Application of kokkium radicicola in cigarette aroma enhancement

Abstract

This invention provides an application of Kocuria rhizophila, wherein the application includes at least one of the following: (1) increasing the aroma of cigarettes; (2) improving the sensory quality of cigarettes; (3) increasing the content of esters, alcohols, aldehydes or ketones in cigarettes; (4) improving the floral and / or honey-sweet aroma of cigarettes; wherein the preservation number of Kocuria rhizophila ATCC 51820 is ATCC 51820.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of Kocuria rhizophila ATCC 51820 in enhancing the aroma of cigarettes. Background Technology

[0002] In recent years, the global tobacco industry has faced policy requirements to reduce tar and harm. Tobacco flavorings can compensate for aroma loss and improve the sensory quality of cigarette products while reducing tar. The development and independent control of tobacco flavorings is one of the main directions for future innovation in the industry's flavoring industry.

[0003] Microbial-mediated bio-fermentation for flavor enhancement has gradually become a research hotspot in the field of tobacco flavorings due to its advantages of naturalness, high efficiency, and safety. During their growth and metabolism, microorganisms can decompose macromolecules such as polysaccharides, proteins, and polyphenols in tobacco and tobacco flavorings, while simultaneously synthesizing various aroma and flavor substances such as esters, alcohols, aldehydes, and ketones, thereby enhancing the quality of tobacco flavorings.

[0004] In related technologies, some extract-based flavoring raw materials, due to their complex composition or excessive acidity, impart certain off-flavors and irritation to cigarettes, thus limiting their application. Tobacco flavoring microorganisms are mostly used for fermentation and flavoring of tobacco extracts, tobacco extracts, and tobacco extract solutions, but there are no research reports on their fermentation and flavoring effects on extract-based tobacco flavorings. Summary of the Invention

[0005] In view of the above, in order to at least partially solve at least one of the aforementioned technical problems, the present invention provides a *Kocuria rhizophila*, deposited at the United States Standard Strains Collection Center (ATCC) with accession number ATCC 51820.

[0006] According to one embodiment of the present invention, an application of Kocuria hizophila ATCC 51820 is provided, comprising at least one of the following: (1) increasing the aroma of cigarettes; (2) improving the sensory quality of cigarettes; (3) increasing the content of esters, alcohols, aldehydes or ketones in cigarettes; and (4) improving the floral and / or honey-sweet aroma of cigarettes.

[0007] According to another aspect of the present invention, an application of Kocuria rhizophila ATCC 51820 is provided, comprising fermenting Kocuria rhizophila ATCC 51820 in a culture medium to produce cigarette flavoring and / or compound cigarette flavoring, and adding the cigarette flavoring and / or compound cigarette flavoring to cigarettes to enhance aroma.

[0008] According to another embodiment of the present invention, a flavoring agent for cigarettes is provided, obtained by fermentation of Kocuria rhizophila ATCC 51820.

[0009] According to another embodiment of the present invention, the application of the above-mentioned flavoring agent or compound flavoring agent for cigarettes is provided in cigarette processing.

[0010] According to another embodiment of the present invention, a compound tobacco flavoring is provided, which is obtained by co-fermentation of Kocuria rhizophila ATCC 51820 and extract-type tobacco flavoring.

[0011] According to another embodiment of the present invention, a cigarette is provided containing the above-described flavoring agents for cigarettes and / or compound flavoring agents for cigarettes.

[0012] According to an embodiment of the present invention, by selecting *Kocuria rhizophila* ATCC51820, it is possible to produce floral components such as styraxyl acetate, styraxyl propionate, phenylethyl 2-methylbutyrate, and phenylethyl isobutyrate, as well as fruity components such as 2-methylbutyric acid, demonstrating a strong ability to synthesize and transform aroma substances. In the fermentation and flavoring application of tobacco extracts, this strain can effectively enhance the floral, fruity, and honey-sweet aromas of the flavorings, reduce off-flavors and irritation, and enhance the richness and harmony of the aroma, making the resulting flavorings more suitable for flavoring tobacco and tobacco products. At the same time, this fermentation and flavoring process is simple to operate, requires no special treatment, and is applicable to various extract-based tobacco flavorings such as chicory extract, vanilla extract, hawthorn extract, and plum extract. It has the advantages of a wide range of applicable raw materials and ease of large-scale production, providing an efficient and feasible biotechnological approach for improving the quality and industrial development of tobacco flavorings. Attached Figure Description

[0013] The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the invention with reference to the accompanying drawings, in which:

[0014] Figure 1 The diagram shows the colony morphology of Kocuria rhizophila ATCC 51820 after activation according to the present invention.

[0015] Figure 2 The diagram shows the aroma characteristics of the fermentation broth of Kocuria rhizophila ATCC 51820 according to the present invention;

[0016] Figure 3 The growth curve (OD) of Kocuria rhizophila ATCC 51820 of the present invention in modified LB liquid medium is shown. 600 )picture;

[0017] Figure 4 The sensory evaluation results of flavored blank cigarettes made from fermentation broth of Kocuria rhizophila ATCC 51820 at different fermentation times are shown in the figure.

[0018] Figure 5 The OD values ​​of Kocuria rhizophila ATCC 51820 of this invention are shown under different inorganic salt formulations and culture conditions. 600 The sensory evaluation results of the fermentation broth and flavored blank cigarettes are shown in the figure, where a represents the OD under different inorganic salt formulations and culture conditions. 600 Figure b shows the sensory evaluation results.

[0019] Figure 6 The OD values ​​of Kocuria rhizophila ATCC 51820 of this invention under different carbon source culture conditions are shown. 600 The sensory evaluation results of the fermentation broth and flavored blank cigarettes are shown in the figure, where a represents the OD under different inorganic salt formulations and culture conditions. 600 Figure b shows the sensory evaluation results.

[0020] Figure 7 The sensory evaluation results of the flavored blank cigarette made with Kocuria rhizophila ATCC 51820 fermented chicory extract are shown in the figure.

[0021] Figure 8 The image shows the sensory evaluation results of flavored blank cigarettes obtained by fermenting chicory extract with Kocuria rhizophila ATCC 51820 at different fermentation temperatures.

[0022] Figure 9 The image shows the sensory evaluation results of flavored blank cigarettes made from fermented chicory extract obtained by fermenting with Kocuria rhizophila ATCC 51820 at different fermentation times.

[0023] Figure 10 The sensory evaluation results of flavored blank cigarettes obtained by fermenting Kocuria rhizophila ATCC 51820 with different amounts of chicory extract are shown in the figure.

[0024] Figure 11 The sensory evaluation results of the blank cigarette flavored with Kocuria rhizophila ATCC 51820 fermented vanilla extract are shown in the figure. Detailed Implementation

[0025] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0027] When using expressions such as "at least one of A, B and C", they should generally be interpreted in accordance with the meaning that is commonly understood by those skilled in the art (e.g., "a system having at least one of A, B and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B and C, etc.).

[0028] Specifically, according to one embodiment of the present invention, the application of Kocuria hizophila ATCC 51820 is provided, including at least one of the following: (1) increasing the aroma of cigarettes; (2) improving the sensory quality of cigarettes; (3) increasing the content of esters, alcohols, aldehydes or ketones in cigarettes; (4) improving the floral and / or honey-sweet aroma of cigarettes.

[0029] According to an embodiment of the present invention, by applying *Kocuria rhizophila* ATCC 51820 to a cigarette flavoring system, the bacteria can generate aroma components such as esters, alcohols, aldehydes, or ketones during their growth and metabolism, enriching the aroma composition of cigarettes and thus increasing their aroma. Furthermore, the transformation or generation of aroma components by *Kocuria rhizophila* ATCC 51820 helps enhance the floral and / or honey-sweet aroma of cigarettes, improving their aroma characteristics and harmony, thereby enhancing their sensory quality. During fermentation, *Kocuria rhizophila* ATCC 51820 can increase the content of esters in cigarettes, contributing to enhanced floral and honey-sweet aroma characteristics; the content of alcohols, aldehydes, or ketones in cigarettes can be increased, helping to enrich the aroma layers and style characteristics of cigarettes, thereby improving their usability and application value.

[0030] In one specific embodiment of the present invention, an application of Kocuria rhizophila ATCC 51820 is provided, comprising: fermenting Kocuria rhizophila ATCC 51820 in a culture medium to produce cigarette flavoring and / or compound cigarette flavoring, and adding the cigarette flavoring and / or compound cigarette flavoring to cigarettes to enhance aroma.

[0031] According to an embodiment of the present invention, the above-mentioned tobacco flavoring is obtained directly by fermenting Kocuria rhizophila ATCC 51820 in a culture medium, producing volatile flavor substances such as esters, alcohols, aldehydes, and ketones that contribute to aroma through its growth and metabolism; the compound tobacco flavoring is obtained by co-fermenting Kocuria rhizophila ATCC 51820 with extract-type tobacco flavoring, through which the strains biotransform, degrade, or modify the macromolecular precursor substances in the extract-type tobacco flavoring, further generating and enriching flavor components with characteristic aromas such as floral, fruity, and honey-sweet aromas.

[0032] According to embodiments of the present invention, extract-based tobacco flavorings refer to natural tobacco flavorings derived from plant raw materials and obtained through extraction processes, such as chicory extract, vanilla extract, hawthorn extract, plum extract, etc., and may also include fruit extracts, flower extracts, spice extracts, and extracts from medicinal and edible plants commonly used in the tobacco industry. These extracts are typically prepared using methods such as water extraction, organic solvent extraction, ultrasound-assisted extraction, reflux extraction, enzymatic extraction, or supercritical fluid extraction. After extraction, the extract is concentrated and filtered to obtain a stock solution suitable for fermentation.

[0033] According to an embodiment of the present invention, the fermentation process of Kocuria rhizophila ATCC 51820 includes amplification culture and fermentation culture: wherein the amplification culture time is 6-72h and the amplification culture temperature is 20-38℃; the fermentation time is 12-72h and the fermentation culture temperature is 20-38℃.

[0034] According to an embodiment of the present invention, the amplification culture time can be 6h-72h. Exemplarily, the amplification culture time can be any two values ​​between 6h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, or more. The amplification culture temperature can be 20℃-38℃. Exemplarily, the amplification culture temperature can be any two values ​​between 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 38℃, or more. The fermentation culture time can be 12h-72h. Exemplarily, the fermentation culture time can be any two values ​​between 12h, 18h, 24h, 36h, 48h, 60h, 72h, or more. The fermentation culture temperature can be 20℃-38℃. For example, the fermentation temperature can be any two values ​​between 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 38℃, or above. The coordinated settings of the amplification culture time, amplification culture temperature, fermentation culture time, and fermentation culture temperature are beneficial to the growth and fermentation metabolism of *Kocuria rhizophila* ATCC 51820, thereby obtaining tobacco flavorings and / or compound tobacco flavorings.

[0035] According to an embodiment of the present invention, the culture medium further includes extractable tobacco flavoring; the amount of extractable tobacco flavoring added is 3%-20% of the mass of the culture medium, preferably 5%-20%; the extractable tobacco flavoring may be selected from one or more of chicory extract, vanilla extract, hawthorn extract, and plum extract. The amount of extractable tobacco flavoring added can be 3%-20% of the mass of the culture medium. Exemplarily, the amount of extractable tobacco flavoring added can be 3%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, or any two of the above values.

[0036] According to an embodiment of the present invention, the extract-type tobacco flavoring, after being added to the culture medium, can serve as a substrate for the fermentation and transformation of Kocuria rhizophila ATCC 51820. Through the fermentation of Kocuria rhizophila ATCC 51820, the variety of aroma substances in the extract-type tobacco flavoring can be increased, promoting the generation and transformation of aroma components, thereby obtaining a compound tobacco flavoring. Applying this compound tobacco flavoring to cigarettes can enrich the aroma composition of the cigarettes, giving them different flavors such as chicory, vanilla, hawthorn, and plum, and improving the sensory quality of the cigarettes.

[0037] According to an embodiment of the present invention, the fermentation medium is a modified LB medium, and based on the original LB medium components (5 g peptone, 5 g glucose, 10 g NaCl, 1.0 g CaCl2, 1000 mL water), 3-10 g / L of glucose and 0.5-2 g / L of CaCl2 are added per liter of medium. Exemplarily, the amount of glucose added can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any two of the above values. The amount of CaCl2 added can be 0.5 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, 2.0 g / L, or any two of the above values. By adding glucose and CaCl2 to LB medium, suitable culture conditions can be provided for the growth and fermentation metabolism of *Kocuria rhizophila* ATCC 51820. Glucose serves as a carbon source for bacterial growth and metabolism, while CaCl2 participates in the bacterial growth process and further activates bacterial enzymes, thereby promoting the conversion of aroma substances. When the fermentation products obtained using this modified LB medium are applied to cigarettes, they can enhance the floral and fruity aroma characteristics and improve the honey-sweet aroma, thus improving the sensory quality of cigarettes.

[0038] According to another embodiment of the present invention, a flavoring agent for cigarettes is provided, obtained by fermentation of Kocuria rhizophila ATCC 51820. Through the direct fermentation metabolism of Kocuria rhizophila ATCC 51820 in modified LB medium, aroma components such as esters, alcohols, aldehydes, or ketones can be directly generated and transformed, enriching the aroma composition of cigarettes, increasing the aroma of cigarettes, thereby increasing the variety of aroma components in the flavoring agent for cigarettes and enriching the aroma characteristics of the flavoring agent for cigarettes.

[0039] According to another embodiment of the present invention, a compound tobacco flavoring is provided, which is obtained by co-fermentation of Kocuria rhizophila ATCC 51820 and extract-based tobacco flavoring. Through the metabolic activity of Kocuria rhizophila ATCC 51820 during fermentation, the components in the extract-based tobacco flavoring can be transformed, generating or enriching aroma-related components, thereby altering the composition of aroma substances in the compound tobacco flavoring and enriching its aroma characteristics.

[0040] According to another embodiment of the present invention, the application of the above-mentioned flavoring agents and / or compound flavoring agents for cigarettes is provided in cigarette processing. These flavoring agents or compound flavoring agents can be used in the cigarette flavoring or blending process to improve the aroma characteristics of the cigarettes. After applying these flavoring agents or compound flavoring agents to cigarettes, the aroma of the cigarettes can be enhanced, improving the floral, fruity, and honey-sweet notes, and improving the sensory quality of the cigarettes, resulting in a richer and more harmonious aroma.

[0041] According to an embodiment of the present invention, the amount of flavoring agent and / or compound flavoring agent added to cigarettes is 2~10 μL / g. Exemplarily, the amount added can be any two values ​​between 2 μL / g, 5 μL / g, 8 μL / g, 10 μL / g, or more. By controlling the amount of flavoring agent and / or compound flavoring agent added, the release intensity of aroma components in cigarettes can be adjusted, thereby regulating the aroma concentration and aroma harmony of cigarettes, making the floral, fruity, and honey-sweet aroma characteristics of cigarettes more balanced, thus improving the sensory quality of cigarettes.

[0042] According to another embodiment of the present invention, a cigarette is provided containing flavoring agents and / or compound flavoring agents. The flavoring agents and / or compound flavoring agents in the cigarette system can alter the composition of aroma components in the cigarette, thereby increasing the cigarette's aroma. This cigarette exhibits floral, fruity, and honey-sweet aroma characteristics during smoking, making the overall aroma of the cigarette richer and more harmonious, while improving the sensory quality of the cigarette.

[0043] Furthermore, this invention achieves the transformation and quality improvement of tobacco flavorings through microbial fermentation. The process conditions are relatively mild and the operation is relatively simple, which is conducive to its promotion and application in the fields of tobacco flavoring processing and cigarette flavoring, and provides a new technical path for the microbial modification of tobacco flavorings.

[0044] The present invention will be further explained below through specific embodiments. Unless otherwise specified, all reagents used are commercially available reagents and all experimental methods used are conventional experimental methods in the art.

[0045] Example 1

[0046] Strain activation: Kocuria rhizophila ATCC 51820, deposited at the U.S. Center for Standard Strains, accession number ATCC 51820.

[0047] Figure 1 The diagram shows the colony morphology of Kocuria rhizophila ATCC 51820 after activation.

[0048] Kocuria rhizophila ATCC 51820 was inoculated onto LB solid medium (5 g peptone, 5 g yeast extract, 10 g NaCl, 18 g agar, 1000 mL water, dissolved and autoclaved at 121°C for 20 min), and then incubated at 30°C for 24 h to complete the activation of the strain. The morphological characteristics of this bacterium are as follows... Figure 1 As shown, the colonies are light yellow and opaque, and are oval or irregular in shape.

[0049] Example 2

[0050] Aroma evaluation of fermentation broth from Kocuria rhizophila ATCC 51820 strain.

[0051] Figure 2 The diagram shows the aroma characteristics of the fermentation broth of Kocuria rhizophila ATCC 51820 according to the present invention.

[0052] Activated Kocuria rhizophila ATCC 51820 was inoculated into modified LB broth (5 g peptone, 5 g glucose, 10 g NaCl, 1.0 g CaCl2, 1000 mL water, dissolved and autoclaved at 121°C for 20 min) and cultured for 36 h. The fermentation broth of Kocuria rhizophila ATCC 51820 was centrifuged and filtered through a microporous membrane for sensory evaluation of aroma. Its aroma characteristics are as follows: Figure 2 As shown. The fermentation broth of this bacterium has a distinct floral and fruity aroma, a certain honey-sweetness, and a slightly sour taste.

[0053] Example 3

[0054] A comparative evaluation of the aroma-enhancing effect of Kocuria rhizophila ATCC51820 fermentation broth cultured on modified LB medium and ordinary LB medium.

[0055] Activated Kocuria rhizophila ATCC 51820 was inoculated into modified LB liquid medium and cultured for 36 h. The fermentation broth of Kocuria rhizophila ATCC 51820 was centrifuged and filtered through a microporous membrane. 5.0 μL of the broth was then uniformly injected into blank cigarettes from Hunan Tobacco as the modified medium group.

[0056] Kocuria rhizophila ATCC 51820 was cultured in ordinary LB medium. After centrifugation and filtration, the fermentation broth was injected evenly into blank cigarettes of Hunan Tobacco at a ratio of 5.0 μL per cigarette as the LB medium group.

[0057] A judging panel composed of seven qualified sensory evaluators conducted a preliminary sensory evaluation based on the "Sensory Evaluation Method for Chinese-style Cigarettes" (YC / T 497-2014). The sensory evaluation method was then fine-tuned. Scoring was primarily based on five aroma characteristics: "roasted aroma," "honey-sweet aroma," "milky aroma," "fruity aroma," and "floral aroma," with a scoring range of 0-5 points. The average sensory evaluation score was taken. The results are shown in Table 1.

[0058] Table 1

[0059]

[0060] Table 1 shows that, compared with the LB medium group, the fermentation broth of Kocuria hizophila ATCC 51820 cultured in the modified medium, when injected into blank cigarettes, significantly enhanced the floral and fruity aromas in the cigarette smoke, and also strengthened the roasted, honey-sweet, and milky aromas. The smoke permeability was better, the irritation was reduced, and the aftertaste was more pleasant.

[0061] Example 4

[0062] The aroma components of the fermentation broth of Kocuria rhizophila ATCC 51820 were analyzed by gas chromatography-mass spectrometry (GC-MS).

[0063] Take 15 mL of the fermentation broth from Example 2, add 7 mL of dichloromethane for extraction twice, combine the dichloromethane layers and concentrate to 0.9 mL, add 0.1 mL of 0.8211 mg / mL phenethyl acetate as an internal standard, and perform GC-MS analysis.

[0064] Gas chromatography (GC) conditions: Agilent HP-5 MS column (30 m × 250 μm × 0.25 μm). Injector temperature: 280 °C. Carrier gas: high-purity helium (99.999%), flow rate: 1.0 mL / min. Temperature program: initial temperature 50 °C, hold for 4 min, then increase to 240 °C at a rate of 2 °C / min, no separation. Injection volume: 1.0 μL.

[0065] Mass spectrometry (MS) conditions: transfer line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; ionization method: electron impact (EI) with electron energy of 70 eV; solvent delay time: 7 min. Full scan mass range (m / z) 35-550 u, GC-MS in full scan mode.

[0066] The GC-MS analysis results of the main aroma components of the fermentation broth of Kocuria rhizophila ATCC 51820 are shown in Table 2 below.

[0067] Table 2

[0068]

[0069] Table 2 shows that the fermentation broth of *Kocuria rhizophila* ATCC 51820 contains ester aroma components such as styraxyl acetate, styraxyl propionate, phenylethyl 2-methylbutyrate, and phenylethyl isobutyrate, with contents of 2.75, 12.71, 1.59, and 1.42 μg / mL, respectively. At low concentrations, styraxyl acetate, styraxyl propionate, phenylethyl 2-methylbutyrate, and phenylethyl isobutyrate exhibit floral and honey-sweet aromas, which can enhance the floral and honey-sweet aromas of tobacco products. 2-Methylbutyrate can enhance fruity and yogurt-like aromas while reducing irritation. Therefore, the cigarette smoke after flavoring with the fermentation broth is rich in floral, fruity, and honey-sweet aromas.

[0070] Example 5

[0071] Optimization of culture time for Kocuria rhizophila ATCC 51820.

[0072] Figure 3 The growth curve (OD) of Kocuria rhizophila ATCC 51820 of the present invention in modified LB liquid medium is shown. 600 )picture;

[0073] Figure 4 The figure shows the sensory evaluation results of flavored blank cigarettes made from the fermentation broth of Kocuria rhizophila ATCC 51820 at different fermentation times according to the present invention.

[0074] Kocuria rhizophila ATCC 51820 was inoculated into modified LB liquid medium and cultured for 12h, 24h, 36h, 48h, and 60h, respectively. The turbidity (OD) value at 600 nm was measured using a UV spectrophotometer. The OD values ​​are shown below. Figure 3 As shown.

[0075] Sensory evaluation was conducted by injecting 5.0 μL of the fermentation broth filtered through membranes at different fermentation times into blank cigarettes. The sensory results are as follows: Figure 4 As shown.

[0076] like Figure 3 , 4The results showed that the growth curve of the strain from 0 to 60 hours was roughly "I"-shaped. The 0-12 hour period was a lag phase, during which the cell concentration increased slowly. The 12-48 hour period was a logarithmic growth phase, with rapid growth, especially from 24 to 48 hours, during which cell metabolism was very active. After 48 hours, growth tended to stabilize. When the culture time was 12 hours, the floral and fruity aromas in cigarettes containing the fermentation broth from the strain were relatively weak. When the culture time of *Kocuria rhizophila* ATCC 51820 was 24 to 36 hours, its application in blank cigarettes significantly enhanced the floral and fruity aromas, while also highlighting the roasted and milky aromas, resulting in a richer cigarette smoke and a significantly reduced irritation. The fermentation broth fermented for 36 hours showed the best effect, with the most significant improvement in overall sensory quality. However, when the culture time exceeded 36 hours, the aroma of cigarettes flavored with the fermentation broth weakened, with a slight off-flavor and a slightly unpleasant aftertaste. Based on the growth curve of the strain and the sensory evaluation results of cigarettes, the optimal fermentation time for Kocuria rhizophila ATCC51820 strain was determined to be 36 h.

[0077] Example 6

[0078] Inorganic salt optimization for Kocuria rhizophila culture ATCC 51820.

[0079] Figure 5 The OD values ​​of Kocuria rhizophila ATCC 51820 of this invention are shown under different inorganic salt formulations and culture conditions. 600 The sensory evaluation results of the fermentation broth and flavored blank cigarettes are shown in the figure, where a represents the OD under different inorganic salt formulations and culture conditions. 600 Figure b shows the sensory evaluation results.

[0080] The liquid culture medium was optimized for inorganic salt composition by replacing CaCl2 in the modified LB medium of Example 2 with KCl, Mg2SO4, and KH2PO4, using ordinary LB medium as a control. Liquid culture was carried out at 30℃ for 36 h, and the absorbance at 600 nm was measured using a UV spectrophotometer. 5.0 μL of the fermentation broth was filtered and injected into blank cigarettes for sensory evaluation. The results are as follows: Figure 5 As shown.

[0081] according to Figure 5 The results showed that replacing inorganic salts with magnesium sulfate and potassium chloride negatively impacted bacterial growth, resulting in poor development. Adding CaCl2 further reduced OD... 600The highest bacterial concentration and best bacterial growth were observed in the fermentation broth. When Mg2SO4, KH2PO4, and KCl were added to blank cigarettes, the aroma quality decreased, with significantly weakened floral, fruity, and milky aromas, and greater irritation and off-flavors, resulting in a poor aftertaste. However, when CaCl2 was added to the fermentation broth and injected into blank cigarettes, the floral and fruity aromas became more prominent, with enhanced honey and roasted notes, and the smoke was smooth with no noticeable irritation or off-flavors. Based on the combined analysis, CaCl2 is the preferred inorganic salt for cultivating this strain.

[0082] Example 7

[0083] Optimization of carbon source for Kocuria rhizophila culture on ATCC 51820.

[0084] Figure 6 The OD values ​​of Kocuria rhizophila ATCC 51820 of this invention under different carbon source culture conditions are shown. 600 The sensory evaluation results of the fermentation broth and flavored blank cigarettes are shown in the figure, where a represents the OD under different inorganic salt formulations and culture conditions. 600 Figure b shows the sensory evaluation results.

[0085] The carbon source formulation of the liquid culture medium was optimized. In Example 2, glucose was replaced with starch, fructose, and sucrose in the modified LB medium. Using standard LB medium as a control, the medium was cultured at 30°C for 36 hours. The absorbance at 600 nm was measured using a UV spectrophotometer. 5.0 μL of the fermentation broth was injected into blank cigarettes via a filter membrane for sensory evaluation. The evaluation results are as follows: Figure 6 As shown.

[0086] Depend on Figure 6 The results showed that glucose was the highest carbon source for bacterial concentration and the best bacterial growth. When starch, fructose, and sucrose were used as carbon sources, the strains did not grow well. When sucrose and fructose were used as carbon sources, the fruity aroma in the smoke was weak, with more off-flavors and irritation, and a bitter aftertaste. When glucose was used as the carbon source, the fruity and floral aromas were more pronounced, with enhanced roasted and honey-sweet notes, increased aroma content in the smoke, and reduced irritation. Based on the combined analysis of these two indicators, glucose can be considered the preferred carbon source for the strains.

[0087] Example 8

[0088] The fermentation and flavoring effect of Kocuria rhizophila ATCC 51820 on chicory extract.

[0089] Figure 7The sensory evaluation results of the flavored blank cigarette made with Kocuria rhizophila ATCC 51820 fermented chicory extract are shown in the figure.

[0090] In this invention, the extract tobacco flavoring includes commercially available chicory extract, vanilla extract, hawthorn extract, and plum extract, etc., wherein the extract tobacco flavoring is prepared by the following method:

[0091] When using chicory extract as a flavoring agent for cigarettes, the chicory extract was first added to a modified LB liquid medium at 10% of the medium mass. The *Kocuria rhizophila* ATCC 51820 culture cultured in Example 2 was inoculated at a 1% inoculum, and the mixture was liquid-cultured at 30°C for 36 hours. One mL of the fermentation broth was centrifuged at 6000 r / min for 7 minutes to obtain the supernatant. This supernatant was then injected into blank cigarettes for sensory evaluation. The sensory evaluation results of the flavoring of cigarettes before and after chicory extract fermentation are shown in Table 3. Figure 7 As shown.

[0092] Table 3

[0093]

[0094] According to Table 3 above and Figure 7 The results showed that chicory extract, when added to cigarette flavoring, enhanced the roasted and honey-sweet aromas, but had little effect on the floral and fruity aromas. Furthermore, the chicory extract contained large molecular impurities such as starch and pectin, resulting in some off-flavors and a slightly irritating sensation. However, chicory extract fermented with *Kocuria rhizophila* ATCC 51820, when added to cigarette flavoring, significantly enhanced the fruity and floral aromas, and also improved the honey and roasted sweet aromas. The off-flavors in the extract were noticeably reduced, the irritation decreased, and the overall effect was significantly improved.

[0095] Example 9

[0096] Analysis of aroma components in chicory extract before and after fermentation with Kocuria rhizophila ATCC 51820.

[0097] Take 15 mL of chicory extract from both before and after co-fermentation with Kocuria rhizophila ATCC 51820 in Example 8, extract twice with 7 mL of dichloromethane, combine the dichloromethane layers and concentrate to 0.9 mL. Add 0.1 mL of 0.8211 mg / mL phenethyl acetate as an internal standard and perform GC-MS analysis. The GC-MS conditions are the same as in Example 3, and the results are shown in Table 4.

[0098] Table 4

[0099]

[0100] Table 4 shows that fermentation of chicory extract increased the content of characteristic sesquiterpenoid components of chicory, such as Jacquinelin, Austricin, Estafiatin, and α-dehydro-aryl-himachalide, enhancing the floral, fruity, and honey-sweet aromas of cigarettes. Increased content of esters, such as styraxyl acetate, styraxyl propionate, and phenylethyl 2-methylbutyrate, enhanced the floral and honey-sweet aromas of cigarettes and improved the richness of the flavor profile. Increased content of 2-methylbutyric acid, ethyl butyrate, ethyl pyruvate, and ethyl 2-propyl acetoacetate improved the fruity aromas of cigarettes and reduced irritation and off-flavors. The increased content of 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) enhances the roasted and honey-sweet aroma of cigarettes.

[0101] Example 10

[0102] Optimization of fermentation temperature for chicory extract fermented with Kocuria rhizophila ATCC 51820.

[0103] Figure 8 The figure shows the sensory evaluation results of flavored blank cigarettes obtained by fermenting chicory extract with Kocuria rhizophila ATCC 51820 at different fermentation temperatures.

[0104] Chicory extract was added to modified LB liquid medium at 10% of the culture medium mass. Kocuria rhizophila ATCC 51820 culture from Example 2 was inoculated at a 1% inoculum, and cultured in liquid medium at 26℃, 28℃, 30℃, and 32℃ for 36 h. 1 mL of fermentation broth was centrifuged at 6000 r / min for 7 min to obtain the supernatant. The supernatant was filtered through a membrane and injected into blank cigarettes for sensory evaluation.

[0105] The results are as follows Figure 8As shown, at lower fermentation temperatures of 26℃ and 28℃, the roasted and honey-sweet aromas of cigarettes infused with fermented chicory extract were enhanced, while the floral and fruity aromas were not significantly improved, with slight off-flavors and a pungent aftertaste. At a higher fermentation temperature of 32℃, although the smoky and fruity aromas of cigarettes infused with fermented chicory extract were enhanced, the off-flavors were heavier, with a certain degree of pungentness and an unpleasant aftertaste. Overall, a fermentation temperature of 30℃ is optimal.

[0106] Example 11

[0107] Fermentation time optimization of chicory extract fermented with Kocuria rhizophila ATCC 51820.

[0108] Figure 9 The image shows the sensory evaluation results of flavored blank cigarettes made from fermented chicory extract obtained by fermenting with Kocuria rhizophila ATCC 51820 at different fermentation times.

[0109] Chicory extract was added to LB liquid medium at 10% of the culture medium mass. Kocuria rhizophila ATCC 51820 cultured in Example 2 was inoculated at a 1% inoculum and cultured in liquid medium at 30°C for 12, 24, 36, and 48 hours. 1 mL of fermentation broth was centrifuged at 6000 r / min for 7 min to obtain the supernatant. The supernatant was filtered through a membrane and injected into blank cigarettes for sensory evaluation.

[0110] The results are as follows Figure 9 As shown, at fermentation times of 12h and 24h, the enhancement of the smoky and fruity aromas in cigarettes injected with the fermentation liquid was not significant, and the overall aroma richness was insufficient. After fermentation time exceeding 36h, the smoke from cigarettes scented with the fermentation liquid had a slightly off-putting smell, and the aftertaste was somewhat unpleasant. An overall fermentation time of 36h is optimal.

[0111] Example 12

[0112] Optimization of the addition amount of Kocuria rhizophila ATCC 51820 fermented chicory extract.

[0113] Figure 10 The image shows the sensory evaluation results of flavored blank cigarettes made from fermentation broth obtained by fermenting Kocuria rhizophila ATCC 51820 with different amounts of chicory extract.

[0114] Chicory extract was added to LB liquid medium at concentrations of 5%, 10%, 15%, and 20% of the medium mass. Kocuria rhizophila ATCC 51820 culture from Example 2 was inoculated at a 1% inoculum, and the mixture was incubated at 30°C for 36 h. One mL of the fermentation broth was centrifuged at 6000 r / min for 7 min to obtain the supernatant. This supernatant was filtered through a membrane and injected into blank cigarettes for sensory evaluation.

[0115] The results are as follows Figure 10 As shown, when the chicory extract content is 5%, the improvement in the smoky and fruity aromas of the cigarettes injected into the fermented liquid is not significant, and the overall aroma richness is not improved enough. When the chicory extract content is 15% and 20%, the aroma richness of the cigarettes flavored with the fermented liquid is acceptable, but there are certain off-flavors and a pungent feeling. The overall addition amount is 10% as optimal.

[0116] Example 13

[0117] The Fermentation and Flavoring Effect of Kocuria rhizophila ATCC 51820 on Vanilla Extract

[0118] Figure 11 The sensory evaluation results of the flavored blank cigarette made with Kocuria rhizophila ATCC 51820 fermented vanilla extract are shown.

[0119] 100 mL of modified LB liquid medium containing vanilla extract prepared using the same method as in Example 8 (vanilla extract added at 10% of the medium mass) was prepared. Kocuria rhizophila ATCC 51820 cultured in Example 2 was inoculated at a 1% inoculum, and liquid fermentation was carried out at 30°C for 36 h. After fermentation, the fermentation broth was centrifuged at 6000 r / min for 7 min, and the supernatant was filtered through a microporous membrane. 5 μL of the supernatant was injected into blank cigarettes for sensory evaluation. The sensory evaluation results are shown in Table 5 and... Figure 11 As shown.

[0120] Table 5

[0121]

[0122] From Table 5 and Figure 11 The results showed that vanilla extract enhanced the milky and fruity aromas of cigarettes to some extent, but its enhancement of floral aromas was not very significant. After co-fermentation with Kocuria rhizophila ATCC 51820, the floral aromas of the cigarettes were significantly enhanced, and the honey and fruity aromas were further strengthened, resulting in good overall harmony.

[0123] Example 14

[0124] The fermentation and flavoring effect of Kocuria rhizophila ATCC 51820 on hawthorn extract.

[0125] Prepare 100 mL of modified LB liquid medium containing hawthorn extract prepared using the same method as in Example 8, wherein the amount of hawthorn extract added is 10% of the mass of the medium. Inoculate the cultured Kocuria hizophila ATCC 51820 from Example 2 at a 1% inoculum and ferment at 30°C for 36 h. After fermentation, centrifuge the fermentation broth at 6000 r / min for 7 min, collect the supernatant, filter it through a microporous membrane, and inject 5 μL into blank cigarettes for sensory evaluation.

[0126] Sensory evaluation results showed that the addition of hawthorn extract significantly enhanced the fruity aroma of cigarettes, but the acidity was more pronounced, while the floral aroma was not significantly improved, and there was a certain degree of irritation and astringency. After co-fermentation with Kocuria rhizophila ATCC 51820, the addition of fermented hawthorn extract significantly enhanced the aroma of cigarettes, further improving the honey and fruity notes, reducing the acidity, and resulting in a richer overall aroma and improved usability.

[0127] Example 15

[0128] The fermentation and flavoring effect of Kocuria rhizophila ATCC 51820 on plum extract.

[0129] Prepare 100 mL of modified LB liquid medium containing plum extract prepared using the same method as in Example 8, wherein the amount of plum extract added is 10% of the mass of the medium. Inoculate the Kocuria hizophila ATCC 51820 liquid culture from Example 2 at a 1% inoculum amount and ferment it in liquid at 30°C for 36 h. After fermentation, centrifuge the fermentation broth at 6000 r / min for 7 min, take the supernatant, filter it through a microporous membrane, and inject 5 μL into blank cigarettes for sensory evaluation.

[0130] Sensory evaluation results showed that the addition of plum extract significantly enhanced the fruity aroma of cigarettes, but the aftertaste was sour with a certain degree of grassy odor, while the improvement in floral and honey-sweet aromas was not significant. After co-fermentation with Kocuria rhizophila ATCC51820, the addition of fermented plum extract significantly enhanced the smoky and honey-sweet aromas of cigarettes, further improved the fruity aroma, reduced the sourness and grassy odor, and resulted in a more pleasant overall aroma and aftertaste, thus improving usability.

[0131] This invention systematically optimizes and verifies the culture conditions, culture medium components, and fermentation effects of *Kocuria rhizophila* ATCC 51820 on different extract tobacco flavorings. It clarifies that this strain exhibits good growth and metabolic activity in a modified LB culture system. The preferred fermentation conditions are 30℃ for 36 h, with glucose as the preferred carbon source and CaCl2 as the preferred inorganic salt additive. Based on these conditions, the fermentation broth of this strain and its co-fermentation products with extract tobacco flavorings significantly improve the sensory quality of cigarettes, particularly in enhancing key aromas such as floral, fruity, honey, and roasted notes. Simultaneously, it effectively reduces irritation and off-flavors, and improves the harmony of the aftertaste. Further GC-MS analysis revealed that the fermentation process enriched various key aroma active components, including esters, furans, pyrazines, and sesquiterpenes, providing a material explanation for the enhanced aroma. Different extracts (chicory, vanilla, hawthorn, and plum) all exhibited a common effect of aroma enhancement and off-flavor reduction after fermentation, indicating that this strain has broad potential for biotransformation and flavor enhancement in tobacco flavorings. Therefore, this invention provides a bio-flavor enhancement technology system based on *Coprinovia rumenophila* fermentation, which can be used to improve the aroma quality and harmony of cigarettes and related tobacco products.

[0132] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of Kocuria rhizophila ATCC 51820, wherein, The application includes at least one of the following: (1) Increase the aroma of cigarettes; (2) Improve the sensory quality of cigarettes; (3) Increase the content of esters, alcohols, aldehydes or ketones in cigarettes; (4) Enhance the floral and / or honey-sweet aroma of cigarettes; The preservation number of Kocuria rhizophila ATCC 51820 is ATCC51820.

2. An application of Kocuria rhizophila ATCC 51820, the application comprising: Kocuria rhizophila ATCC 51820 is fermented in a culture medium to produce cigarette flavoring and / or compound cigarette flavoring, which is then added to cigarettes to enhance aroma.

3. Use according to claim 2, wherein, The fermentation process of the Kocuria rhizophila ATCC 51820 includes amplification culture and fermentation culture: The amplification culture time is 6-72 hours, and the amplification culture temperature is 20-38℃; The fermentation time is 12-72 hours, and the fermentation temperature is 20-38℃. The fermentation carbon source includes starch, fructose, and sucrose, preferably glucose.

4. The application according to claim 2, wherein, The culture medium also includes extracts of tobacco flavorings; The amount of the extract-type tobacco flavoring added is 3%-20% of the mass of the culture medium, preferably 5%-20%; The extract-type tobacco flavoring is selected from one or more of chicory extract, vanilla extract, hawthorn extract, and plum extract.

5. The application according to claim 2 or 4, The culture medium is a modified LB medium, with 3-10 g / L glucose and 0.5-2 g / L CaCl2 added per liter of LB medium.

6. A flavoring agent for cigarettes, obtained by fermentation of Kocuria rhizophila ATCC 51820.

7. A compound tobacco flavoring, obtained by co-fermentation of Kocuria rhizophila ATCC 51820 and extract-based tobacco flavoring.

8. The application of a cigarette flavoring as described in claim 6 or a compound cigarette flavoring as described in claim 7 in cigarette processing.

9. Use according to claim 8, wherein, The amount of the flavoring agent for cigarettes and / or the compound flavoring agent for cigarettes added to cigarettes is 2-20 μL / g.

10. A cigarette containing the flavoring agent for cigarettes as claimed in claim 6 or the compound flavoring agent for cigarettes as claimed in claim 7.

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