Rhodotorula mucilaginosa H04 and tobacco flavor prepared from same

Abstract

The invention belongs to the technical field of tobacco strains, and particularly relates to a rhodotorula mucilaginosa H04 strain and application thereof in preparation of tobacco flavor. The rhodotorula mucilaginosa H04 provided by the invention has excellent aroma producing performance and can produce tobacco characteristic aroma substances such as alcohols, aldehydes, esters and pyrazines at high yield. The rhodotorula mucilaginosa H04 has good tolerance to nicotine, can still keep high growth activity under the condition that the nicotine concentration is as high as 8 mg / mL, and is suitable for being used for preparing tobacco flavor; the tobacco flavor prepared by the invention contains a plurality of natural aroma components, has good coordination with the original aroma of tobacco, and can significantly improve the aroma richness and comfort of tobacco products; the highest beta-glucosidase activity of the rhodotorula mucilaginosa H04 reaches 464.0 + / -24.4 U / mL, and the laccase activity reaches 60.4 + / -3.2 U / mL, so that the release of bound aroma precursor substances in tobacco is facilitated, and the aroma expression is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of tobacco processing technology, specifically relating to a strain of Rhodotorula mucilaginosa H04 and its application in the preparation of tobacco flavorings. Background Technology

[0002] The aroma quality of tobacco products is one of the important indicators for evaluating their quality. Currently, the main method to improve the aroma quality of tobacco is to add flavorings, and flavorings derived from natural fermentation are of great interest due to their natural aroma and high safety. Traditional tobacco flavorings are mostly derived from plant extracts or chemical synthesis, which have problems such as monotonous aroma, high cost, and safety risks.

[0003] In recent years, the preparation of natural tobacco flavorings using microbial fermentation technology has become a research hotspot due to its ability to produce flavor compounds with complex structures, natural aromas, and high safety. Non-Saccharomyces yeasts have been proven to have abundant aroma-producing capabilities in food fermentation. Among them, Rhodotorula mucilaginosa, a common non-Saccharomyces yeast, has the ability to secrete various extracellular enzymes (such as β-glucosidase, laccase, and pectinase), which can hydrolyze bound aroma precursors in tobacco, releasing free aroma compounds. However, current research on the application of Rhodotorula mucilaginosa in tobacco flavoring preparation is limited, especially regarding its growth characteristics and aroma-producing capabilities in specific tobacco environments (such as nicotine-containing environments).

[0004] While existing technologies include the use of Rhodotorula mucilaginosa in tobacco products, such as CN110786534 A which discloses the use of carotenoids produced by Rhodotorula mucilaginosa to improve the aroma of tobacco extracts, and CN120477419 A which discloses the use of fermentation supernatant containing Rhodotorula mucilaginosa for the body aroma of cigar tobacco leaves; CN 110771942A uses Rhodotorula mucilaginosa as a fermentation strain for solid-state fermentation of tobacco stems and shreds, which increases the content of reducing sugars and total sugars, and improves the utilization rate of tobacco stems in cigarettes. CN 118813429 A discloses an enhanced fermentation method using *Rhodotorula glutinis* on cigar tobacco leaves. Compared to the control group using sterile water fermentation, the yeast fermentation group significantly increased the content of volatile aroma components, including chlorophyll degradation products such as phytosterol acetate, carotenoid degradation products (such as dihydroactinol, 4,7,9-magnacitriene-3-one), Maillard reaction products (including 6-ethyl-5,6-dihydro-2H-pyran-3-one, acetone-1 (3-pyridyl)), and phenylalanine degradation products such as phenylethanol. This resulted in a 43% increase in the total content of volatile aroma components, while simultaneously reducing off-flavors and irritation, and making the smoke more permeable. However, the existing technology still has the following defects: (1) There is a lack of high aroma-producing yeast strains suitable for the tobacco environment; (2) The tobacco system contains a high concentration of nicotine, which has a strong inhibitory effect on most microorganisms. Existing aroma-producing yeasts are limited in growth in nicotine-containing environments, making it difficult to effectively exert their aroma-producing function and resulting in low aroma-producing efficiency. The aroma-producing capacity of the reported Rhodotorula glutinis strains is limited, especially the yield of alcohols, aldehydes, esters and pyrazines, which are crucial for the formation of tobacco characteristic aromas. Therefore, screening microorganisms with high aroma-producing performance, rich tobacco aroma and good aroma coordination (Rhodotorula glutinis H04 in this application, which has been published in an article entitled: Isolation and Screening of Rhodotorula glutinis, Analysis of Aroma-producing Characteristics and Application in Yellow Wine Fermentation) and developing them for the preparation of tobacco flavorings is of great significance for improving the quality of tobacco products. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Rhodotorula glutinis H04 with high aroma production performance, high nicotine tolerance, high production of tobacco characteristic aroma alcohols, aldehydes, esters and pyrazines, and good aroma coordination, as well as a method for preparing tobacco flavorings using this strain, thereby solving the problems of poor aroma production capacity, poor aroma coordination and single aroma of existing Rhodotorula glutinis for tobacco.

[0006] This invention provides a strain of Rhodotorula mucilaginosa H04, which was deposited on July 1, 2024 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241401, located at Wuhan University, Wuhan, China.

[0007] This application provides a tobacco flavoring agent, which is prepared by fermentation of the red yeast H04 as described in claim 1.

[0008] Preferably, the application includes the following steps: inoculating Rhodotorula glutinis H04 into a seed culture medium and fermenting to obtain a seed liquid; The seed culture was inoculated into the fermentation medium for fermentation; after fermentation, the culture was centrifuged and the supernatant was collected. The supernatant was concentrated under reduced pressure and dried to obtain tobacco flavoring.

[0009] Preferably, the fermentation parameters for the seed culture medium are: 25℃ for 24-48 hours.

[0010] Preferably, the seed culture is inoculated into the fermentation medium at an inoculation rate of 5-10%.

[0011] Preferably, the fermentation parameters of the fermentation medium are: fermentation at 25℃ and 150rpm for 48-72 hours.

[0012] Preferably, the fermentation medium contains: glucose, peptone, yeast extract, KH2PO4, MgSO4, and nicotine.

[0013] Preferably, the fermentation medium contains 30-35 g / L glucose, 10-15 g / L peptone, 5-8 g / L yeast extract, 2-6 g / L KH2PO4, 1-5 g / L MgSO4, 2-5 g / L nicotine, and pH 5.5-6.0.

[0014] Preferably, the centrifugation parameters are: 8000 rpm for 10 minutes.

[0015] In another aspect, the present invention provides the application of the above-mentioned Rhodotorula glutinis H04 in the preparation of tobacco flavorings.

[0016] Beneficial effects (1) The red yeast H04 provided by the present invention has excellent aroma production performance and can produce high levels of tobacco characteristic aroma substances such as alcohols, aldehydes, esters and pyrazines. The optimal growth temperature is 25℃, which has better low temperature tolerance and reduces the impact of high temperature fermentation on tobacco aroma components.

[0017] (2) Rhodotorula glutinis H04 has good tolerance to nicotine and can maintain high growth activity even under nicotine concentrations as high as 8 mg / mL, making it suitable for preparing tobacco flavorings.

[0018] (3) The tobacco flavoring prepared by the present invention contains a variety of natural aroma components, which are well coordinated with the natural aroma of tobacco and can significantly improve the aroma richness and comfort of tobacco products.

[0019] (4) The highest β-glucosidase activity of Rhodotorula glutinis H04 reached 464.0±24.4 U / mL, and the laccase activity reached 60.4±3.2 U / mL, which is conducive to the release of bound aroma precursors in tobacco and enhances aroma performance. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of Rhodotorula glutinis H04.

[0021] Figure 2 The growth curves of different Rhodotorula glutinis are shown.

[0022] Figure 3 This is a graph showing the temperature tolerance of Rhodotorula glutinis.

[0023] Figure 4 This is a diagram showing the pH tolerance of Rhodotorula glutinis. Detailed Implementation

[0024] The following embodiments are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of the present invention, but the scope of protection of the present invention is not limited thereto. This section provides a more detailed description of the present invention in conjunction with specific implementation examples, and its technical features and advantages will be clearly demonstrated in the description. It should be noted that the embodiments described are merely illustrative examples and are not intended to limit the scope of the claims of the present invention. Any detailed adjustments, equivalent substitutions, or adaptive improvements based on the core principles of the present invention fall within the substantive protection scope of the present invention.

[0025] Experimental methods: Strain isolation and purification: Weigh 10.0 g of sample, add 100 mL of PBS, and incubate at 30℃ and 150 r / min for 30 min. Take 1 mL of the above culture solution and dilute it to 10⁻³~10⁻⁷. Take 100 μL of each dilution and spread it evenly on YPD solid medium. Incubate at 30℃ for 36-48 h. Select single colonies with the characteristics of R. mucilaginosa and streak them for purification more than 3 times until no other morphological colonies are visible on the plate.

[0026] Molecular biological identification of the strain: ITS sequence was amplified, and the amplification product was sent to Qingdao Weilan Technology Co., Ltd. for sequencing. The sequenced sequence was compared with the NCBI database for homology analysis.

[0027] Growth curve determination of Rhodotorula mucilaginosa isolate: The yeast was inoculated at a 1% inoculum in YPD liquid medium and cultured at 30℃ and 150 r / min for 48 h. The absorbance value at a wavelength of 600 nm was measured every 4 h to plot the growth curve of the strain.

[0028] pH tolerance analysis of Rhodotorula mucilaginosa: The isolated strain was inoculated at a rate of 1% into YPD liquid medium with different pH values ​​(2.0-6.0), and cultured at 30℃ and 150 r / min for 48 h on a shaker. The absorbance value at a wavelength of 600 nm was measured.

[0029] Temperature tolerance analysis of Rhodotorula mucilaginosa: The isolated strain was inoculated into YPD liquid medium at a 1% inoculum and cultured at 150 r / min for 48 h in eight gradients from 10.0℃ to 45.0℃ with a growth gradient of 5.0℃. The absorbance value at a wavelength of 600 nm was measured.

[0030] Nicotine acceptability analysis of Rhodotorula mucilaginosa: The isolated strains were inoculated at a rate of 1% into YPD liquid medium with different concentrations of nicotine (3-8 mg / ml), and cultured at 30℃ and 150 r / min for 48 h on a shaker. The absorbance value at a wavelength of 600 nm was measured.

[0031] Determination of BGL enzyme activity and laccase activity in Rhodotorula mucilaginosa isolate: BGL enzyme activity and laccase activity assays were performed according to the instructions of the kits, and a standard curve was established. The formula for calculating enzyme activity is as follows: BGL(U / mL) = (x V_antitotal) / (V_sample) T) In the formula: Vsample, the volume of crude enzyme solution added to the reaction system, 0.1 mL; Vreaction total, the total volume of the reaction system, 1 mL; T: reaction time, 0.5 h; x, obtained from the standard curve.

[0032] Example 1: Isolation, screening and identification of Rhodotorula glutinis JH-6 (H04) Appearance identification: The nine strains obtained from screening were numbered JH-1 to JH-9 respectively. The colonies of the nine strains were all orange-red, slightly glossy, round, viscous, smooth and moist, with uniform color in the center and edge, and easy to pick up (Table 1).

[0033] Molecular biological identification: The ITS gene sequence of the strain was uploaded to the NCBI database for BLAST homology comparison. The sequence showed 100% coverage and 99.53-99.84% similarity to the already identified R. mucilaginosa sequence KY488461.1. Therefore, all nine selected strains were identified as R. mucilaginosa. Strain information is shown in Table 1.

[0034] Table 1. Results of strain sequence alignment

[0035] Example 2: Growth curve of Rhodotorula mucilaginosa strain The growth status of the nine strains was measured every 4 hours over 48 hours, and the growth curves of the strains were determined. There were no significant differences in the lag phase, logarithmic growth phase, and stationary phase among the different strains; all strains were in the lag phase from 0 to 4 hours, and in the logarithmic growth phase from 4 to 28 hours. After 28 hours, the OD value changed little, reaching the stationary phase. (See details below.) Figure 2 ...

[0036] Example 3: BGL and laccase activities of Rhodotorula mucilaginosa strain Table 2 shows that among the screened Rhodotorula glutinis strains, JH-9, JH-8, JH-6, and JH-2 had relatively high BGL enzyme activities, all above 400.0 U / mL. JH-5, JH-7, JH-1, and JH-3 were next, with enzyme activities between 300.0 and 400.0 U / mL. JH-4 had the lowest activity. Most non-NONs can synthesize and release BGL; however, NONs exhibit very strong strain specificity, leading to interspecies and intraspecies differences in BGL enzyme activity. Furthermore, the location and polymerization mode of BGL also differ among different Rhodotorula glutinis cells. BGL can hydrolyze glycoside-bound aroma substances to release glucose and free aroma substances. As shown in Table 3, the Rhodotorula mucilaginosa strains with good laccase activity are JH-6, JH-9, and JH-5, among which JH-6 has the highest laccase activity, reaching 60.4±3.2 U / mL. Combining BGL enzyme activity and laccase activity, the results indicate that Rhodotorula mucilaginosa strain JH-6, i.e., H04, has a high enzyme production capacity.

[0037] Table 2. Rhodotorula glutinis R. mucilaginosa β -Glucosidase activity

[0038] Table 3. Rhodotorula glutinis R. mucilaginosa laccase Enzyme activity

[0039] Example 4: Tolerance of Rhodotorula mucilaginosa strain The stress factors encountered by Rhodotorula mucilaginosa may vary under different fermentation environments, with temperature, pH, and nicotine being the main stressors faced by Rhodotorula mucilaginosa in tobacco fermentation environments. Therefore, the fermentation performance of nine Rhodotorula mucilaginosa strains was assessed by measuring their tolerance to tobacco fermentation environments.

[0040] (1) Temperature tolerance like Figure 3 As shown, the OD value first increases and then decreases with temperature, exhibiting better growth within the range of 10℃ to 35℃. Biomass reaches its highest level at 25℃, indicating that the optimal growth temperature is 25℃. At 10℃, the OD value of the strain is above 1, indicating relatively good growth at low temperatures. Growth is gradually inhibited above 40℃. Temperature affects the quantity and activity of microorganisms during fermentation. Higher fermentation temperatures lead to the volatilization of aroma components, loss of flavor substances, and impact on the quality of the final product. The Rhodotorula mucilaginosa strain screened in this study exhibits better low-temperature tolerance.

[0041] (2) Acid tolerance like Figure 4 As shown, the biomass of each Rhodotorula mucilaginosa strain initially increased and then decreased with decreasing pH. Above pH 4.0, all strains exhibited significantly faster growth and higher biomass, indicating that this condition is favorable for strain growth. The best growth was observed at pH 5.0, while growth was inhibited when the pH dropped below 4.0. Acidity (pH) affects the activity of various enzymes and the formation of metabolites in microbial metabolic pathways. A suitable pH is beneficial for inhibiting the growth and reproduction of contaminating microorganisms and promoting the growth and fermentation of Rhodotorula mucilaginosa. The results indicate that Rhodotorula mucilaginosa exhibits good acid tolerance, with an optimal pH range of 4.0–6.0.

[0042] (3) Nicotine tolerance As shown in Table 3, compared with other Rhodotorula mucilaginosa yeasts, Rhodotorula mucilaginosa H-6 (H04) exhibited the highest growth activity at all nicotine concentrations. Its activity was basically unaffected at nicotine concentrations of 3-5 mg / mL, and remained high at 8 mg / mL. In contrast, JH-9, JH-8, and JH-2, which had high β-glucosidase activity, showed significantly reduced activity at nicotine concentrations of 6-8 mg / mL. This indicates that Rhodotorula mucilaginosa H-6 (H04) possesses the unique characteristics of simultaneously high β-glucosidase activity and tolerance to high nicotine levels, making it particularly suitable for fermentation in high-nicotine environments to prepare tobacco flavorings.

[0043] Table 4. R. mucilaginosa Nicotine tolerance

[0044] Example 5: Comparison of aroma-producing effects of Rhodotorula glutinis JH-6 (H04) and JH-9 Fermentation medium with the same composition (containing 5 g / L nicotine) was prepared, and strains JH-6 and JH-9 were fermented at 25℃ for 48 hours. The content of major aroma substances in the fermentation broth (μg / mL) was analyzed, and the results are shown in the table below: As shown in Table 5, the aroma-producing capacity of Rhodotorula glutinis JH-6 (H04) is much higher than that of Rhodotorula glutinis JH-9. The yields of important aldehydes (isovaleraldehyde, hexanal, etc.), alcohols (n-pentanol, isovalerol, etc.), pyrazines (such as 2-methylpyrazine, 2,5-dimethylpyrazine), and esters (vinyl acetate) in tobacco fermentation are all much higher in JH-6 than in JH-9.

[0045] Table 5. Aroma Production Effect Analysis of Rhodotorula glutinis JH-6 and JH-9

[0046] Example 6: Preparation of Tobacco Flavorings and Application in Tobacco Flavoring The fermentation broth prepared by fermenting red yeast JH-6 (H04) and JH-9 in Example 5 was sprayed onto flue-cured tobacco shreds at a ratio of 0.2%, sealed and stored for 24 hours, and then subjected to sensory evaluation.

[0047] Sensory evaluation results showed that the aroma of tobacco with added JH-6 flavoring was significantly richer, the smoke was smoother and more delicate, the irritation was reduced, and the aftertaste was clean and comfortable. The overall quality was significantly better than that of tobacco with added JH-9 flavoring (the aroma was less intense and had a certain degree of irritation).

Claims

1. A strain of Rhodotorula mucilaginosa H04, characterized in that, The Rhodotorula glutinis H04 was deposited on July 1, 2024 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241401, located at Wuhan University, Wuhan, China.

2. A tobacco flavoring, characterized in that, The tobacco flavoring is prepared by fermentation of the red yeast H04 as described in claim 1.

3. The tobacco flavoring as described in claim 2, characterized in that, The tobacco flavoring is prepared by the following steps: inoculating Rhodotorula glutinis H04 into a seed culture medium and fermenting to obtain a seed liquid; The seed culture was inoculated into the fermentation medium for fermentation; after fermentation, the culture was centrifuged and the supernatant was collected. The supernatant was concentrated under reduced pressure and dried to obtain tobacco flavoring.

4. The tobacco flavoring as described in claim 3, characterized in that, The parameters for the seed culture medium fermentation are: 25℃ for 24-48 hours.

5. The tobacco flavoring as described in claim 3, characterized in that, Inoculate the seed culture into the fermentation medium at an inoculation rate of 5-10%.

6. The tobacco flavoring as described in claim 3, characterized in that, The fermentation parameters for the fermentation medium are: fermentation at 25℃ and 150rpm for 48-72 hours.

7. The tobacco flavoring as described in claim 3, characterized in that, The fermentation medium contains: glucose, peptone, yeast extract, KH2PO4, MgSO4, and nicotine.

8. The tobacco flavoring as described in claim 3, characterized in that, The fermentation medium contains 30-35 g / L glucose, 10-15 g / L peptone, 5-8 g / L yeast extract, 2-6 g / L KH2PO4, 1-5 g / L MgSO4, 3-5 g / L nicotine, and pH 5.5-6.

0.

9. The tobacco flavoring as described in claim 3, wherein the centrifugation parameters are 8000 rpm for 10 minutes.

10. The use of the Rhodotorula glutinis HO4 according to claim 1 in the preparation of tobacco flavorings.

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