melon flavoring in this embodiment of the invention are shown; where 1 is an internal standard and 2 is L-citrulline.

[0056] Figure 5 This is a GC-MS chromatogram of the total ion flow of volatile components of fermented melon flavoring in an embodiment of the present invention. Detailed Implementation

[0057] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0058] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0059] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] The Bacillus velezensis NS42 of this invention is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 30646. The deposit date was May 16, 2024, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0061] The LB liquid culture medium formula is: 10 g / L tryptone, 10 g / L yeast extract, and 5 g / L sodium chloride.

[0062] The LB solid medium formula is: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 20 g / L agar.

[0063] Example 1: Isolation and screening of Bacillus belyssus NS42

[0064] Under aseptic conditions, the roots, stems, and leaves of fresh tobacco plants (Qinyan 99) from Baoji, Shaanxi Province, were washed with sterile water, soaked in 1% sodium hypochlorite for 10 minutes, and then soaked in 75% ethanol for 2 minutes for disinfection. No microbial growth was detected after washing with sterile water, indicating successful sample surface disinfection. 20g of sample was weighed, ground into a homogenate, and 200mL of physiological saline was added. The mixture was shaken at 200 rpm and 20°C for 30 minutes, and then filtered through two layers of gauze. The filtrate was spread onto LB agar and incubated at 30°C for 28 hours. Single colonies with varying morphologies were then collected and preserved for later use.

[0065] After the above-preserved strains were enriched and activated in LB medium, they were inoculated into the seed melon solution and cultured at 30℃ and 150r / min for 36h. After fermentation, a preliminary sensory evaluation was carried out, and the strains with obvious aroma changes were marked and preserved as NS42.

[0066] Example 2 Identification of Bacillus belyssus NS42

[0067] NS42 cells were streaked onto LB agar and incubated at 30°C for 24 hours. After Gram staining, morphological observation was performed. The bacterial cell morphology is as follows: Figure 1 As shown. The *Bacillus belyssus* NS42 described in this invention is a Gram-positive bacterium with a size of 0.7-1.0 μm × 2.2-3.6 μm, rod-shaped, and flagellated. On LB agar plates, its colony morphology is milky white, raised, round, with neat edges and a smooth, moist surface. It can grow well under both aerobic and anaerobic conditions.

[0068] NS42 was inoculated into LB liquid medium and cultured overnight at 30°C and 210 rpm. After centrifugation at 12000 rpm for 5 min, the supernatant was discarded to obtain bacterial cells. Genomic DNA of NS42 strain was extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using the 16S universal primers 27F: AGAGTTTGATCM TGGCTCAG (SEQ ID No:2); 1492R: TACGGYTACCTTGTTACGACTT (SEQ ID No:3).

[0069] 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× Buffer 2.5 μL, ultrapure water 19.8 μL.

[0070] PCR reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; extension at 72℃ for 10 min.

[0071] The amplified products were analyzed by electrophoresis on a 0.8% agarose gel, and the successfully amplified DNA fragments were detected. These fragments were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, yielding the 16S rDNA sequence of the bacterium. The 16S rDNA sequence was compared with the NCBI database using BLAST, and strain NS42 was identified as *Bacillus velezensis*. A phylogenetic tree was constructed using MEGA 11.0 software, as shown below. Figure 2 As shown. By Figure 2 It can be seen that Bacillus velezensis NS42 belongs to the Bacillus velezensis clade.

[0072]

[0073] The whole genome of strain NS42 was sequenced using the Illumina NovaSeq and Pac Bio Sequel sequencing platforms. Reads <500bp were filtered out, and the whole genome circle of Bacillus belyssum is shown below. Figure 3 As shown.

[0074] Example 3: A method for preparing a flavoring agent for biologically-derived melon seeds.

[0075] The preparation method of a bio-tobacco seed flavoring in this embodiment includes the following steps:

[0076] (1) Pretreatment: Select fresh, unrotten mature seed melons, wash and cut them, separate the seed melon pulp and seed melon peel, remove the seeds from the seed melon pulp, juice and filter to obtain the original seed melon juice; treat the seed melon under low temperature conditions to ensure the activity of the effective ingredients in the seed melon and improve the extraction efficiency.

[0077] (2) Ultrasonic extraction: The seed melon juice prepared in step (1) is mixed with water and glucose in a mass ratio of 1:20:1 and ultrasonically treated. The ultrasonic equipment is set to a temperature of 55℃, a power of 500KW, and an extraction time of 60min to obtain a seed melon solution.

[0078] (3) Solution inactivation: The seed melon solution obtained in step (2) was inactivated; sterilized at 121℃ for 30 min, and then cooled to room temperature;

[0079] (4) Preparation of seed culture of Bacillus belyss NS42: Bacillus belyss was activated on LB plates, and single colonies were picked and inoculated into LB liquid medium to prepare seed culture.

[0080] (5) Microbial fermentation treatment: Inoculate the inactivated seed cucumber solution in step (3) with the Bacillus vesicles NS42 seed liquid obtained in step (4), the inoculation amount of Bacillus vesicles NS42 seed liquid is 1% of the volume percentage of the seed cucumber solution; carry out fermentation treatment at 28℃, stirring speed 210r / min, culture time is 3d, and seed cucumber microbial fermentation broth is obtained after fermentation.

[0081] (6) Purification: Using 75% (V / V) ethanol as the extractant, the fermentation broth of the seed melon microorganisms was mixed with ethanol at a volume ratio of 1:10 to perform a first ethanol extraction on the fermentation broth of the seed melon microorganisms in step (5); then the mixture was filtered through a 60-mesh filter cloth and centrifuged at 10,000 rpm for 10 min to remove the seed melon residue and Bacillus belesii cells; using 90% (V / V) ethanol as the extractant, the fermentation broth of the seed melon microorganisms was mixed with ethanol at a ratio of 1:10 to perform a second ethanol extraction to obtain the fermentation extract of the seed melon;

[0082] (7) The fermented extract of seed melon obtained in step (6) was filtered using a 200-mesh filter membrane and the filtrate was concentrated by rotary distillation under vacuum. The concentration temperature was 30℃, the vacuum degree was 35mbar, and the rotation speed was 75rpm. The fermented extract of seed melon was concentrated under vacuum to 1.20±0.005g / mL to obtain seed melon tobacco flavoring.

[0083] Example 4: Component Analysis of Seed Melon Flavoring for Bio-tobacco

[0084] The non-volatile active ingredient L-citrulline in the melon seed flavoring of Example 3 before and after fermentation in NS42 was detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). 1 g of concentrated flavoring was taken, and 0.1 mL of L-ornithine (1 μg / mL) internal standard solution was added. The mixture was extracted with 25 mL of 0.1% formic acid aqueous solution by ultrasonication, centrifugation was performed, and the supernatant was filtered through a 0.22 μm organic phase filter membrane for analysis. The chromatographic column was a hydrophilic Poroshell 120HILIC-Z column (2.1 mm × 150 mm, 2.7 μm); mobile phase A was 0.1% formic acid-10 mM ammonium formate aqueous solution, and mobile phase B was 0.1% formic acid-acetonitrile solution; the flow rate was 0.3 mL / min; the gradient elution program was: 0–2 min: 5% A, 5 min: 30% A, 7 min: 60% A; 7.1 min: 5% A; 10 min: 5% A; the injection volume was 1 μL; the column temperature was 40℃; both standards and samples were in 0.1% formic acid aqueous solution, and quantification was performed using the internal standard method. Mass spectrometry conditions: electrospray ionization (ESI), drying gas temperature 250℃, drying gas flow rate 7.0 L / min, nebulizer gas pressure 30 psi, sheath gas temperature 325℃, sheath gas flow rate 11.0 L / min. The mass spectrometry parameters for each substance in multiple reaction monitoring (MRM) mode are shown in Table 1.

[0085] Control Samples: Control Sample 1 was the original juice of the seed melon. 1g of the original juice from Example 3 was weighed, and 0.1mL of L-ornithine (1μg / mL) internal standard solution was added. The mixture was extracted with 25mL of 0.1% formic acid aqueous solution using ultrasound, centrifuged, and the supernatant was filtered through a 0.22μm organic phase filter membrane for analysis. Control Sample 2 was 1g of flavoring prepared under the same process conditions as in Example 3, without fermentation, and treated under the same conditions.

[0086] Table 2 shows the comparison of L-citrulline content in melon seed flavoring before and after treatment with Bacillus vesicles NS42. The table represents the average of three measurements, and the RSD of the results is <10%.

[0087] Table 1. Mass Spectrometry Parameter Settings for Compounds

[0088]

[0089] Table 2. Detection results of characteristic components of flavoring for melon seed tobacco.

[0090]

[0091]

[0092] The volatile aroma components of the melon seed flavoring in Example 3 before and after fermentation on NS42 were qualitatively detected using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). The aroma substance content was calculated using a semi-quantitative method. 4g of concentrated flavoring was placed in a 20mL headspace sample vial, 5μL of 2-ethyl-1-hexanol (0.02mg / mL) internal standard solution was added, along with 1g of anhydrous NaCl, and the mixture was shaken well. A DVB / PDMS / CarbonWR-80μm (50μm / 30μm) extraction head was used. After incubation at 60℃ for 30min to reach equilibrium, extraction was performed for 30min, followed by desorption at 250℃ for 5min via the injection port. Chromatographic column: Agilent DB-5 (60m × 0.25mm, 0.25μm), injection port temperature 250℃, splitless mode, carrier gas: helium (99.9% purity), carrier gas flow rate: 1mL / min; temperature program: initial temperature 50℃, hold for 5 min, increase to 280℃ at a rate of 6℃ / min, hold for 5 min; end the process. Mass spectrometry ionization mode: electron impact ionization (EI), full scan mode, scan range 50–500 m / z.

[0093] Control sample: The control sample is a fragrance prepared under the same process conditions without fermentation according to the steps in Example 3, and treated under the same conditions.

[0094] The volatile components of the seed melon flavoring were identified and screened before and after treatment with Bacillus vesicles NS42. The relative contents of some substances are shown in Table 3. The table shows the average values ​​of three determinations.

[0095] Table 3. Results of volatile component detection for melon seed tobacco flavoring.

[0096]

[0097]

[0098] Note: "-" indicates not detected.

[0099] Table 2 shows that the L-citrulline content of the seed-fed flavoring from *Bacillus vesalis* fermented in this invention increased by 27.3%, a significant change compared to the unfermented flavoring, and achieving highly efficient enrichment compared to the original seed-fed juice. Table 3 shows that the seed-fed flavoring from *Bacillus vesalis* fermented in this invention has a high content of aldehydes and alcohols, and some flavor components changed significantly after fermentation. Specifically, (E)-4-nonenal, (E)-2-nonenal, nonanal, Z-3-nonen-1-ol, and 1-nonanol increased by 22.73%, 16.10%, 15.18%, 31.45%, and 23.89%, respectively.

[0100] Sour melon seeds are rich in various active ingredients, among which L-citrulline, as a precursor to L-arginine and nitric oxide, plays a vital role in blood circulation, athletic performance, and overall health by supporting the two core systems of nitric oxide and urea cycles. Cardiovascularly, it can improve cardiovascular health, assist in the treatment of hypertension, arteriosclerosis, and other diseases, and help maintain normal cholesterol levels. In terms of physical function, it can enhance exercise endurance, combat fatigue, and provide antioxidant effects. Regarding the immune and nervous systems, it can improve immune system function, enhance mental clarity, and help reduce stress and alleviate feelings of depression. This invention, through a combination of microbial fermentation, distillation, and vacuum concentration techniques, produces a sour melon seed flavoring that ensures highly efficient enrichment of the active ingredients in sour melon seeds. The content of active ingredients in the prepared sour melon seed flavoring is significantly higher than that of the control sample, thereby enhancing its health benefits.

[0101] Among the volatile components of the flavorings used in melon seed tobacco, (E)-4-nonenal has fruity and melon aromas, with a slight grassy and fatty note; (E)-2-nonenal has a typical cucumber and melon peel aroma, and is one of the key aroma compounds in many melons, providing the characteristic "freshness" of melons; nonanal has citrus peel, rose, and fatty aromas; Z-3-nonen-1-ol has cucumber and melon aromas, with hints of green leaves and light floral notes; 1-nonanol has sweet orange, waxy, and slightly oily aromas, with a slight floral note. The flavor chemistry of melon seed tobacco highlights its "refreshing and sweet aftertaste" characteristics.

[0102] UPLC-MS / MS chromatogram of non-volatile characteristic component (L-citrulline) of flavoring in fermented melon tobacco is shown below. Figure 4 The total ion chromatogram of volatile components by GC-MS detection is shown below. Figure 5 .

[0103] Example 5: Application and Effect Evaluation of Biological Seed Melon Tobacco Flavoring in Cigarettes

[0104] In this embodiment, the bio-seed melon tobacco flavoring prepared in Example 3 above is sprayed onto the surface of tobacco shreds, processed into cigarettes, and then the cigarettes are subjected to sensory quality evaluation.

[0105] (1) Application of bio-seed melon flavoring in cigarettes:

[0106] Take 0.01% of the bio-seed melon flavoring by weight of cigarette tobacco, dilute it with ethanol and spray it onto the surface of the tobacco blend formulated by Gansu Tobacco Industry Co., Ltd., and equilibrate it in a constant temperature and humidity chamber at 70%±2.0 and 22℃±2 for 48 hours. After the moisture balance is completed, cigarettes are rolled and sensory evaluation is carried out.

[0107] (2) Sensory evaluation:

[0108] The experimental cigarettes and the blank control group cigarettes were subjected to sensory evaluation by nationally certified tasters to analyze their efficacy. The fermented melon seed flavoring imparted prominent aroma characteristics of freshness, fruitiness, and sweetness to the cigarette products, enriching their flavor profile, smoke characteristics, and aroma profile. The results are shown in Table 4. The treated tobacco shreds exhibited better delicacy, smoothness, and roundness, improved smoke texture, reduced irritation and dryness, increased cleanliness and sweetness in the taste, and enhanced clarity. This melon seed flavoring delivers a fresh, sweet, and slightly green melon-fruit aroma.

[0109] Table 4. Analysis of the efficacy of melon-flavored cigarettes

[0110]

[0111]

[0112] The effects of microbial fermentation on the sensory quality of cigarettes were investigated, as shown in Table 5. Microbial fermentation significantly improved the aroma, increasing the total sensory quality score of cigarettes by 0.82 points, with the aroma index showing the most significant increase of 0.4 points. The results indicate that microbial fermentation significantly improves the sensory quality of cigarettes; the treated tobacco's harmony, aroma, and aftertaste scores were all higher than the control, with increased aroma harmony, a cleaner aftertaste, and a significantly improved smoking quality.

[0113] Table 5. Impact of Sensory Quality of Cigarettes

[0114] project luster aroma Harmony Mixed gases Irritating Aftertaste Total Score Comparison 5.00 28.00 5.00 10.50 17.50 22.00 88.00 deal with 5.00 28.40 5.00 10.54 17.68 22.20 88.82

[0115] In summary, the bio-flavoring of melon seeds prepared by fermentation using Bacillus vesiculus NS42 of this invention increases the L-citrulline content, thus ensuring and enhancing the health benefits of melon seeds. The increased content of aroma compounds such as (E)-4-nonenal, (E)-2-nonenal, nonanal, Z-3-nonen-1-ol, and 1-nonanol significantly enhances the refreshing and natural sensation. Sensory evaluation of cigarette flavoring shows that this flavoring has a harmonious and unique aroma, effectively blending with the natural aroma of tobacco to impart a fresh and sweet fragrance to cigarettes. It effectively complements the fresh, fruity, and sweet aromas, significantly improving the richness of the smoke aroma, enhancing the cigarette's texture, and producing a smooth and delicate smoke. Simultaneously, it reduces throat irritation and dryness, resulting in an overall improved smoking experience.

[0116] The microbial fermentation method in this invention ensures that tobacco flavorings are developed in a more targeted, greener, more environmentally friendly, and more energy-efficient direction. At the same time, the detection method used in the invention is more suitable for the analysis of components in complex matrices, providing technical support for the study of the aroma mechanism and quality control of flavorings, and laying a theoretical foundation for the development and application of core technologies for natural aromatic raw materials.

[0117] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Bacillus velezensis, characterized in that, Its accession number is CGMCCNo.30646.

2. The Bacillus belye according to claim 1, characterized in that, The nucleotide sequence of the Bacillus belyss 16S rDNA is shown in SEQ ID NO:

1.

3. A microbial inoculant, characterized in that, The microbial agent contains Bacillus belye as described in any one of claims 1-2.

4. The use of Bacillus berberis as described in any one of claims 1-2 or the microbial agent as described in claim 3 in the preparation of melon seed flavoring.

5. The application according to claim 4, characterized in that, The efficacy of the Bacillus belyssus includes at least one of the following: (I) Increase the content of L-citrulline, a characteristic component in seed melon; (II) Increase the content of (E)-4-nonenal, (E)-2-nonenal, nonanal, Z-3-nonen-1-ol, and 1-nonanol.

6. A method for preparing a melon seed flavoring, characterized in that, Includes the following steps: (1) Wash and cut fresh, unrotten, mature seed melons, separate the pulp and peel, remove the seeds from the pulp, juice and filter to obtain the original juice of the seed melon; (2) Mix the seed melon juice obtained in step (1) with water and glucose evenly, and then treat it with ultrasound to obtain a seed melon solution. (3) The seed melon solution obtained in step (2) is subjected to inactivation treatment; (4) Activate the Bacillus berberis according to any one of claims 1-2 or the microbial agent according to claim 3 on LB plates, and pick a single colony to inoculate into LB liquid medium to prepare seed liquid; (5) Inoculate the Bacillus berberis seed liquid obtained in step (4) into the inactivated seed melon solution in step (3), and ferment to obtain seed melon microbial fermentation liquid; (6) The seed melon microbial fermentation broth in step (5) is subjected to a first alcohol extraction, filtration, and a second alcohol extraction to obtain the seed melon fermentation extract; (7) The fermented extract of seed melon obtained in step (6) is filtered and concentrated to obtain seed melon flavoring.

7. A type of melon seed flavoring for tobacco, characterized in that, It is prepared using the method described in claim 6 for preparing melon seed flavoring.

8. A tobacco product, characterized in that, The tobacco product is made with the melon seed flavoring as described in claim 7.

9. The tobacco product according to claim 8, characterized in that, The tobacco seed flavoring is added to tobacco products at a weight 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, re-dried tobacco leaves, tobacco flavorings, tobacco extracts, flue-cured tobacco leaves, and cigar tobacco leaves.

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