Use of a yeast strain

Abstract

The application discloses application of a yeast strain. The yeast strain is Saccharomycopsis fibuligera Y1402, and the preservation number is CGMCC No. 23667. The strain has the characteristics of nicotine tolerance, and also has the functions of producing phenylethanol, 3-methylthiopropanol and esters. The strain can efficiently produce 3-methylthiopropanol through tobacco leaf fermentation. The application provides a new way for improving quality and fragrance of tobacco leaves.

Description

Technical Field

[0001] This invention belongs to the field of yeast fermentation aroma production technology, specifically involving the application of an aroma-producing yeast strain in tobacco fermentation aroma production. Background Technology

[0002] Yeast is a very important microbial community in fermented foods. It can produce a variety of flavor substances, such as esters, alcohols, and sulfur-containing compounds, which play an important role in improving the quality and aroma of fermented foods. Therefore, in order to screen for strains that can enhance the flavor, aroma, and quality of tobacco, this invention identifies yeast as the target microorganism. Yeast strains are screened from the environment using traditional methods, and their nicotine tolerance is explored. These strains are then applied to the fermentation of tobacco leaves to screen for strains that can improve the quality and aroma of tobacco, thus providing excellent strains for tobacco quality and aroma enhancement. Summary of the Invention

[0003] The purpose of this invention is to provide an application of a yeast strain (Saccharomycopsis fibuligera) Y1402, with accession number CGMCC No. 23667. This strain is nicotine resistant and exhibits outstanding production of phenylethanol, 3-methylthiopropanol, and esters. It can efficiently produce 3-methylthiopropanol during tobacco fermentation.

[0004] Application of a yeast strain (Saccharomycopsis fibuligera) Y1402 in the production of at least one phenylethanol or ester.

[0005] Furthermore, the esters include at least one of ethyl hexanoate, ethyl octanoate, and isoamyl acetate.

[0006] Before producing the above substances, the yeast is activated. Preferably, the yeast is inoculated into YPD culture medium and activated for 22-26 hours at a temperature of 26-30℃ and a rotation speed of 150-200 r / min.

[0007] Further optimization: Yeast was inoculated into YPD culture medium and activated for 24 hours at a temperature of 28℃ and a rotation speed of 180r / min.

[0008] Methods for producing phenylethanol include: inoculating activated yeast cells into a phenylethanol conversion medium and culturing them.

[0009] Furthermore, the phenylethanol conversion medium includes, but is not limited to, the following formulation: glucose 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 5 g / L, yeast extract 5 g / L, L-phenylalanine 5 g / L, sterilized at 121°C for 20 min.

[0010] Furthermore, the activated yeast was inoculated into phenylethanol conversion medium at an inoculation rate of 0.1-0.3% and cultured for 46-50 h at a temperature of 26-30℃ and a rotation speed of 150-200 r / min.

[0011] Furthermore, the three activated yeast strains were inoculated into phenylethanol conversion medium at an inoculation rate of 0.2% and cultured for 48 h at a temperature of 28°C and a rotation speed of 180 r / min.

[0012] Methods for producing esters include: inoculating activated yeast into a culture medium, adding ester precursor substances, and continuing to culture.

[0013] Furthermore, some important ethyl esters were synthesized in 50 mL of YPD medium. The specific procedure involved adding bacteria and precursors to the YPD medium. After inoculating the activated strain into fresh YPD medium and culturing for 24 h, precursors were added. Combinations of 4% alcohol (v / v) (including ethanol, phenylethanol, and isoamyl alcohol) and 0.02% acid (v / v) (including acetic acid, lactic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, and decanoic acid) were prepared and cultured for another 36 h. The supernatant of the fermentation broth was then extracted 1:1 with n-hexane. After extraction, anhydrous sodium sulfate or sodium sulfite was added to remove water. The ester concentrations were determined using GC-MS, with three replicates for each reaction, and the average concentration of each ester was measured.

[0014] The present invention also provides the application of the above-mentioned yeast Y1402 in the preparation of 3-methylthiopropanol by adding tobacco leaves.

[0015] Furthermore, tobacco leaves were added for fermentation culture, with a liquid volume of 12.5-37.5 mL / 250 mL, an L-methionine concentration of 4-8 g / L, and a culture time of 36-60 h.

[0016] Furthermore, the culture medium formula is as follows: glucose 50-70 g / L, yeast extract 0.2-0.6 g / L, L-methionine 4-8 g / L, potassium dihydrogen phosphate 8 g / L, dipotassium hydrogen phosphate 6 g / L, sodium chloride 2 g / L, zinc sulfate 0.03 g / L, magnesium chloride 0.01 g / L, and 0.02 g / L ferrous chloride sterilized by passing through a 0.22 μm filter membrane.

[0017] Furthermore, adjust the pH to 4.5-5.5; the culture temperature to 28-36℃; the rotation speed to 200-250 rpm; the tobacco leaf inoculum amount to 0.1-3%; the tobacco leaf mesh size to 40-60 mesh; and the bacterial inoculum amount to 0.05-0.15%.

[0018] The present invention also provides the application of the yeast Y1402 in enhancing the aroma of tobacco, that is, centrifuging the fermentation liquid obtained in the above application, collecting the supernatant, and spraying it onto the tobacco.

[0019] Alternatively, a yeast suspension of Y1402 can be sprayed onto unsterilized tobacco shreds, incubated at 28-32℃ for 24-36 hours, then extracted with sterile water, centrifuged, and the supernatant can be sprayed onto the tobacco shreds.

[0020] The yeast strain (Saccharomycopsis fibuligera) Y1402 used in this invention has the accession number CGMCCNo.23667.

[0021] This strain is nicotine resistant and has outstanding ability to produce phenylethanol, 3-methylthiopropanol and esters. It can efficiently produce 3-methylthiopropanol when fermented with tobacco leaves.

[0022] This invention optimized the technical scheme for producing 3-methylthiopropanol from tobacco leaves through single-factor experiments, Plackett-Burman experiments, steepest ramp experiments, and response surface methodology. High yields of 3-methylthiopropanol were obtained, providing a new approach for improving the quality and aroma of tobacco leaves.

[0023] Biological Preservation Information

[0024] The yeast strain of this invention is *Saccharomycopsis fibuligera* Y1402, which was deposited on October 26, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 23667.

[0025] The colony characteristics and biochemical properties of the *Saccharomyces cerevisiae* Y1402 strain are as follows: On solid YPD medium, the colonies are milky white, small, with regular edges, a slightly raised center, a smooth, moist, and somewhat viscous surface, oval-shaped cells, budding at the apex, and without mycelium (see...). Figure 19 It produces a strong aroma during fermentation, can tolerate 9% (v / v) ethanol, can tolerate 15% (w / v) NaCl, has a pH growth range of 2 to 13, and a growth temperature range of 20 to 40℃.

[0026] The capsule-forming yeast Y1402 can be used to produce 3-methylthiopropanol. The fermentation preparation of 3-methylthiopropanol can achieve a yield of 2.0 to 4.07 g / L.

[0027] Molecular biological identification

[0028] To identify the aforementioned yeast strains, the molecular biological assays performed included the following steps:

[0029] (1) Bacterial culture and collection

[0030] The activated strain was inoculated into YPD liquid medium and cultured at 28℃ and 180 r / min for 48 h. The bacterial cells were then collected by centrifugation at 10000 r / min for 10 min.

[0031] (2) Extraction of yeast strain genomic DNA and amplification of 26S rDNA

[0032] The extraction method for yeast genomic DNA followed the method described in the fungal DNA extraction kit. Amplification primers were used: using the extracted total yeast DNA as a template, the 26S universal primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') were used to amplify the 26S rDNA D1 / D2 region sequence. The PCR reaction mixture consisted of 2.5 μL LA PCR Buffer, 1 μL each of primers, 2 μL dNTPs, 0.2 μL LAtaq enzyme, and 2 μL DNA, adjusted to a final volume of 25 μL with ddH2O.

[0033] PCR amplification program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles, with a final extension at 72℃ for 10 min. The PCR amplification products of yeast strain DNA were examined by electrophoresis on a 1% agarose gel.

[0034] (3) Sequencing and phylogenetic tree construction

[0035] The 26S amplification product was purified and sequenced to obtain the original sequence of the PCR amplified fragment of the strain. The sequence was proofread using BioEdit sequence mapping software, referring to the forward sequence map. The proofread 26S rDNA D1 / D2 region sequence was then used to perform a BLAST search in the GenBank nucleic acid sequence database to compare the similarity between the tested strain and the corresponding sequences in known yeast strains.

[0036] Based on homology search results, multiple sequences of the test strain and related strains were aligned and analyzed using MEGA 6.0 biological software, and a phylogenetic tree was constructed using the Neighbour-Joining method. (See [link to documentation]). Figure 20 .

[0037] After BLAST sequence alignment of the sequencing results of yeast Y1402 in NCBI, MEGA7 software was used to align the sequences of strains with high homology with strain Y1402 and construct a phylogenetic tree through Neighbor-Joining. Yeast Y14012 is most closely related to Saccharomycopsis fibuligera KU956952.1. Based on morphological observation, physiological and biochemical characteristics and molecular biology, yeast Y1402 was identified as Saccharomycopsis fibuligera. Attached Figure Description

[0038] Figure 1 : Standard curve of nicotine ultraviolet absorption;

[0039] Figure 2 Flowchart of tobacco fermentation process;

[0040] Figure 3 Results of phenylethanol production by 3 yeast strains;

[0041] Figure 4 Results of 3 yeast strains producing 3-methylthiopropanol;

[0042] Figure 5 Validation results of 3-methylthiopropanol production by yeast Y1402;

[0043] Figure 6 The effect of tobacco leaf addition amount on 3-methylthiopropanol concentration;

[0044] Figure 7 The effect of tobacco leaf mesh size on the concentration of 3-methylthiopropanol;

[0045] Figure 8 The effect of glucose concentration on 3-methylthiopropanol concentration;

[0046] Figure 9 Effect of yeast extract concentration on 3-methylthiopropanol concentration;

[0047] Figure 10 The effect of L-methionine concentration on 3-methylthiopropanol concentration;

[0048] Figure 11 Effect of culture temperature on the concentration of 3-methylthiopropanol;

[0049] Figure 12 The effect of initial pH of the culture medium on the concentration of 3-methylthiopropanol;

[0050] Figure 13 Effect of incubation time on the concentration of 3-methylthiopropanol;

[0051] Figure 14 Effect of rotational speed on the concentration of 3-methylthiopropanol;

[0052] Figure 15 Effect of liquid volume on the concentration of 3-methylthiopropanol;

[0053] Figure 16 Effect of inoculum size on 3-methylthiopropanol concentration;

[0054] Figure 17 Response surface and contour plot of the effect of the interaction between various factors on the yield of 3-methylthiopropanol;

[0055] Figure 18 Solid-state fermentation of tobacco shreds using yeast Y1402;

[0056] Figure 19 The colony morphology (a) and cell morphology (b) of the Y1402 strain of *Saccharomyces cerevisiae* on WL differential medium were shown at 10×100x magnification.

[0057] Figure 20 Phylogenetic tree of the Y1402 strain of *Saccharomyces cerevisiae* in this invention. Detailed Implementation

[0058] The following examples are intended to further illustrate the present invention, but not to limit it.

[0059] The main instruments and equipment used in the embodiments of this invention are as follows:

[0060] BL-2200H Electronic Analytical Balance, Shimadzu International Trading (Shanghai) Co., Ltd.; PHS-3C pH Meter, Shanghai Precision Scientific Instruments Co., Ltd.; DL-CJ-2ND I Clean Bench, Beijing Donglian Haer Instrument Manufacturing Co., Ltd.; LHS-100CL Constant Temperature and Humidity Incubator, Shanghai Yiheng Scientific Instruments Co., Ltd.; YQX-SG46-280S Autoclave, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; TU-19 UV-Vis Spectrophotometer, Beijing Puxi General Instrument Co., Ltd.; TENSUC Constant Temperature Shaker, Shanghai Tiancheng Experimental Instrument Manufacturing Co., Ltd.; Microfuge 2R Low Temperature Centrifuge, Beijing Tianlin Hengtai Technology Co., Ltd.; CKX41-F32F Electron Microscope, OLYMPUS Corporation (Japan); Tprofessional PCR System, Biometra Corporation; UV-HPLC System, Agilent Technologies, USA; GC-MS System, Agilent Technologies, USA.

[0061] Experimental reagents and materials used in the embodiments of this invention:

[0062] Nicotine standard (purity ≥99%) was purchased from Beijing Spectrometry Co., Ltd.; tobacco leaves were from Changsha, Hunan; 3-methylthiopropanol standard was purchased from Sigma Reagent Co., Ltd.; methanol was chromatographic grade; all other reagents were domestically produced analytical grade reagents.

[0063] Culture medium:

[0064] YPD medium (g / L): yeast extract 10, peptone 20, glucose 20, sterilized at 121℃ for 20 min.

[0065] WL medium (g / L): glucose 50, yeast extract 5, peptone 5, potassium dihydrogen phosphate 0.55, potassium chloride 0.425, calcium chloride 0.125, ferric chloride 0.0025, magnesium sulfate 0.125, manganese sulfate 0.0025, bromocresol green 0.022, agar 20, pH 6.5, sterilized at 121℃ for 20 min.

[0066] Nicotine liquid medium (g / L): dipotassium hydrogen phosphate 13.3, potassium dihydrogen phosphate 4, magnesium sulfate 0.2 g, 0.5 mL trace element solution (manganese sulfate 0.008, calcium chloride 0.05, copper chloride 0.05, ferrous sulfate 0.004, zinc sulfate 0.1, sodium molybdate 0.1, diluted to 1 L with 0.1 mol / L hydrochloric acid), sterilized at 121℃ for 20 min. Nicotine was filtered through a 0.22 μm filter membrane and mixed 1:1 with the sterilized enrichment medium (initial nicotine medium concentration was 0.05 g / L).

[0067] Phenylacetic acid conversion medium (g / L): glucose 20, magnesium sulfate 0.5, potassium dihydrogen phosphate 5, yeast extract 5, L-phenylalanine 5, sterilized at 121℃ for 20 min.

[0068] 3-Methylthiopropanol conversion medium (g / L): glucose 30, yeast extract 0.8, L-methionine 4, potassium dihydrogen phosphate 8, dipotassium hydrogen phosphate 6, sodium chloride 2, zinc sulfate 0.03, magnesium chloride 0.01, pH adjusted to 5, sterilized at 121℃ for 20 min. 0.02 g ferrous chloride was sterilized by passing through a 0.22 μm filter membrane.

[0069] Example 1: Screening of yeast strains and determination of nicotine adaptability

[0070] 1) Yeast strains were isolated from different environments using traditional microbial isolation methods. The specific method is as follows: Take 1g of sample and dilute it 10 times with sterile water. In a sterile environment, the bacterial suspension was serially diluted to 10⁻⁶. -5 10 -6 and 10 -7Spread 0.1 mL of each graded dilution onto YPD plates and incubate at 30°C for 2 days. Pick single colonies with white, raised, opaque structures resembling yeast and streak them onto YPD plates to obtain pure yeast culture. Store in glycerol tubes for later use.

[0071] 2) To investigate the adaptability of yeast strains to tobacco applications and analyze their nicotine tolerance, the specific methods are as follows:

[0072] Yeast strain activation: The selected yeast strains were inoculated into YPD medium and activated at 28℃ and 180r / min for 24h.

[0073] Screening of nicotine-adaptable yeast strains: Activated yeast seed culture was inoculated at a rate of 5% into 15 mL test tubes containing 2 mL of nicotine liquid culture medium. The cultures were incubated at 28℃ and 180 r / min for 72 h. After incubation, the biomass was measured at 560 nm using a UV spectrophotometer to analyze nicotine adaptability. Uninoculated nicotine liquid culture medium served as a blank control. Nicotine residues were determined using UV-HPLC, and the nicotine degradation rate of the strains was calculated. This determined the nicotine degradation ability of the baijiu-derived microorganisms. The formula for calculating the nicotine degradation rate is shown in Equation 1.

[0074] Nicotine degradation rate (%) = (XY / X) * 100 (Equation 1)

[0075] Where: X — initial concentration of nicotine in the fermentation broth (g / L);

[0076] Y – Residual concentration of nicotine in the fermentation broth after degradation (g / L).

[0077] Determination of nicotine content

[0078] (1) Determination of nicotine content by high performance liquid chromatography

[0079] The fermentation broth was centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and the residual nicotine was detected by high performance liquid chromatography.

[0080] Specific high-performance liquid chromatography (HPLC) method: C-18 reversed-phase column (ZORBAX Eclipse Plus C-18) 4.6×250mm, 5μm; mobile phase methanol-Na2HPO4 buffer (0.2mol / L, 0.46% triethylamine, pH=6.5)=60∶40; flow rate: 0.5mL / min; detection wavelength: 259nm; column temperature: 30℃; injection volume: 10μL; nicotine detection time: 9.5min.

[0081] (2) Drawing the nicotine standard curve

[0082] Accurately weigh 0.01 g of nicotine standard, dilute to 1 mL with pure water, and prepare a 10 g / L stock solution. Dilute this stock solution to obtain standard solutions of 0 g / L, 0.005 g / L, 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1 g / L. Analyze the standard solutions using high-performance liquid chromatography (HPLC). Plot a standard curve with nicotine concentration on the x-axis and peak area on the y-axis. (See figure). Figure 1 .

[0083] The ability of microorganisms to grow in a culture medium with nicotine as the sole carbon and nitrogen source proves their ability to utilize nicotine. Eighty-five yeast strains were screened and inoculated into nicotine liquid medium for nicotine tolerance analysis, examining their ability to degrade and tolerate nicotine. The degradation of nicotine by the yeast strains is shown in Table 1. Twenty yeast strains showed good growth in the nicotine liquid medium and were able to degrade nicotine, with degradation rates ranging from 0.3% to 11%. F12507 showed the highest degradation rate at 10.8%, and after cultivation, compared to the control, the medium became turbid with slight sedimentation at the bottom. F1504 showed the lowest degradation rate at 0.3%, with slight turbidity in the medium. Most yeast strains exhibited nicotine degradation rates above 2%. Based on the nicotine tolerance of these 20 strains, and considering preliminary sensory analysis during the nicotine tolerance screening process, it was found that 10 yeast strains could produce pleasant flavor compounds; therefore, these 10 yeast strains were selected for subsequent solid-state fermentation.

[0084] Table 1. Nicotine degradation in yeast

[0085]

[0086]

[0087] Example 2: Fermentation treatment of tobacco shreds with yeast

[0088] 1) Strain activation and inoculum preparation

[0089] Ten nicotine-adapted yeast strains were inoculated into YPD medium and activated for 24 hours at 28°C in a constant temperature shaker at 180 rpm. After activation, they were transferred to fresh YPD medium and cultured for 36 hours for inoculum preparation. The specific method for inoculum preparation is as follows:

[0090] T1: The cultured bacterial suspension (including live bacteria and their produced enzymes) is recorded as bacterial agent T1 without any treatment and is directly sprayed onto tobacco.

[0091] T2: Centrifuge the bacterial suspension cultured in YPD medium at 5000 r / min for 5 min, discard the supernatant, collect the bacterial sludge, resuspend the bacterial sludge in physiological saline and centrifuge again. Repeat the above operation 3 times. Record the bacterial agent obtained after resuspension as T2 and spray it on tobacco.

[0092] 2) Fermentation of tobacco shreds

[0093] Remove stems and impurities from the tobacco leaves. Weigh 20g of tobacco shreds and place them in an iron tray covered with 8 layers of gauze for sterilization. Under aseptic conditions, evenly spray the prepared test strain inoculants T1 and T2 onto the surface of the tobacco shreds. When spraying the test strain inoculants, be careful to turn the tobacco shreds to ensure that the inoculants can evenly contact the tobacco shreds. Wrap the evenly mixed tobacco shreds with 8 layers of gauze and place them in a constant temperature and humidity incubator at 28℃ and a relative humidity of 50% to 60% for 72 hours (see procedure). Figure 2 Sterilized YPD medium and physiological saline were used as blank controls for T1 and T2 bacterial agents, respectively.

[0094] 3) Sensory evaluation of fermented tobacco

[0095] The treated tobacco shreds were dried at 50°C to constant weight, placed in a preservation bag and kept at constant temperature and humidity for 24 hours, and then made into cigarettes using a small cigarette machine according to cigarette standards. The cigarettes were then provided to the sensory laboratory of Hunan Tobacco Industry Co., Ltd., where multiple sensory evaluation experts conducted evaluations according to the industry standard "YC / T 138-1998". The results are shown in Table 2.

[0096] Table 2 Sensory evaluation results of yeast-fermented tobacco

[0097]

[0098]

[0099] Ten yeast strains all had varying degrees of impact on tobacco shreds after fermentation. Microbial fermentation can cause changes in cigarette quality. Compared with the control, tobacco shreds fermented with yeast Y1511 had a clear aroma but no special aroma; tobacco shreds treated with F12507 showed significant improvements in both aroma quality and quantity, with a clear aroma and a distinct roasted sweetness; compared with the control, tobacco shreds treated with YHM-G had a slight off-flavor and a slightly astringent feel when smoking, but the irritation and strength were significantly reduced, and the aroma quality and quantity were improved, resulting in an enhanced smoking quality and a distinct cigar aroma; tobacco shreds treated with strain F1607 had poor combustibility and no special aroma; tobacco shreds treated with strain Y1007... The tobacco treated with strain Y1402 showed an increased sweet aroma and significantly improved texture. The aroma quality and quantity of the tobacco treated with strain Y1402 were both increased compared to the control, with slightly more pronounced off-flavors, a slightly improved taste and irritation, and a clear aftertaste. Both the aroma and the smoke exhibited a unique fragrance, resembling esters and roasted notes. The tobacco treated with yeast strain 3 (YPD group) showed little change in aroma quality and quantity compared to the control, but exhibited a slightly astringent taste, a slightly heavier off-flavor, and a woody aroma. The tobacco treated with physiological saline and yeast strain 3 showed a significantly improved smoking result compared to the control, with a clear aroma and a clean aftertaste. In summary, the tobacco treated with yeast strains Y1402, YHM-G, and strain 3 showed the best sensory results and the highest overall score. Furthermore, the irritation of the tobacco treated with all three strains was reduced, and a unique aroma was observed. Therefore, the volatile flavor compounds of the tobacco treated with strains Y1402, YHM-G, and strain 3 were determined.

[0100] Example 3: Analysis of the aroma-producing characteristics of yeast strains

[0101] (1) Activation of strains

[0102] Three yeast strains, YHM-G, YHM-3, and Y1402, were inoculated into YPD culture medium and activated for 24 h at a temperature of 28℃ and a rotation speed of 180 r / min.

[0103] (2) Analysis of the ability of yeast to produce phenylethanol

[0104] Three activated yeast strains were inoculated into phenylethanol conversion medium at an inoculation rate of 0.2% and cultured at 28°C and 180 r / min for 48 h. The yield of phenylethanol synthesized by the three yeast strains was determined by high performance liquid chromatography.

[0105] (3) Analysis of the ability of yeast to produce 3-methylthiopropanol

[0106] Three activated yeast seed cultures were inoculated into sterilized 3-methylthiopropanol conversion medium at an inoculation rate of 0.2%. The cultures were then incubated for 48 hours in a shaker at 30°C and 200 rpm. After the culture was completed, the yield of 3-methylthiopropanol synthesized by the three yeast strains was determined by high performance liquid chromatography.

[0107] (4) Analysis of ester production in yeast

[0108] The synthesis of some important ethyl esters was carried out in 50 mL YPD medium. The specific operation was carried out in the following three ways: (1) adding bacteria directly to YPD medium; (2) adding only the precursors for ester synthesis to YPD medium; (3) adding bacteria and precursors for ester synthesis to YPD medium. After the activated strain was inoculated into a new YPD medium and cultured for 24 h, the precursors were added. 4% alcohol (v / v) (including ethanol, phenylethanol and isoamyl alcohol) and 0.02% acid (v / v) (including acetic acid, lactic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid and decanoic acid) were combined respectively. After culturing for 36 h, the supernatant of the fermentation broth was extracted with n-hexane at a ratio of 1:1. After extraction, anhydrous sodium sulfate or sodium sulfite was added to remove water. The concentration of esters was determined by GC-MS. Each reaction was set up in 3 parallels, and the average concentration of each ester was determined.

[0109] Determination of aroma substance content in yeast fermentation broth

[0110] 1) Determination of phenylethanol content in fermentation broth

[0111] High performance liquid chromatography (HPLC) parameters: C-18 reversed-phase column (ZORBAX Eclipse Plus C-18, 4.6×250mm, 5μm); mobile phase: methanol:water = 50:50; flow rate: 0.55mL / min; detection wavelength: 260nm; column temperature: 35℃; injection volume: 10μL; HPLC retention time of phenylethanol: 13min.

[0112] 2) Determination of 3-methylthiopropanol content in fermentation broth

[0113] High performance liquid chromatography (HPLC) parameters: C-18 reversed-phase column (ZORBAX Eclipse Plus C-18, 4.6×250mm, 5μm); mobile phase: methanol:water = 30:70; flow rate: 0.7mL / min; detection wavelength: 215nm; column temperature: 30℃; injection volume: 10μL; retention time of 3-methylthiopropanol: 9.3min.

[0114] 3) Determination of ester content in fermentation broth

[0115] Gas chromatography conditions: DB-Wax column (30m×0.25mm×0.25μm); carrier gas: helium, flow rate 1mL / min; injection method: splitless injection; injection volume: 2μL, split ratio 30:1; temperature program: initial temperature 50℃, hold for 5min, increase to 150℃ at 2℃ / min, then increase to 230℃ at 4℃ / min, hold for 10min, and finally increase to 250℃ at 3℃ / min, hold for 10min.

[0116] Mass spectrometry conditions: EI+; ionization voltage: 70 eV; scan range: 35–450 amu; ion source temperature: 230 °C.

[0117] Phenylacetyl alcohol possesses a rose-like aroma and is a relatively abundant aroma compound in tobacco. In tobacco, it can reduce tobacco irritation, harmonize the smoke, and enhance the sweet, floral fragrance. The ability of three yeast strains to produce phenylacetyl alcohol was investigated. Figure 3 As shown, all three yeast strains can produce phenylethanol, with yeast YHM-G having the highest yield of 1.41 g / L.

[0118] 3-Methylthiopropanol is a very important aroma compound contributing to roasted flavors. The ability of three yeast strains to produce 3-methylthiopropanol was investigated. Figure 4 It can be seen that all three yeast strains can produce 3-methylthiopropanol, among which yeast Y1402 has the strongest ability to produce 3-methylthiopropanol, with a yield of 1.73 g / L.

[0119] Various esters in tobacco possess the aroma characteristics of flue-cured tobacco. Ethyl esters can provide fruity, floral, and waxy aromas and can mellow the smoke. This invention investigated the ester production capacity of three yeast strains. Qualitative analysis of the synthesized esters revealed that all three yeast strains could synthesize ethyl hexanoate, ethyl octanoate, and isoamyl acetate, but none could synthesize ethyl lactate, ethyl butyrate, ethyl valerate, or ethyl phenylacetate.

[0120] In summary, the three yeast strains can produce phenylethanol, 3-methylthiopropanol, ethyl acetate, ethyl hexanoate, and other esters. Notably, tobacco leaves treated with yeast Y1402 have a roasted nut aroma, and 3-methylthiopropanol happens to have roasted aroma characteristics. Therefore, 3-methylthiopropanol was selected as the target product for further research.

[0121] Validation experiment on the production of 3-methylthiopropanol by yeast

[0122] The ability of three yeast strains to produce the target product was verified based on the selected target product (see [link]). Figure 5 All three yeast strains could produce 3-methylthiopropanol, with yeast Y1402 having the highest yield of 2.0 g / L. Therefore, yeast Y1402 was selected as the target strain for subsequent research on 3-methylthiopropanol.

[0123] The optimized liquid fermentation medium consisted of: glucose 40–50 g / L, yeast extract 0.9 g / L, L-methionine 4 g / L, potassium dihydrogen phosphate 8 g / L, dipotassium hydrogen phosphate 6 g / L, sodium chloride 2 g / L, zinc sulfate 0.03 g / L, magnesium chloride 0.01 g / L, and pH 5.0.

[0124] The optimized fermentation conditions were pH=5, time 60h, and yeast extract concentration 0.9g / L. The final yield of 3-methylthiopropanol produced by fermentation of Saccharomycopsis fibuligera Y1402 reached 4.07g / L.

[0125] Example 4: Single-factor optimization of 3-methylthiopropanol production by yeast Y1402 in tobacco matrix

[0126] 3-Methylthiopropanol conversion medium was used as the basic culture medium.

[0127] Initial culture conditions: tobacco leaf mesh size 40, tobacco leaf addition 5%, Tween-80 2 g / L. The effects of culture medium composition (glucose concentration, yeast extract concentration, L-methionine concentration, tobacco leaf mesh size, tobacco leaf addition) and culture conditions (culture temperature, rotation speed, pH, culture time, liquid volume, inoculum size) on the production of 3-methylthiopropanol by the strain were investigated. The design of each factor level is shown in Table 3. Each level was set up with 3 replicates, and the optimal experimental level was used as the center value for subsequent experiments.

[0128] Table 3 Single-factor optimization design of fermentation conditions

[0129]

[0130] Figure 6 The results showed that the yield of 3-methylthiopropanol was highest when the tobacco leaf addition was 1%. This is likely because the carbon and nitrogen sources in tobacco provide nutrients for yeast growth, promoting the conversion of 3-methylthiopropanol. When the tobacco leaf addition was greater than 1%, the yield of 3-methylthiopropanol gradually decreased and stabilized. This may be because as the amount of tobacco leaf added increased, the amount of nicotine and other substances in the tobacco leaf that inhibit the growth of the strain gradually increased, thus weakening the strain's ability to convert and produce 3-methylthiopropanol, thereby reducing its yield. Based on the significance analysis, there was no significant difference between the addition amount of 3% and the blank control. In order to make fuller use of the yeast strain to treat tobacco leaves and explore the characteristics of 3-methylthiopropanol production by the strain in the tobacco substrate, a tobacco leaf addition amount of 3% was selected for subsequent research.

[0131] The mesh size of tobacco leaves affects the contact area between yeast and tobacco leaves, thus influencing the growth and metabolism of yeast. Therefore, the effect of tobacco leaf mesh size on the yield of 3-methylthiopropanol was investigated. Figure 7 The results showed that different finenesses of tobacco powder had significant effects on the production of 3-methylthiopropanol by the strain. As the mesh size of the tobacco leaves increased, the yield of 3-methylthiopropanol first increased and then decreased. The highest yield of 3-methylthiopropanol was achieved when tobacco leaves with a mesh size of 40-60 were added. This may be because moderately pulverizing the tobacco leaves is beneficial to the function of the yeast. Therefore, 40-60 mesh was selected as the optimal mesh size of tobacco leaves.

[0132] Glucose, as a substance that provides the carbon skeleton for the synthesis of target products and the energy necessary for life activities during microbial growth and metabolism, can be directly utilized by microorganisms. Figure 8 It can be seen that the concentration of 3-methylthiopropanol first increases and then decreases with increasing glucose concentration, and the yield is highest when the glucose concentration is 60 g / L.

[0133] Yeast extract provides a nitrogen source for cell growth and metabolism. Figure 9 The study showed the effect of yeast extract on the synthesis of 3-methylthiopropanol. The highest yield of 3-methylthiopropanol was achieved when the yeast extract concentration was 0.4 g / L. As the yeast extract concentration increased, the yield of the target product first decreased and then tended to stabilize. Excessive concentration inhibited the activity of certain enzymes in the Ayle pathway, thus leading to a decrease in the yield of 3-methylthiopropanol. Therefore, a yeast extract concentration of 0.4 g / L was the most suitable.

[0134] In the Ashley pathway, the substrate for the bioconversion synthesis of 3-methylthiopropanol is L-methionine. As more L-methionine is added to the conversion medium, more 3-methylthiopropanol is synthesized; therefore, the concentration of L-methionine was investigated. Figure 10 It was found that the yield of 3-methylthiopropanol increased sharply when the L-methionine concentration was in the range of 0-6 g / L. When the concentration was greater than 6 g / L, the yield of the target product decreased slightly, and the yield of 3-methylthiopropanol was the highest when the L-methionine concentration was 6 g / L. Increasing the L-methionine concentration can promote the formation of 3-methylthiopropanol and other sulfur-containing compounds. Therefore, the optimal concentration of L-methionine was selected as 6 g / L.

[0135] Culture temperature has a very important impact on the growth and metabolism of microorganisms. Microorganisms need to pass through the cell membrane to obtain nutrients from the outside world, and the fluidity of the cell membrane is related to temperature, thus affecting the metabolic growth of microorganisms. In addition, temperature also affects the enzyme activity in the synthesis and conversion of 3-methylthiopropanol. Figure 11The results showed that strain Y1402 could still produce 1 g / L of 3-methylthiopropanol at 20℃, indicating that the strain has a certain tolerance to low temperatures. As the temperature gradually increased, the yield of 3-methylthiopropanol first increased and then decreased, reaching its maximum at 32℃. A certain amount of 3-methylthiopropanol could still be synthesized at 40℃. Therefore, the optimal fermentation temperature for the strain is 32℃, which is also beneficial for its subsequent application in actual production.

[0136] The acid-base balance of the internal environment of microbial cells is inextricably linked to the pH value of the culture medium. pH can regulate cellular physiological functions and metabolism; the migration of charges on the microbial cell membrane and the dissociation of ions in the system are both affected by pH. Figure 12 It can be seen that as the pH value increases, the yield of 3-methylthiopropanol increases and then decreases, with the highest yield at pH=5. This may be because a suitable acidity or alkalinity promotes the formation of the cell biofilm, the barrier to microbial growth, thus promoting microbial growth and increasing the yield of the target product, reaching its maximum at pH=5. This indicates that yeast is suitable for growth and metabolism in a slightly acidic environment. Excessive acidity or alkalinity will inhibit the growth and metabolism of yeast. Therefore, pH=5 was chosen as the optimal pH for the conversion to 3-methylthiopropanol.

[0137] As the culture time increased, the yield of 3-methylthiopropanol gradually increased, with a sharp increase in the target product yield within 0-24 hours, reaching its maximum at 60 hours. When the culture time continued to increase, the yield of 3-methylthiopropanol gradually decreased. This may be because, with the increase in culture time, some toxic metabolites of the microorganisms gradually accumulate and have a toxic effect on the cells. Simultaneously, with the extension of fermentation time, the microbial cells gradually age, leading to insufficient ability of the strain to synthesize the target product. Figure 13 ).

[0138] Depend on Figure 14 It can be seen that as the rotation speed increases, the yield of 3-methylthiopropanol first increases and then decreases, with the highest yield at 225 r / min. Rotation speed is directly proportional to the dissolved oxygen level of the cells; however, excessively high rotation speeds lead to the rapid consumption of nutrients by the bacteria, resulting in insufficient ability for the bacteria to synthesize the target product later. Conversely, excessively low rotation speeds result in slow turbulent flow in the Erlenmeyer flask, which is detrimental to dissolved oxygen levels in the bacterial cells and thus negatively impacts the growth and metabolism of the strain. Therefore, 225 r / min was chosen as the optimal level.

[0139] Depend on Figure 15It can be seen that the yield of 3-methylthiopropanol is highest when the liquid volume is 25mL / 250mL. As the liquid volume increases, the yield of 3-methylthiopropanol decreases. The liquid volume is related to the dissolved oxygen level of the cells and affects the fermentation efficiency for converting the target product. Although the dissolved oxygen level and the liquid volume are negatively correlated, too little liquid volume will cause liquid evaporation during shaking and the adhesion of substances such as tobacco leaves in the culture medium to the bottle wall, thereby reducing the yield of the target product. Too much liquid volume will lead to insufficient dissolved oxygen level in the cells, which will reduce the fermentation efficiency and thus reduce the yield of 3-methylthiopropanol.

[0140] Figure 16 The results showed that the yield of 3-methylthiopropanol initially decreased and then stabilized with increasing inoculum size, reaching its maximum at an inoculum size of 0.1%. Excessive inoculum size can deplete nutrients in the culture medium too quickly, potentially weakening the strain's ability to synthesize the target product. Furthermore, excessive inoculum size can also lead to competition between strains, affecting their growth. Conversely, insufficient inoculum size can prolong the fermentation period and reduce fermentation efficiency. Therefore, an inoculum size of 0.1% is optimal.

[0141] Example 5: Plackett–Burman Experiment Optimization of 3-Methylthiopropanol Production by Yeast Y1402 in Tobacco Substrate

[0142] Based on the single-factor optimization results of Example 4, this experiment used a Plackett-Burman experimental design with N=12 to investigate nine factors other than the amount of tobacco leaves added and the mesh size of tobacco leaves. Each factor was selected with two levels, +1 and -1. Therefore, the optimal level of each investigated factor was taken as the center value, and the two adjacent values ​​were taken as -1 and +1 levels, respectively. The yield of 3-methylthiopropanol was used as the response value. The coding levels are shown in Table 4.

[0143] Table 4. Factor Levels in the Plackett-Burman Experimental Design

[0144]

[0145] Table 5. Results of the Plackett-Burman experiment

[0146]

[0147] The regression equation is:

[0148] MTP output = 2.88 + 0.038A + 0.2561B + 0.247C - 0.02800D + 0.0187E - 0.00200F - 0.05047G + 0.03095H - 3.52I

[0149] According to the regression equation, all these factors have a certain impact on the yield of 3-methylthiopropanol. Among them, the cultivation time, temperature, rotation speed, and inoculum size have a positive effect on the yield of 3-methylthiopropanol, while the other factors have a negative effect. The results of the ANOVA in Table 6 and the significance test results in Table 7 can be used to analyze the degree of influence of each factor on the yield of 3-methylthiopropanol. Table 6 shows that the P-value corresponding to the model is 0.002 (P<0.01), proving that the model is significant. Table 7 shows that the liquid volume, L-methionine concentration, cultivation time, glucose concentration, inoculum size, and rotation speed have a significant impact on the yield of 3-methylthiopropanol. Therefore, these five factors were selected as the steepest yield ramp-up test subjects, while the optimal levels of yeast extract concentration, rotation speed, and cultivation temperature were selected for the ramp-up test.

[0150] Table 6. Analysis of variance of the regression equation for 3-methylthiopropanol production

[0151]

[0152] Table 7. Results of Plackett-Burman significance analysis

[0153]

[0154] Example 6: Steepest Climbing Test Optimization of 3-Methylthiopropanol Production by Yeast Y1402 in Tobacco Matrix

[0155] Based on the Plackett-Burman experimental design results, the steepest climbing experiment was designed. The direction and step size of the change of factors that reached the significance level were designed to approach the optimal region of the response value. Other factors were designed with reference to the single-factor optimal value. Five groups of experiments were set up, with three parallels in each group. Finally, the highest level of the response value was found as the center point of the subsequent response surface experiment.

[0156] Based on the Plackett-Burman test results, and combined with the regression coefficients in Table 7, the step size and direction of the climbing test were determined. The results of the steepest climbing test are shown in Table 8.

[0157] Table 8. Experimental Design and Results for the Steepest Climb

[0158]

[0159] Table 8 shows that the highest response value, i.e., the yield of 3-methylthiopropanol, was achieved in the third group of experiments. Specifically, the highest yield of 3-methylthiopropanol (3.87 g / L) was obtained when the liquid volume was 24.5 mL, the L-methionine concentration was 6 g / L, the culture time was 44 h, the glucose concentration was 60 g / L, and the inoculum size was 0.09%. Therefore, the data from the third group were used as the center point for the response surface methodology.

[0160] Example 7: Response surface methodology optimization for the production of 3-methylthiopropanol by yeast Y1402 in tobacco matrix

[0161] Based on the results of the steepest ascent experiment and the Plackett-Burman experiment, three factors—volume volume (mL), L-methionine concentration (g / L), and incubation time (h)—and their corresponding center points were determined. The center point was used as the 0 level. The Box-Benhnken center combination experiment coding levels were obtained, with volume volume (mL), L-methionine concentration (g / L), and incubation time (h) as variables. The response surface methodology was designed using Design-Expert.V.8.0.6.1 software, as shown in Table 9.

[0162] Table 9 Response Surface Experimental Design Factors Levels

[0163]

[0164] The results are shown in Table 10:

[0165] Table 10 Box-Behnken Results

[0166]

[0167]

[0168] Table 11 shows the analysis of variance for the response surface regression model. A "Prob>F" value less than 0.05 indicates the model is significant. From Table 11, P = 0.0182 < 0.05, proving the model is significant. The regression equation shows that R... 2 =0.9331, indicating that the model can explain 93.31% of the response value variation, demonstrating a good fit to the experiment and small error. Furthermore, the lack-of-fit term P = 0.0755 > 0.05, therefore it is not significant, indicating a good correlation between the actual and predicted values. This model can be used to predict the yield of 3-methylthiopropanol. From the correlation coefficients in the table, it can be seen that the linear term and interaction term of the regression equation are not significant, while A in the quadratic term... 2 and C 2 Both are significant.

[0169] Table 11 Analysis of Variance for the Regression Model

[0170]

[0171] Interaction analysis among factors

[0172] The three-dimensional response surface plot was generated using Design-Expert 8.0.6 software to intuitively reflect the interaction between the three factors of liquid volume, L-methionine concentration and culture time on the yield of 3-methylthiopropanol. Figure 17 The results showed that the interaction between liquid volume and L-methionine concentration, culture time and L-methionine concentration was weak, but overall it would still have a certain impact on the yield of 3-methylthiopropanol.

[0173] According to the model calculations, when the optimal conditions are set (24.5 mL liquid volume, L-methionine concentration of 6 g / L, and culture time of 44 h), the predicted yield of 3-methylthiopropanol is 3.94 g / L. To verify its accuracy, three replicate experiments were conducted under the optimal conditions, and the actual value of 3.99 g / L was close to the predicted value of 3.94 g / L, demonstrating that the model can accurately predict the optimal conditions for 3-methylthiopropanol production. Compared with relevant studies at home and abroad, the yield of 3-methylthiopropanol is at a relatively high level.

[0174] Example 8: Tobacco quality improvement and aroma enhancement through yeast Y1402 fermentation treatment

[0175] 1) Liquid fermentation

[0176] Fermentation was carried out using 3-methylthiopropanol conversion medium under the optimized conditions described above (3% tobacco addition, 40-60 mesh size, 24.5 mL / 250 mL liquid volume, 6 g / L L-methionine concentration, 44 h culture time, 32 °C temperature, initial pH 5.0, 225 r / min rotation speed, 60 g / L glucose concentration, 0.4 g / L yeast extract concentration, and 0.09% inoculum). The mixture was centrifuged at low temperature, and the supernatant was collected. The fermentation broth was then sprayed onto the tobacco for cigarette making and smoking evaluation.

[0177] 2) Solid-state fermentation

[0178] According to the procedure Figure 18 The method involves spraying a 20% inoculum of yeast Y1402 onto unsterilized tobacco (20g, 40-60 mesh), incubating at 30℃ for different times, then extracting with sterile water for 30 minutes at a solid-liquid ratio of 1:10, centrifuging, taking the supernatant, spraying it onto the tobacco, and then rolling and tasting the cigarettes.

[0179] Liquid fermentation was conducted under optimized conditions for the production of 3-methylthiopropanol. The supernatant from the fermentation broth was collected by centrifugation and sprayed onto tobacco shreds for smoking evaluation. Results showed that the treated group had superior aroma texture and richness compared to the control group without the inoculated strain, but exhibited a pickled vegetable-like sour taste. Therefore, studies were conducted on different concentrations of L-methionine. The results indicated that excessive L-methionine was the cause. Smoking evaluation revealed that a methionine concentration of 2.0 g / L yielded the best effect and the highest overall score.

[0180] In addition, the strain was used to directly ferment tobacco shreds for different times, and the fermentation liquid extracted with sterile water was sprayed onto the tobacco shreds to enhance the aroma. The results showed that the tobacco shreds extract after yeast treatment for different times could also enhance the quality of tobacco products, with the samples fermented for 24-36 hours showing the most significant effect.

Claims

1. A strain of cladocyst-coated yeast ( Saccharomycopsis fibuligera The application of Y1402 in the production of at least one of phenylethanol, ethyl hexanoate, ethyl octanoate and isoamyl acetate; the strain's preservation number is CGMCC No. 23667.

2. The application according to claim 1, characterized in that, The method for producing phenylethanol includes: inoculating activated sacchariform yeast into a phenylethanol conversion medium and culturing it.

3. The application according to claim 1, characterized in that, The method for producing ethyl hexanoate, ethyl octanoate, and isoamyl acetate includes: inoculating activated *Saccharomyces cerevisiae* into a culture medium, adding precursor substances, and continuing the culture.

4. Application of a *Saccharomyces cerevisiae* strain Y1402 in the preparation of 3-methylthiopropanol by adding tobacco leaves; the strain's preservation number is CGMCC No. 23667.

5. The application according to claim 4, characterized in that, Add tobacco leaves for fermentation culture, with a liquid volume of 12.5-37.5 mL / 250 mL, an L-methionine concentration of 4-8 g / L, and a culture time of 36-60 h.

6. The application according to claim 4, characterized in that, Culture medium formulation: glucose 50-70 g / L, yeast extract 0.2-0.6 g / L, L-methionine 4-8 g / L, potassium dihydrogen phosphate 8 g / L, dipotassium hydrogen phosphate 6 g / L, sodium chloride 2 g / L, zinc sulfate 0.03 g / L, magnesium chloride 0.01 g / L, 0.02 g / L ferrous chloride sterilized by passing through a 0.22 μm filter membrane.

7. The application according to claim 4, characterized in that, Adjust pH to 4.5-5.5; culture temperature 28-36℃; rotation speed 200-250 rpm; tobacco leaf inoculum size 0.1-3%; tobacco leaf mesh size 40-60 mesh; bacterial culture inoculum size 0.05-0.15%.

8. The application of a single strain of *Saccharomyces cerevisiae* Y1402 in enhancing the aroma of tobacco, characterized in that... The fermentation broth obtained in any one of claims 4-7 is centrifuged, the supernatant is collected, and sprayed onto tobacco shreds; the strain's preservation number is CGMCCNo.23667.

9. The application of a single strain of *Saccharomyces cerevisiae* Y1402 in enhancing the aroma of tobacco, characterized in that... The suspension of *Saccharomyces cerevisiae* Y1402 was sprayed onto unsterilized tobacco shreds and cultured at 28-32℃ for 24-36 hours. Then, it was extracted with sterile water, centrifuged, and the supernatant was sprayed onto the tobacco shreds. The strain's preservation number is CGMCC No. 23667.

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