Saccharomyces cerevisiae LSY-1 and application thereof in preparation of honey-flavor raw dark green tea

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to saccharomyces cerevisiae LSY-1 and application thereof in preparation of honey-flavor raw dark green tea. The preservation number of the saccharomyces cerevisiae LSY-1 is CCTCC (China Center For Type Culture Collection) NO: M20241224. When the saccharomyces cerevisiae is applied to raw dark green tea fermentation, the prepared raw dark green tea has the characteristics of rich honey fragrance, sweet and mellow taste and the like. The strain disclosed by the invention can provide unique flavor for corresponding products in the fermentation production of raw dark green tea, and has a relatively good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of brewer's yeast LSY-1 and application thereof in the preparation of honey-aroma dark tea. Background Art

[0002] Dark tea (rough tea) is the raw material for making dark tea, and its aroma and flavor directly influence the flavor of the finished product. Traditional dark tea production is typically carried out in a natural environment. After withering and rolling, the tea leaves are piled into piles approximately 1 meter in height. Fermentation is then completed through natural inoculation of environmental microorganisms, and the finished product is obtained after drying. This prolonged fermentation process results in a dark tea with a duller aroma than other teas and a tendency to develop unpleasant odors such as rancidity. Furthermore, the traditional fermentation method creates a complex microbial composition, resulting in varying quality between batches of dark tea under different fermentation conditions.

[0003] The preparation of dark tea still needs to be improved. Summary of the Invention

[0004] The present invention isolates a highly active, safe, and efficient honey-aroma brewing yeast strain, LSY-1, from the dark tea fermentation process. This strain secretes multiple hydrolases, including β-glucosidase and esterase, and maintains high activity and stability over a wide temperature and pH range. LSY-1 is used to produce honey-aroma dark tea. Temperature-controlled fermentation solves the technical challenges of dark tea production, such as unstable processes, inconsistent finished product quality, difficulty ensuring quality, and unstable tea aroma.

[0005] The brewer's yeast LSY-1 (Saccharomyces cerevisiae LSY-1) of the present invention was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, zip code 430072) on June 14, 2024, and its deposit number is: CCTCC NO: M 20241224.

[0006] The present invention also provides a microbial preparation, comprising the Saccharomyces cerevisiae LSY-1.

[0007] The present invention also provides application of the brewer's yeast LSY-1 or the microbial preparation in tea fermentation.

[0008] Preferably, the tea leaves include but are not limited to black tea.

[0009] The present invention also provides a method for preparing honey-scented dark tea, comprising:

[0010] 1) preparing a starter culture using the Saccharomyces cerevisiae LSY-1;

[0011] 2) mixing the fermentation agent of step 1) with the tea leaves and performing solid-state fermentation.

[0012] Preferably, the concentration of Saccharomyces cerevisiae LSY-1 in the starter is 1.0×10 6 to 1.0×10 7 CFU / mL.

[0013] Preferably, the method for preparing the starter comprises: inoculating the brewer's yeast LSY-1 (Saccharomyces cerevisiae LSY-1) into a YPD medium for subculture to obtain an activated first-generation culture; then transferring 3-5 mL of the activated first-generation culture to 150-250 mL of a YPD tea soup (0.2-0.5% tea leaves) liquid culture medium, culturing at 28° C. and 120 rpm for 48 hours, and waiting for a large number of brewer's yeast LSY-1 (Saccharomyces cerevisiae LSY-1) cells to grow in the culture medium, inoculating the culture medium with a YPD tea soup (0.5-1% tea leaves) liquid culture medium, subculture at 28° C. for 48 hours to obtain yeast cells, washing the yeast cells twice, collecting them, and then resuspending them in sterile water to obtain the starter.

[0014] Preferably, in step 2), the volume mass ratio of the fermentation agent to the tea leaves is 1:(50-80) in mL / g.

[0015] Preferably, the step 2) comprises: spraying purified water into the dry tea dregs to moisten them according to a weight ratio of dry tea dregs to water of 100:(40-50), sterilizing the dried tea dregs, and cooling; then, uniformly mixing the fermentation agent with the tea dregs according to a volume mass ratio of the fermentation agent to the tea dregs of 1:(50-80) in mL / g, fermenting at 28-30° C. for 20-30 h, and drying (e.g., 80-90° C.) to obtain the finished product.

[0016] Preferably, during the fermentation process, the water content of the tea leaves is controlled at 40%-50%.

[0017] The present invention also provides a honey-scented dark tea, which is prepared by the above-mentioned preparation method.

[0018] Experiments show that dark tea fermented with Saccharomyces cerevisiae LSY-1 has a less astringent taste and a sweeter, mellower taste. The aroma exhibits a rich sweet and honey aroma. The aroma score and total sensory evaluation score are significantly better than those of the original sample before fermentation and the traditional pile-fermentation sample.

[0019] The experiment showed that 47 volatile compounds were detected in dark tea samples fermented by Saccharomyces cerevisiae LSY-1. After inoculation with Saccharomyces cerevisiae LSY-1, the content of alcohol compounds and hydrocarbon compounds decreased, while the content of ester compounds increased. 28 differential volatile components were obtained through principal component analysis. The results showed that the aroma showed significant differences after 12 hours of fermentation. These components include Pentanal (n-valeraldehyde) with a sweet and refreshing aroma, 2-Methyl-1-butanol (2-methylbutanol) with a sweet apple aroma, Phenylethyl with a sweet rose aroma. Alcohol (phenylethyl alcohol), 1-Pentanol (n-pentanol) with cider aroma, Dodecanal (dodecane) with fresh aroma, (2E,6Z)-nona-2,6-dienal (trans, cis-2,6-nonadienal), 4-Methyl-3-penten-2-one (4-methyl-3-penten-2-one) with honey sweet aroma, 1-Pentadecene (1-pentadecene) with beeswax aroma, o-Xylene (o-xylene) with high sharp sweet aroma, 2-(2-butoxyethoxy)-Ethanol (diethylene glycol butyl ether) with rose sweet aroma, and (R)-4,4,7a-Trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (dihydroactinol) with tropical fruit sweet aroma, all of which have an important influence on the formation of sweet wine aroma and honey aroma of dark tea. According to the Spearman correlation calculation method, rho≤-0.5 and rho≥0.5 were used to establish a correlation cluster heat map. It was found that (E)-2-Nonenal (trans-2-nonanal) with a sweet aroma, 1-Octen-3-ol (1-octen-3-ol) with a mushroom aroma, 2-Acetylpyrrole (2-acetylpyrrole) with a roasted nut sweet aroma, 2-Methyl-butanal (2-methylbutanal) with a malt sweet aroma, 2,2,6-trimethyl-Cyclohexanone (2,2,6-trimethylcyclohexanone) with a mint aroma, 3-Methylbutanal (isovaleraldehyde) with a malt aroma, Geraniol (geraniol) with a geranium aroma, Heptanal (heptanal) with an almond aroma, n-Decanal (decanal) with a sweet orange aroma, and Triethyl phosphate (triethyl phosphate) with a honey sweet aroma were positively correlated with brewer's yeast.

[0020] The experiment showed that a total of 275 non-volatile differential components were screened out from dark tea samples fermented by Saccharomyces cerevisiae LSY-1, including 18 amino acids and their derivatives, 6 nucleic acids and nucleotides, 34 lipids, 43 organic acids, 12 carbohydrates and their conjugates, 16 alkaloids, 15 flavonoids, and 131 other categories.

[0021] The brewer's yeast LSY-1, isolated and screened from the natural fermentation process of dark tea, is highly safe. When used in dark tea fermentation, this yeast produces dark tea with a rich honey aroma, a lasting nectar fragrance, and a sweet and mellow taste. The strain can significantly improve the flavor of dark tea and holds great promise for future applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony morphology of Saccharomyces cerevisiae LSY-1 (YPD medium).

[0023] Figure 2 This is the GC-MS total ion chromatogram of a dark tea sample fermented by Saccharomyces cerevisiae LSY-1 for 24 hours.

[0024] Figure 3 The quantity (A), type and content composition (B) of volatile components in fermentation piles inoculated with different yeasts for 24 hours.

[0025] Figure 4 This is a cluster heat map of the different volatile components of dark tea inoculated with different yeasts and fermented for 24 hours.

[0026] Figure 5 This is the LC-MS total ion chromatogram of dark tea produced by solid-state fermentation of Saccharomyces cerevisiae LSY-1 for 24 hours.

[0027] Figure 6 This is a heat map of the differences in non-volatile components of dark tea fermented by brewer's yeast at different fermentation times. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] YPD medium: 1% yeast extract (10 g / L), 2% peptone (20 g / L), 2% glucose (20 g / L), 1.5%-2% agar powder (15-20 g / L), make up to 1 L with distilled water, autoclave (121°C, 20 minutes), and aliquot for later use.

[0030] YPD tea soup (0.5% tea leaves) liquid culture medium: 1% (10 g / L) yeast extract, 2% (20 g / L) peptone, 2% (20 g / L) glucose, 0.5% (5 g / L) green tea. Make up to 1 L with distilled water, sterilize by autoclaving (121°C, 20 minutes), and aliquot for later use.

[0031] Example 1 Screening, Identification and Preservation of Saccharomyces cerevisiae

[0032] 1. Screening of brewer's yeast

[0033] In March 2024, the strain was obtained through isolation and purification from the dark tea pile production process of the Hunan Anhua No. 1 Tea Factory in Anhua County, Yiyang City, Hunan Province. Through initial screening of the strain's fermentation ability in tea soup culture medium and re-screening of solid-state enrichment fermentation, the strain LSY-1 was obtained, which can give the dark tea a honey flavor.

[0034] 2. Identification of Saccharomyces cerevisiae

[0035] 1. Morphological identification

[0036] After culturing strain LSY-1 in YPD medium for 24-48 hours, colonies will appear round or nearly round with neat edges and typically 2-5 mm in diameter. Initially, they are milky white or creamy in color, with a smooth, moist surface. The colonies are uniform in texture, with a smooth, glossy (creamy) surface and no wrinkles or fuzzy structures.

[0037] Microscopically, the cells are spherical or elliptical, with a diameter of approximately 5-10 μm and uniform size. The cell wall is thin, the cytoplasm is uniform, and vacuoles may be observed. The mother cell produces daughter cells by budding, forming a "bud" or short chain-like structure (see Figure 1 ). No pseudohyphae or hyphae.

[0038] 2. Molecular identification

[0039] The 18S rDNA sequence (ITS / D1 / D2 sequencing) of strain LSY-1 was sequenced (SEQ NO. 1) and compared to the NCBI GenBank database. Multiple sequence alignment was performed using ClusterX software. The results showed that the 18S rDNA gene sequences of strain LSY-1 and Saccharomyces cerevisiae naturally clustered, with a similarity of 99%. Therefore, strain LSY-1 was identified as Saccharomyces cerevisiae.

[0040] The 18S rDNA sequence is as follows:

[0041]

[0042] Based on the above morphological observations, physiological and biochemical identifications and 18S rDNA sequence analysis results, the strain was determined to be Saccharomyces cerevisiae of the family Saccharomycesceriaceae and was named Saccharomyces cerevisiae LSY-1.

[0043] 3. Preservation of Saccharomyces cerevisiae LSY-1

[0044] Saccharomyces cerevisiae LSY-1 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on June 14, 2024, and its deposit number is: CCTCC NO: M 20241224.

[0045] Example 2 Preparation of dark tea fermented by brewer's yeast

[0046] In order to verify the performance of brewer's yeast in dark tea fermentation, the strain was used to produce dark tea by solid-state fermentation. The dark tea production process of the present invention is divided into two parts: the preparation of the starter and the preparation of honey-flavored dark tea inoculated with brewer's yeast.

[0047] 1. Preparation of Saccharomyces cerevisiae LSY-1 starter culture

[0048] Saccharomyces cerevisiae LSY-1 is inoculated into a YPD medium for subculture to obtain an activated first generation. 3 mL of the activated first generation is then transferred to 150 mL of a YPD tea soup (0.5% tea leaves) liquid culture medium, and cultured at 28° C. and 120 rpm for 48 hours. After a large number of Saccharomyces cerevisiae LSY-1 cells grow in the culture medium, they are inoculated into a YPD tea soup (1% tea leaves) liquid culture medium, and subcultured at 28° C. for 48 hours to obtain yeast cells. The yeast cells are washed twice and collected, and then resuspended in sterile water to obtain the Saccharomyces cerevisiae LSY-1 starter culture.

[0049] 2. Fermentation process of honey-flavored dark tea

[0050] 500g of dark tea leaves were placed in a glass bottle, sprayed evenly with 200mL of water to moisten them, and sterilized at 121℃ for 10min. After cooling to room temperature, 10mL of the brewer's yeast LSY-1 starter (brewer's yeast suspension, 10 7 CFU / mL).

[0051] The blank control was a sample fermented without yeast at the same tidal volume; the positive control was a sample fermented with Debaryomyces fabri and Debaryomyces roxburghii at the same tidal volume. Debaryomyces fabri and Debaryomyces roxburghii were prepared into starter cultures according to the method described above, and then dark tea was fermented according to the same process described above.

[0052] Debaryomyces fabryi was isolated and purified by our research group, and the strain was identified by morphology and molecular biology.

[0053] Debaryomyces robertsiae LSY-2, isolated and purified by our research group, was deposited with the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China, 430072) on June 14, 2024, with the accession number M 20241223. This strain is also known as Debaryomyces robertsiae.

[0054] The glass bottles were fermented in a microbial incubator at 28°C for 30 hours, with samples taken every 6 hours. After production, the composition and flavor characteristics of the different finished products were tested and analyzed accordingly.

[0055] 1. Sensory quality analysis of dark tea fermented by brewer’s yeast

[0056] Sensory evaluation was conducted according to the national standard method for sensory evaluation (GB / T 23776-2018, "Tea Sensory Evaluation") on dark tea samples fermented at different fermentation times, without yeast, and with different yeasts. As shown in Table 1, dark tea samples fermented with Saccharomyces cerevisiae LSY-1 exhibited a reduced astringency and a sweet, mellow taste; their aroma exhibited a rich sweet and honeyed aroma.

[0057] Table 1 Sensory quality of dark tea with different fermentation times for samples fermented with yeast and without yeast

[0058]

[0059] Note: In Table 1, the cerevisiae yeast refers to Saccharomyces cerevisiae LSY-1.

[0060] Table 2 Sensory quality scores of dark tea with different fermentation treatments for 24 hours

[0061] Sample number shape aroma Soup color taste leaf bottom Total score Original sample 85 82 89 80 80 82.85 No yeast added sample 90 85 90 84 90 86.95 Saccharomyces cerevisiae samples 91 96 88 94 91 93.95 Barry yeast fermentation samples 91 90 80 90 91 88.8 Debaryomyces roegensis fermentation samples 91 90 80 91 91 89.1

[0062] Note: In Table 2, the cerevisiae yeast refers to Saccharomyces cerevisiae LSY-1.

[0063] Table 2 shows that the aroma score and the total sensory evaluation score are significantly better than the original sample and the traditional fermentation sample before treatment.

[0064] 2. Analysis of volatile and non-volatile quality components

[0065] 2.1 Detection method

[0066] (1) Solid phase microextraction method

[0067] Tea aroma was prepared using headspace solid-phase microextraction (HS-SPME). Weigh 0.2 g of ground tea sample, add 5 mL of boiling water to a 20 mL extraction vial, and add 5 μL of 25 μL / mL ethyl decanoate as an internal standard. The vial was quickly sealed and allowed to equilibrate for 5 minutes. Afterward, the extraction head was inserted and equilibrated in a 60°C water bath for 60 minutes. The SPME fiber was desorbed at 250°C for 5 minutes in the gas chromatography-mass spectrometry (GC-MS) inlet before GC-MS analysis.

[0068] (2) GC-MS chromatography and mass spectrometry conditions

[0069] A DB-5MS flexible quartz capillary column (30.0 m × 0.25 μm × 0.25 μm) was used. The inlet temperature was 250°C, and the carrier gas was helium (>99.999% purity) with splitless injection at a pressure of 99.9 kPa and a linear velocity of 36.3 cm / sec. The initial temperature was 40°C, held for 2 min, then increased at a rate of 4°C / min to 100°C, held for 2 min, then at a rate of 2°C / min to 120°C, held for 4 min, then at a rate of 3°C / min to 180°C, held for 2 min, and finally at a rate of 10°C / min to 230°C, held for 2 min. The ion source was EI, with a source temperature of 230°C and an ionization energy of 70 eV. The detector voltage was 0.25 kV, and a full-spectrum scan was performed over the mass range of 40-400 m / z.

[0070] 2.2 Results Analysis

[0071] Figure 2 This is the GC-MS total ion chromatogram of a dark tea sample fermented by Saccharomyces cerevisiae LSY-1 for 24 hours. Figure 3The quantity (A), type and content composition (B) of volatile components in fermentation piles inoculated with different yeasts for 24 hours. Figure 4 This is a cluster heat map of the different volatile components of dark tea inoculated with different yeasts and fermented for 24 hours.

[0072] Figure 3 Among them, Alcohols are alcohols, Aldehydes are aldehydes, Esters are esters, Ketones are ketones, Hydrocarbons are hydrocarbons, Heterocyclic oxygen compounds are oxygen-containing heterocyclic compounds, Nitrogen compounds are nitrogen-containing compounds, and Others are others.

[0073] Figure 3 and Figure 4 In the table, CK is blank control, Sc is Saccharomyces cerevisiae LSY-1, Df is Debaryomyces fabri, Dr is Debaryomyces rothei, and Content is content.

[0074] The volatile components of dark tea samples inoculated with Saccharomyces cerevisiae LSY-1 were analyzed using HS-SPME-GC-MS. Sixty-two volatile compounds were detected. After inoculation with Saccharomyces cerevisiae, the content of alcohol and hydrocarbon compounds decreased, while the content of ester compounds increased. PCA and HCA models were constructed for the samples fermented with Saccharomyces cerevisiae, with R2X(cum)=0.993, R2Y(cum)=1, and Q2(cum)=0.999. Principal component analysis (PCA) screened 28 differentially expressed volatile components, revealing significant aroma differences after 12 hours of fermentation. These components included pentanal (n-valeraldehyde), which has a sweet, refreshing aroma, 2-Methyl-1-butanol (2-methylbutanol), which has an apple-sweet aroma, and phenylethyl benzoate (Phenylethyl benzoate), which has a rose-sweet aroma. Alcohol (phenylethyl alcohol), 1-Pentanol (n-pentanol) with cider aroma, Dodecanal (dodecane) with fresh aroma, (2E,6Z)-nona-2,6-dienal (trans, cis-2,6-nonadienal), 4-Methyl-3-penten-2-one (4-methyl-3-penten-2-one) with honey sweet aroma, 1-Pentadecene (1-pentadecene) with beeswax aroma, o-Xylene (o-xylene) with high sharp sweet aroma, 2-(2-butoxyethoxy)-Ethanol (diethylene glycol butyl ether) with rose sweet aroma, and (R)-4,4,7a-Trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (dihydroactinol) with tropical fruit sweet aroma, all of which have an important influence on the formation of sweet wine aroma and honey aroma of dark tea. According to the Spearman correlation calculation method, rho≤-0.5 and rho≥0.5 were used to establish a correlation cluster heat map. It was found that (E)-2-Nonenal (trans-2-nonanal) with a sweet aroma, 1-Octen-3-ol (1-octen-3-ol) with a mushroom aroma, 2-Acetylpyrrole (2-acetylpyrrole) with a roasted nut sweet aroma, 2-Methyl-butanal (2-methylbutanal) with a malt sweet aroma, 2,2,6-trimethyl-Cyclohexanone (2,2,6-trimethylcyclohexanone) with a mint aroma, 3-Methylbutanal (isovaleraldehyde) with a malt aroma, Geraniol (geraniol) with a geranium aroma, Heptanal (heptanal) with an almond aroma, n-Decanal (decanal) with a sweet orange aroma, and Triethyl phosphate (triethyl phosphate) with a honey sweet aroma were positively correlated with brewer's yeast.The apple-scented 2-Methyl-1-butanol 2(-methylbutanol), the lime-leaf-scented 3,7-dimethyl-1,5,7-octatrien-3-ol(dihydrolinalool), and the orange-flower-scented Nerol(nerol) were negatively correlated with brewer's yeast.

[0075] After inoculation with the yeast Fabri de Barre, the following products were synthesized: Nerol (nerol) with orange blossom aroma, trans-2-Heptenal (trans-2-heptenal) with grassy aroma, Camphor (camphor) with camphor aroma, (E)-1-(2,6,6-Trimethyl-1,3-cyclohexadien-1-yl)-2-buten-1-one (damasketone) with rosehip aroma, trans-2-Undecenal (trans- The content of (Z)-Nerolidol (trans-nerolidol) with sandalwood aroma and 4-(2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one (4-[2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl]-3-buten-2-one) with mango aroma is relatively high, which makes the aroma of black tea fermented by Bridgbaryi yeast tend to be light floral.

[0076] After the introduction of Debaryomyces roeberii, the contents of Tetradecanal (n-tetradecanal) with wax aroma, (+)-2-Bornanone (D-camphor) with wood aroma, (E)-2-Dodecen-1-ol (trans-2-dodecen-1-ol) with cucumber aroma, 1-Pentadecene (pentadecene) with wood resin aroma, Phenylethyl alcohol (phenylethanol), trans-Geranylacetone (trans-geranylacetone) with cucumber aroma, 2,6,6-trimethyl-1-Cyclohexene-1-carboxaldehyde (β-cyclocitral) with lemongrass aroma, 2,6,6-trimethylcyclohexa-1,3-diene-1-carbaldehyde (safranal) with saffron medicinal aroma, and 2,4-Heptadienal (2,4-heptadienal) with cucumber peel aroma increased significantly. The aroma of black tea fermented by Debaryomyces roeberii tends to be green. Therefore, from the perspective of aroma component differences, the dark tea fermented by Debaryomyces fabri and Debaryomyces roeblingi does not have the material basis required for the rich honey-sweet aroma of the dark tea fermented by the brewer's yeast of the present invention.

[0077] Table 3 Volatile compound content of different yeast fermentation samples (24h fermentation)

[0078]

[0079]

[0080]

[0081] Note: In Table 3, the cerevisiae yeast refers to Saccharomyces cerevisiae LSY-1.

[0082] 3. Analysis of non-volatile quality components

[0083] 3.1 Detection Method

[0084] (1) Sample pretreatment

[0085] Accurately weigh the ground sample into a 2 mL centrifuge tube, add 600 μL of methanol containing 2-chloro-L-phenylalanine (4 ppm), and vortex for 30 seconds; add steel balls, place in a tissue grinder, and grind at 55 Hz for 60 seconds; ultrasonicate at room temperature for 15 minutes; centrifuge at 2000 rpm at 4°C for 10 minutes, take the supernatant and filter it through a 0.22 μm membrane. The filtrate is added to the detection bottle for LC-MS detection.

[0086] (2) Ultra-high performance liquid chromatography conditions:

[0087] Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid chromatography system was used. An HSS T3 (2.1 × 100 mm, 1.8 μm) column (Waters, Milford, MA, USA) was used at a flow rate of 0.3 mL / min and a column temperature of 40°C. The injection volume was 2 μL. In positive ion mode, the mobile phase consisted of 0.1% formic acid in acetonitrile (B2) and 0.1% formic acid in water (A2). The gradient elution program was as follows: 0–1 min, 10% B2; 1–5 min, 10%–98% B2; 5–6.5 min, 98% B2; 6.5–6.6 min, 98%–10% B2; 6.6–8 min, 10% B2. In negative ion mode, the mobile phase was acetonitrile (B3) and 5 mM ammonium formate water (A3), and the gradient elution program was: 0-1 min, 10% B3; 1-5 min, 10%-98% B3; 5-6.5 min, 98% B3; 6.5-6.6 min, 98%-10% B3; 6.6-8 min, 10% B3.

[0088] (3) Mass spectrometry conditions:

[0089] Data were collected using a Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA) with an electrospray ionization (ESI) source in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325°C, and a full scan was performed at a resolution of 70,000. The primary ion scan range was m / z 100 to 1000. Secondary fragmentation was performed using HCD with a collision energy of 30 eV and a secondary resolution of 17,500. The top three ions were fragmented, and dynamic exclusion was used to remove unnecessary MS / MS information.

[0090] 3.2 Results Analysis

[0091] Figure 5 This is the LC-MS total ion chromatogram of dark tea produced by solid-state fermentation of Saccharomyces cerevisiae LSY-1 for 24 hours. Figure 5 In the figure, the horizontal axis Retnetion Time (mins) is the retention time (minutes), the vertical axis Relative intensity (persentage%) is the relative intensity (percentage%), and the Total Ion Chromatogram is the total ion chromatogram.

[0092] According to the screening conditions of VIP>1 and p<0.05 in the PLS-DA model, a total of 275 differential compounds were screened out in the sample group inoculated with Saccharomyces cerevisiae LSY-1, including 18 amino acids and their derivatives, 6 nucleic acids and nucleotides, 34 lipids, 43 organic acids, 12 carbohydrates and their conjugates, 16 alkaloids, 15 flavonoids, and 131 other compounds.

[0093] Figure 6 The heat map of the differences in non-volatile components of dark tea fermented by Saccharomyces cerevisiae at different fermentation times. Figure 6 The total amount of flavonoids shown in the figure showed a trend of first decreasing and then increasing. For example, the content of astringent cyanidins and (-)-epigallocatechin 3-(4-methyl-gallate) remained stable after 12 hours. The content of baicalein, which has a distinct bitterness, showed a continuous downward trend with increasing fermentation time.

[0094] The total amount of amino acids decreased with increasing fermentation time, while the content of some amino acids increased. The levels of the umami enhancer Glutamylhydroxyproline, the rich umami flavor Glutamyl-Gamma-glutamate, and S-(phenylacetothiohydroximoyl)-L-cysteine ​​accumulated continuously with the fermentation process. The sweet N-alpha-Acetyl-L-lysine content surged to its peak after 24 hours of fermentation. The content of substances such as L-Allothreonine, which has a cooling effect, initially decreased and then increased.

[0095] The content of most organic acids showed a downward trend, such as the bitter pantothenic acid, astringent caffeic acid, and sour gluconic acid. The sharp and sour cis-aconitic acid and rancid hexadecanedioic acid ((R)-2-hydroxy-4-methylvaleric acid) accumulated most after 12 hours of fermentation, but their content decreased significantly after 24 hours.

[0096] The content of carbohydrates and their conjugates generally decreased with increasing fermentation time, with only the fruity-sweet primeverose showing a continuous increase. The content of indican (3-indolyl-beta-D-pyranoglucoside), which has a pungent, sulfur-bitter taste, peaked at 12 hours and then declined. Nucleosides and nucleotides also showed a downward trend, with most showing a significant decrease after 12 hours. The content of bitter alkaloids decreased with increasing fermentation time, while the content of sweet-savory propiobetaine, green-bitter N-(3-Methylbut-2-EN-1-YL)-9H-purin-6-amine, and protopine increased significantly after 24 hours. Among lipids, 10 non-volatile compounds decreased significantly with fermentation, primarily due to the increase in umami and salty phosphocholine compounds.

[0097] In summary, sensory evaluation results and the conversion of flavor compounds (volatile aroma components and non-volatile flavor components) indicate that the Saccharomyces cerevisiae LSY-1 strain obtained by the present invention can impart a rich honey aroma and enhance the sweet and mellow flavor of dark tea when used in the production of dark tea. It is a strain with excellent performance and suitable for dark tea fermentation. Using the strain of the present invention for dark tea fermentation is in line with the development model of modern industry.

[0098] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Saccharomyces cerevisiae LSY-1, deposited with CCTCCNO: M20241224.

2. A microbial preparation, characterized in that: The invention comprises the Saccharomyces cerevisiae LSY-1 according to claim 1.

3. Use of the cerevisiae LSY-1 according to claim 1 or the microbial preparation according to claim 2 in tea fermentation.

4. The use according to claim 3, characterized in that The tea leaves include but are not limited to black tea.

5. A method for preparing honey-scented dark tea, characterized in that: include: 1) preparing a starter culture using the Saccharomyces cerevisiae LSY-1 according to claim 1; 2) mixing the fermentation agent of step 1) with the tea leaves and performing solid-state fermentation.

6. The method for preparing honey-scented dark tea according to claim 5, characterized in that: The concentration of Saccharomyces cerevisiae LSY-1 in the fermentation agent is 1.0×10 6 to 1.0×10 7 CFU / mL.

7. The method for preparing the honey-scented dark tea according to claim 5 or 6, characterized in that: The method for preparing a starter comprises: inoculating the brewer's yeast LSY-1 (Saccharomyces cerevisiae LSY-1) into a YPD medium for subculture to obtain an activated first-generation culture; then taking 3-5 mL of the activated first-generation culture and transferring it to 150-250 mL of a YPD tea soup (0.2-0.5% tea leaves) liquid culture medium, culturing it at 28° C. and 120 rpm for 48 hours, and waiting for a large number of brewer's yeast LSY-1 (Saccharomyces cerevisiae LSY-1) cells to grow in the culture medium; then inoculating it into a YPD tea soup (0.5-1% tea leaves) liquid culture medium, subculture it at 28° C. for 48 hours to obtain yeast cells, washing the yeast cells twice, collecting them, and then resuspending them in sterile water to obtain the starter.

8. The method for preparing the honey-scented dark tea according to any one of claims 5 to 7, characterized in that: In the step 2), the volume mass ratio of the starter to the tea leaves is 1:(50-80) in mL / g.

9. The method for preparing honey-scented dark tea according to any one of claims 5 to 8, characterized in that: The step 2) comprises: spraying purified water into the dry tea leaves to moisten them at a weight ratio of dry tea leaves to water of 100:(40-50), sterilizing the dried tea leaves, and cooling the dried tea leaves; then, mixing the fermentation agent and the dried tea leaves uniformly at a volume mass ratio of the fermentation agent to the dried tea leaves of 1:(50-80) in mL / g, fermenting the mixture at 28-30° C. for 20-30 hours, and drying the mixture to obtain the finished product; Preferably, during the fermentation process, the water content of the tea leaves is controlled at 40%-50%.

10. A honey-scented dark tea, characterized in that: Prepared by the method according to any one of claims 5 to 9.

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