Staphylococcus capitis W54, microbial preparation, tobacco flavor and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an aroma-producing staphylococcus capitis W54 (Staphylococcus capitis W54) separated from white spirit yeast, a bacterial agent and application of the bacterial agent in preparation of tobacco flavor. The strain is preserved in the China General Microbiological Culture Collection Center on December 11, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.33029. The staphylococcus capitis W54 is added into the tobacco powder fermentation culture medium after high-temperature sterilization for fermentation, and the variety and number of aroma components of fermentation liquor are obviously increased. And carrying out primary extraction, filtration and secondary extraction on the fermentation liquor to obtain a fermentation extracting solution, and then filtering and concentrating to obtain the novel tobacco flavor. When the tobacco flavor is added to a cigarette, a smoke panel test result shows that compared with a contrast, the aroma quality and the aroma amount of the cigarette are improved, the aroma is full, the coordination of the aroma is improved, the body fluid engendering feeling is improved, and the sensory quality is remarkably improved. Therefore, the aroma-producing staphylococcus capitis W54 has great application potential in the field of improving the tobacco quality.

Description

Technical Field

[0001] The present application relates to the intersection of microbial technology and tobacco processing, and specifically to a scent-producing Staphylococcus capitis W54 isolated from Baijiu Daqu, a microbial preparation, a tobacco flavoring and its application. Background Art

[0002] In recent years, with the continuous expansion of global tobacco production, the treatment of tobacco waste has become a key challenge for the sustainable development of the industry. It is estimated that millions of tons of waste tobacco leaves and tobacco dust are generated worldwide each year. Their traditional treatment methods (such as landfill and incineration) not only cause waste of resources, but also pose environmental pollution risks. Therefore, it is urgent to develop high-value comprehensive utilization technologies for tobacco waste. At present, the resource utilization of waste tobacco leaves at home and abroad is mainly concentrated in three major directions: one is the extraction of high-value-added chemical raw materials (such as solanesol, nicotine, leaf protein, etc.); the second is the combination of biotechnology to prepare organic fertilizers or functional substances; and the third is recycling for tobacco flakes, pulp and other cigarette materials. Among them, microbial fermentation technology has attracted much attention in the field of tobacco flavor development because of its green and environmentally friendly and efficient conversion characteristics.

[0003] With the deepening promotion of the concept of "reducing tar and reducing harm", the reduction of tar content in cigarettes has led to a significant loss of tobacco's original aroma components. Although traditional chemical synthetic flavors can make up for the aroma defects, their artificial addition is prone to problems such as poor coordination and residual odor, making it difficult to meet consumers' demand for natural feeling and quality stability. Studies have shown that microbial metabolism can directionally transform sugars, proteins and polyphenols in the tobacco matrix to produce natural aroma components such as esters, ketones, and aldehydes. These products are naturally compatible with the original aroma of tobacco. In the existing technology, there have been attempts to use waste tobacco to ferment with a single strain of bacteria to prepare flavors, but there are generally bottlenecks such as a single type of aroma substances, large fluctuations in content, and insignificant aroma enhancement effects. The reasons are: 1) High-efficiency aroma-producing strains have not been screened based on the characteristics of tobacco waste components; 2) The fermentation process parameters are not sufficiently compatible with the tobacco matrix, resulting in a low conversion rate of precursor substances; 3) There is a lack of systematic analysis of the metabolic pathways of the strains, making it difficult to directionally regulate the aroma components.

[0004] Furthermore, existing microbial fermentation flavoring technologies primarily focus on the direct application of general-purpose aroma-producing fungi (e.g., yeast and lactic acid bacteria), ignoring the inhibitory effects of complex components in tobacco waste (e.g., alkaloids and cellulose) on the metabolic activity of strains, resulting in insufficient abundance of aroma-producing substances in fermentation products. Furthermore, the lack of targeted strain domestication and molecular identification during flavor preparation further restricts standardized production and quality stability.

[0005] Based on this, it is urgent to screen efficient degradation-aroma-producing synergistic strains and construct a microbial fermentation aroma-producing technology system based on tobacco waste as the matrix to achieve the efficient conversion of tobacco waste into natural tobacco flavors. Summary of the Invention

[0006] In view of this, the main purpose of the present invention is to provide an aroma-producing Staphylococcus capitis strain that can effectively improve the aroma quality of tobacco and its application.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention discloses a Staphylococcus capitis strain W54, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCCNO.33029 and a deposit date of December 11, 2024. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0009] The strains were isolated and the aroma-producing fungi were screened using liquor koji as the raw material. The specific method was as follows: 5 g of completely crushed liquor koji was weighed and placed in a 250 mL conical flask. 95 mL of normal saline was added, and glass beads were added. The flask was placed in a shaker at 37 ° C for 30 min. The liquor koji extract was diluted 10 times in a gradient manner, and the dilution factor was 10. -4 , 10 -5 , 10 -6 , 10 -7 Spread 0.1 mL of the diluted solution directly onto nutrient agar (10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, 15 g / L agar, pH adjusted to 6.8-7.2). Place the plate with the diluted solution in an incubator and culture (37°C, 1-2 days). Select bacteria with good growth and significant morphological differences and streak purification for 2-3 generations. The isolated and purified strains were plated onto nutrient agar slants and stored at 4°C.

[0010] The selected strains were added to a fermentation medium containing waste tobacco, and fermented. Through sensory evaluation and inhalation, strains that were distinguishable from the control and exhibited significantly enhanced aroma were rescreened. The aroma-producing Staphylococcus capitis W54 was identified through fermentation comparison, olfactory evaluation, and sensory evaluation. Molecular identification and phylogenetic tree analysis confirmed its identification as Staphylococcus capitis.

[0011] A microbial preparation comprising the above-mentioned Staphylococcus capitis W54; preferably, the concentration of the Staphylococcus capitis W54 is ≥1.0×10 8 cfu / mL; more preferably, the concentration of Staphylococcus capitis W54 is 1.0×108 ~1.0×10 10 cfu / mL; preferably, the microbial preparation is a liquid or powder.

[0012] A method for producing the aforementioned microbial preparation comprises culturing the aforementioned Staphylococcus capitis W54 at 30-37°C for 24-48 hours to obtain the microbial preparation. Preferably, the culture medium used in the culturing of the aforementioned Staphylococcus capitis W54 is a nutrient agar liquid medium composed of 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, 15 g / L agar, and a pH of 6.4-6.6. The stirring speed during the culturing of the aforementioned Staphylococcus capitis W54 is preferably 180-220 rpm to accelerate the growth rate of the aforementioned Staphylococcus capitis W54.

[0013] A tobacco flavoring agent is provided, wherein the above-mentioned Staphylococcus capitis W54 or the above-mentioned microbial preparation is inoculated into a fermentation medium for fermentation. After fermentation, the fermentation liquid is extracted to obtain a fermentation extract, and the fermentation extract is then filtered and concentrated to obtain the tobacco flavoring agent. The composition of the fermentation medium is as follows: the ratio of tobacco dust mass to deionized water volume is 1:10 to 1:20 g / mL; the fermentation conditions are: fermentation in a shaker at a speed of 180 to 220 rpm, a temperature of 30 to 37°C, and a time of 24 to 72 hours; the concentration conditions are: a temperature of 50 to 60°C and a speed of 50 to 60 rpm. The tobacco dust in the fermentation medium can be waste tobacco dust, which is provided by the Technology Center of Jilin Tobacco Industry Co., Ltd.; the microbial preparation is Staphylococcus capitis W54 at a concentration of 1.0×10 8 ~1.0×10 10 cfu / mL liquid, the inoculation amount of the microbial preparation is 5% to 10% of the volume of the fermentation medium. After the fermentation is completed, when the fermentation liquid is extracted to obtain the fermentation extract, the fermentation liquid is sequentially subjected to a first extraction, filtration, a second extraction, and filtration, and the filtrates obtained from the first extraction and the second extraction are combined to obtain the fermentation extract. When the fermentation liquid is subjected to a first extraction and a second extraction, the solvent used is ethanol with a volume fraction of 80% to 90%, the volume of the solvent used is 2 to 4 times that of the fermentation liquid, the extraction temperature is 85 to 95°C, and the extraction time is 2 to 3 hours. The density of tobacco flavoring is 1.0 to 1.2 g / cm 3Compared with the blank tobacco fermentation medium, the fermentation extract used to prepare tobacco flavoring showed increased contents of phenylacetic acid, megastigmatrienone, β-ionone, damascenone, caryophyllene, (-)-isolongifoliol, trumpet-ol, 3,5,5-trimethylcyclohex-2-en-1-one, spirulinatone, and (+)-citronellal, which improved the flavor of the tobacco fermentation liquid and increased the aroma quantity and quality, thereby improving the sensory quality of the tobacco fermentation liquid, and was beneficial to enhancing the permeability of the cigarette and the sense of saliva production from the smoke.

[0014] A use of the Staphylococcus capitis W54 or the microbial preparation or the tobacco flavoring in improving the aroma quality of tobacco. Preferably, the application is manifested in increasing the content of components such as phenylacetic acid, megastigmatrienone, β-ionone, damascenone, caryophyllene, (-)-isolongitol, hornblende alcohol, 3,5,5-trimethylcyclohex-2-en-1-one, spirulinatone and (+)-citronellal in the tobacco fermentation liquid. When the tobacco flavoring is used to improve the aroma quality of tobacco, the tobacco flavoring is diluted, and the dilution is added to the tobacco stick to balance the moisture. Specifically, the tobacco flavoring is injected into the tobacco stick with a micro syringe, and a sensory evaluation is performed after the moisture is balanced. The amount of tobacco flavoring added is 0.05 to 0.10% of the mass of the tobacco.

[0015] The invention screens out a strain with obvious aroma-producing effect from white wine Daqu, enriches the strain library resources of tobacco aroma-producing fungi, and simultaneously utilizes the strain to ferment waste tobacco dust to prepare tobacco flavoring, thereby obtaining a special tobacco flavoring with outstanding aroma quantity and aroma quality. Specialty tobacco flavorings contain high levels of alcohols and ketones. Among them, megastigmatrienone is the main component of tobacco's natural aroma, with a sweet, tobacco-like and dried fruity aroma that harmonizes with the tobacco's aroma and masks other odors. Damascenone has rich floral and fruity properties, imparting a sweet, rose-like and citrus-like aroma to tobacco, significantly enhancing the aroma quality and layering of the smoke, making the aroma released during tobacco combustion more uniform and lasting, and improving the aftertaste experience. Caryophyllene, (-)-isolongitol, hornblendeol, 3,5,5-trimethylcyclohex-2-en-1-one, spirulinatone, and (+)-helitenone have woody aromas that can form a complex aroma with the tobacco's natural flavor, reducing the tobacco's pungency and monotony, enhancing the layering of the tobacco's aroma, making it softer on the palate and leaving a light, elegant fragrance. Furthermore, heterocyclic compounds such as pyridine and pyrrole have nutty and cocoa-like aromas, which modify the tobacco's aroma and enhance the quality of the smoke. Tobacco flavorings prepared by fermentation of Staphylococcus aureus W54 can improve the permeability of cigarettes and enhance the salivation effect of smoke. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a colony morphology diagram of the aroma-producing fungus W54 provided in Example 2 of the present invention.

[0017] Figure 2 This is a Gram staining microscopic examination image of the aroma-producing fungus W54 provided in Example 2 of the present invention.

[0018] Figure 3 This is the phylogenetic tree constructed based on the 16S rDNA gene sequence in Example 2 of the present invention.

[0019] Figure 4 This is the growth curve of Staphylococcus capitis W54 in tobacco fermentation medium in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions of the preferred embodiments of the present application in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection claimed in this application.

[0021] Example 1: Isolation and screening of Staphylococcus capitis W54

[0022] Strain isolation: Weigh 5g of completely crushed liquor yeast and put it into a 250mL conical flask, add 95mL of normal saline and glass beads, and shake it in a shaker at 37℃ for 30min. Dilute the liquor yeast extract in 10-fold gradients, and take the dilution factor of 10 as the dilution factor. -4 , 10 -5 , 10 -6 , 10 -7 Spread 0.1 mL of the diluted solution directly onto nutrient agar (10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, 15 g / L agar, pH adjusted to 6.8-7.2). Place the plate with the diluted solution in an incubator and culture (37°C, 1-2 days). Select bacteria with good growth and significant morphological differences and streak purification for 2-3 generations. The isolated and purified strains were plated onto nutrient agar slants and stored at 4°C.

[0023] Initial screening of aroma-producing fungi: The isolated strains were inoculated into nutrient agar liquid seed culture medium and cultured in a shaker (35°C, 200 r / min) for 36 h. The aroma-producing fungi were initially screened by olfactory identification.

[0024] Fermentation: 8% seed culture was inoculated into tobacco fermentation medium and fermented in a shaker at 35°C and 200 rpm for 72 hours. Changes in the fermentation broth's aroma were observed and recorded during the incubation process. A blank control group was inoculated with 8% deionized water into tobacco fermentation medium and fermented under the same conditions for 72 hours.

[0025] Rescreening of aroma-producing fungi: The fermentation broth with a different aroma from the control group was removed for olfactory identification. The fermentation broth was centrifuged to obtain the supernatant, which was then prepared using 75% (volume fraction) ethanol as the solvent to create a flavoring solution. A blank flavoring solution was prepared using the blank control culture medium as the base. This solution was injected into blank cigarette samples using a cigarette flavoring injector at an injection volume of 6 μL / cigarette. The blank control group received the same volume of blank flavoring solution. After equilibration at 25°C and 60% relative humidity for 48 hours, a professional panel evaluated the solution and identified the strain with the best aroma-producing properties, Staphylococcus capitis W54. The sensory evaluation results of the fermentation broth of the aroma-producing Staphylococcus capitis W54 strain are shown in Table 1.

[0026] Table 1 Sensory evaluation results of cigarettes

[0027]

[0028] As shown in Table 1, the control group's cigarettes had insufficient aroma quality and volume, with a strong presence of off-flavors and irritation, and a bitter aftertaste. Adding the fermentation broth of the W54 strain increased the aroma quality and volume of the cigarettes, with floral and creamy notes, reduced irritation, a harmonious aftertaste, and a more mellow and sweet aftertaste.

[0029] Analysis of aroma components of aroma-producing fungus fermentation samples: The aroma components of the fermentation broth of the W54 strain obtained above and the blank control group were determined by headspace solid phase microextraction. The results are shown in Table 2.

[0030] Table 2 Volatile aroma components (μg / g)

[0031]

[0032]

[0033] Note: “ / ” means “not detected”

[0034] Analysis of Table 2 shows that the fermentation liquid of strain W54 has a high content of aroma substances, and phenylacetic acid has a honey-like sweetness; megastigmatrienone is the main component of the natural aroma of tobacco, with a sweet tobacco-like aroma and dried fruit aroma, which can be coordinated with the aroma of tobacco, can cover up the miscellaneous smell, and give the tobacco leaves a sweet aroma; β-ionone has a sweet violet aroma, which can increase the sweet flavor of tobacco leaves; 4-hydroxy-β-dihydrodamascone has a strong floral and fruity aroma, which can give tobacco a sweet aroma similar to rose and citrus, significantly improving the aroma quality and layering of the smoke, making tobacco burning more pleasant. The aroma released during burning is more uniform and lasting, improving the aftertaste experience; caryophyllene, (-)-isolongifoliol, hornblendeol, 3,5,5-trimethylcyclohex-2-en-1-one, spirulinatone and (+)-citronellal have a woody aroma, which can form a complex aroma with the original flavor of tobacco, reduce the spiciness and monotony of tobacco, enhance the layering of tobacco aroma, make the taste softer, and leave a light and elegant fragrance; heterocyclic compounds such as pyridine and pyrrole have the aroma of nuts and cocoa, which has a good modifying effect on the aroma of tobacco and can improve the quality of the tobacco flavor.

[0035] Example 2: Microbiological Identification of Staphylococcus capitis W54

[0036] Morphological identification: The W54 strain obtained by the above screening was inoculated onto a nutrient agar solid culture medium plate and cultured at 35°C for 2 days before observing the colony morphology. Figure 1 As shown, colonies of Staphylococcus capitis W54 are usually opaque and round, about 2-3 mm in diameter, milky white, moist and drop-shaped, with a smooth, protruding surface and neat edges. The bacteria were examined under a microscope after Gram staining. Figure 2 As shown, the individual cells are purple (Gram-positive), spherical, and aggregated into grape-like shapes, and were preliminarily identified as Staphylococci.

[0037] Cultural characteristics: The strain W54 was inoculated into nutrient agar liquid culture medium and cultured at different temperatures. Studies have shown that the minimum growth temperature of W54 is 15°C, the maximum growth temperature is 45°C, and the optimal growth temperature is 30-37°C. When inoculated into nutrient agar liquid culture medium with different pH values and cultured at a constant temperature of 35°C, the highest growth pH of the W54 strain was 9.0, the lowest growth pH was 4.0, and the optimal growth pH was 6.5.

[0038] Molecular Biological Identification: A single colony of purified Staphylococcus capitis W54 was inoculated into 10 mL of nutrient agar medium and cultured at 35°C for 14–16 h. The culture was then centrifuged (8000 rpm for 15 min) to collect the cells. Genomic DNA from the culture was extracted using a genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.) and used as a template for PCR amplification of the 16S rRNA gene using the universal bacterial identification primers 27F (3'-AGAGTTTGATCCTGGCTCAG-3') and R1492 (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system (50 μL) consisted of 25 μL of 2× Taq Master Mix, 1.0 μL of each upstream and downstream primer, 5 μL of template DNA, and ddH2O to make up to 50 μL. PCR amplification conditions were as follows: 95°C pre-denaturation for 4 min; 30 cycles of 95°C denaturation for 30 s, 55°C annealing for 50 s, and 72°C extension for 40 s; 72°C extension for 10 min, and storage at 4°C. PCR products were recovered using a column-based PCR product purification kit (Sangon Biotech (Shanghai) Co., Ltd.) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were BLAST-compared on NCBI and analyzed using MEGA 11 software to determine the taxonomic status of the strain. A phylogenetic tree was constructed using MEGA 11 software, as shown in Figure 2. Figure 3 The obtained 16S rDNA nucleotide sequence of Staphylococcus capitis W54 is shown in SEQ ID NO: 1, and the sequence length is 1487 bp.

[0039] Based on the above-mentioned microbiological characteristics such as colony, bacterial morphology, culturology and its genetic characteristics 16S rDNA, W54 was identified as Staphylococcus capitis. The strain was deposited in the General Microbiology Center of China Culture Collection Administration on December 11, 2024, and its deposit number is CGMCC NO.33029.

[0040] Example 3: Growth curve of Staphylococcus capitis W54 in tobacco fermentation medium

[0041] The activated Staphylococcus capitis W54 was inoculated into the tobacco powder fermentation medium at an inoculum volume of 8% (v / v), and cultured at 35°C and 200 r / min for 60 h. The OD value of the culture solution was measured at 600 nm every 4 h. 600 The growth curve of Staphylococcus capitis W54 in tobacco fermentation medium was obtained by plotting the value against time. Figure 4As shown, Staphylococcus capitis W54 grew rapidly in tobacco residue fermentation medium, entered the logarithmic growth phase in 12 hours, and reached the stationary phase in 40 hours.

[0042] Example 4: Preparation of tobacco flavorings by fermenting tobacco waste culture medium with Staphylococcus capitis W54

[0043] Preparation of seed solution: Inoculate the fragrant Staphylococcus aureus W54 into nutrient agar liquid seed culture medium and culture it in a shaker (35°C, 200 rpm) for 24 hours for expansion. Adjust the bacterial concentration of the seed solution to 1.0×10 9 cfu / mL, and prepared into microbial liquid preparation.

[0044] Fermentation: The above microbial preparation was inoculated into tobacco powder fermentation medium at an inoculation rate of 8%, and then fermented at a constant temperature in a shaking incubator.

[0045] The fermentation medium consisted of 5 g of waste tobacco and 100 mL of water. The fermentation temperature was 35° C., the fermentation time was 72 h, and the shaker speed was 200 rpm. After the fermentation was completed, the fermentation liquid was centrifuged (10,000 rpm for 15 min) to obtain the supernatant.

[0046] Extraction: The fermentation broth is subjected to a primary extraction, filtration, and a secondary extraction. The solvent used in the secondary extractions is 85% ethanol by volume, the volume ratio of fermentation broth to solvent used is 1:3, and the extraction conditions are: temperature 90°C, time 2.5 hours. After the primary extraction, the extract is filtered through four layers of gauze, and the same volume of solvent as in the primary extraction is added to the filter residue for a secondary extraction, and the extract is filtered. The filtrates from the two extractions are combined to obtain a fermentation extract.

[0047] Concentration: The fermentation extract obtained above was filtered, and the filtrate was concentrated under reduced pressure to prepare a density of 1.10 g / cm 3 The concentration temperature is 55°C and the rotation speed is 55 rpm.

[0048] Example 5: Application of tobacco flavorings

[0049] The tobacco flavoring obtained in Example 4 was subjected to a flavoring evaluation of the cigarettes. 1 mL of the prepared tobacco flavoring was weighed and diluted to 10 mL with 75% ethanol. The cigarettes were then injected with a microsyringe at a flavoring amount of 0.07% of the tobacco mass. The cigarettes were then placed in an environment at a temperature of (25 ± 1) ° C and a relative humidity of (68 ± 2)% to balance moisture for 48 hours. The control group (CK) was treated with a concentrated extract of the fermentation broth of blank tobacco, and all the steps were the same as those for adding tobacco flavoring. After moisture balance was complete, a sensory evaluation panel was conducted on the two types of cigarettes. The panel consisted of eight people and evaluated the results after smoking according to the National Standard of the People's Republic of China GB 15269.4-2011, thereby judging the use value of the tobacco flavoring. The evaluation results are shown in Table 3.

[0050] Table 3 Sensory evaluation results of cigarettes after adding tobacco flavoring

[0051]

[0052] As can be seen from Table 3, compared with the control group, the aroma of the smoke produced by the tobacco treated with tobacco flavorings is significantly increased, the fragrance is more intense, the irritation is reduced, the permeability of the cigarette is enhanced, and the sense of salivation and strength are also improved, which is consistent with the composition and performance of the tobacco fermentation liquid shown in Table 2.

[0053] The embodiment of the present invention obtains a strain of aromatic Staphylococcus aureus W54 by a targeted screening method, and ferments and cultures it with tobacco dust as a culture medium, significantly increasing the content of ingredients such as phenylacetic acid, megastigmatrienone, β-ionone, damascenone, caryophyllene, (-)-isolongifoliol, trumpet alcohol, 3,5,5-trimethylcyclohex-2-ene-1-one, spirulinatone and (+)-citronene in the tobacco dust fermentation liquid, thereby improving the flavor of the tobacco dust fermentation liquid and significantly increasing the aroma volume. Using the tobacco flavoring prepared in the embodiment to treat cigarettes can increase the aroma volume, aroma quality, permeability, salivation and strength of the cigarettes, reduce irritation, reduce foreign gases, make the smoke softer, and make the aftertaste more coordinated. Therefore, the aromatic Staphylococcus aureus W54 provided in the embodiment of the present invention has great application potential in the field of improving tobacco quality. This invention establishes a microbial fermentation and aroma production technology system using tobacco waste as a substrate. By screening highly efficient degradation-and aroma-producing synergistic strains, optimizing solid-state and liquid-phase fermentation processes, and analyzing the metabolic mechanisms of key strains, it achieves efficient conversion of tobacco waste into natural tobacco flavorings. Through innovative strain screening methods and process control strategies, this invention overcomes existing technological bottlenecks and provides a new path for green transformation and high-value utilization in the tobacco industry.

[0054] Based on the above-mentioned embodiments of the present application, in the absence of explicit negation or conflict, the technical features of one embodiment may be beneficially combined with one or more other embodiments.

[0055] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application. The scope of the present application is defined by the appended claims.

Claims

1. A Staphylococcus capitis strain W54, whose deposit number is CGMCC NO.33029.

2. A microbial preparation, characterized in that: comprising the Staphylococcus capitis W54 of claim 1; Preferably, the concentration of Staphylococcus capitis W54 is ≥1.0×10 8 cfu / mL; more preferably, the concentration of Staphylococcus capitis W54 is 1.0×10 8 ~1.0×10 10 cfu / mL; Preferably, the microbial preparation is a liquid or powder.

3. A method for producing the microbial preparation according to claim 2, characterized in that: Culturing the Staphylococcus capitis W54 according to claim 1 at 30-37° C. for 24-48 hours to obtain the microbial preparation; Preferably, the culture medium used in the culture process of Staphylococcus capitis W54 is a nutrient agar liquid culture medium with a pH of 6.4 to 6.

6.

4. A tobacco flavoring, characterized in that: The Staphylococcus capitis W54 according to claim 1 or the microbial preparation according to claim 2 is inoculated into a fermentation medium for fermentation, and after the fermentation is completed, the fermentation liquid is extracted to obtain a fermentation extract, and then the fermentation extract is filtered and concentrated to obtain the tobacco flavoring; The composition of the fermentation medium is as follows: the ratio of tobacco dust mass to deionized water volume is 1:10 to 1:20 g / mL; the fermentation conditions are as follows: fermentation in a shaker at a rotation speed of 180 to 220 rpm, a temperature of 30 to 37° C., and a time of 24 to 72 hours; and the concentration conditions are as follows: a temperature of 50 to 60° C. and a rotation speed of 50 to 60 rpm.

5. The tobacco flavoring according to claim 4, wherein: The concentration of the microbial preparation is 1.0×10 8 ~1.0×10 10 cfu / mL liquid, and the inoculation amount of the microbial preparation is 5% to 10% of the volume of the fermentation culture medium.

6. The tobacco flavoring according to claim 4, wherein: After the fermentation is completed, the fermentation liquid is extracted to obtain a fermentation extract. The fermentation liquid is sequentially subjected to primary extraction, filtration, secondary extraction, and filtration. The filtrates obtained from the primary extraction and the secondary extraction are combined to obtain a fermentation extract.

7. The tobacco flavoring according to claim 6, wherein: When the fermentation liquid is subjected to primary and secondary extraction, the solvent used is ethanol with a volume fraction of 80% to 90%, the volume of the solvent used is 2 to 4 times that of the fermentation liquid, the extraction temperature is 85 to 95° C., and the extraction time is 2 to 3 hours.

8. The tobacco flavoring according to claim 4, wherein: The density of tobacco flavoring is 1.0~1.2g / cm 3 .

9. Use of the Staphylococcus capitis W54 according to claim 1, the microbial preparation according to claim 2, or the tobacco flavoring according to claim 4 in improving the aroma quality of tobacco.

10. The use according to claim 9, characterized in that: When the tobacco flavoring according to claim 4 is used to improve the aroma quality of tobacco, the tobacco flavoring is diluted and the diluent is added to the cigarette to balance the moisture.

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