Ethyl acetate high-yield yeast and application thereof

Abstract

The application provides a high-yield ethyl acetate Saccharomyces cerevisiae and application thereof, the Saccharomyces cerevisiae is named Saccharomyces cerevisiae GJY6-9, is preserved in China typical culture collection center, and the preservation number is CCTCC NO: M2026897, and the preservation date is May 8, 2026.The strain screened by the application produces flavoring substances when producing fruit wine, and the flavoring substances typical esters are ethyl acetate, and the sugar alcohol conversion rate is high.When the above strain is fermented, the product with more pure and more mellow flavor can be obtained.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a yeast strain that produces high levels of ethyl acetate and its applications. Background Technology

[0002] Ethyl acetate is a key fruity flavor component in fruit wines and vinegars, playing a vital role in enhancing the sensory quality of the products. Citrus fruits are rich in vitamin C, carotenoids, flavonoids, and various volatile flavor compounds. Using citrus fruits as a raw material for fermenting fruit wines and vinegars can fully utilize citrus resources, increase farmers' income, and endow the products with unique functionality and flavor. However, currently, domestic fruit wine and vinegar fermentation still mainly uses traditional strains such as common brewing yeast. Although the fermentation efficiency is high, the characteristic citrus aroma is largely lost in the products, resulting in low market competitiveness and severely restricting the development of the citrus deep processing industry.

[0003] Previous analysis of flavor compound attenuation patterns and olfactory threshold experiments revealed that ethyl acetate is a key contributor to the characteristic flavor esters of citrus, and its content is positively correlated with the typicality of the citrus flavor in the product. However, traditional brewing yeasts have insufficient ethyl acetate synthesis capacity during citrus juice fermentation.

[0004] Currently, the screening of high-ethyl acetate-producing Saccharomyces cerevisiae for citrus juice systems faces challenges such as unclear screening targets, lack of high-throughput methods, poor genetic stability, and insufficient process adaptability. Therefore, there is an urgent need to breed a Saccharomyces cerevisiae strain with strong fermentation ability in citrus juice, high ethyl acetate synthesis ability, and genetic stability to improve the characteristic flavor and quality of citrus wine and vinegar. Summary of the Invention

[0005] In view of the above, the present invention provides a yeast strain that produces high levels of ethyl acetate and its applications.

[0006] The technical solution of this invention is as follows: A high-ethyl acetate-producing Saccharomyces cerevisiae GJY6-9 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026897, deposited on May 8, 2026, at Wuhan University, Wuhan, China.

[0007] Preferably, when the brewing yeast prepares fruit wine, only ethyl acetate is produced among the esters of the flavor substances generated.

[0008] More preferably, the yield of ethyl acetate reaches 250 mg / L or higher.

[0009] Preferably, when the brewing yeast is used to prepare fruit wine, the sugar alcohol conversion rate is above 95%.

[0010] The application of the high-ethyl acetate-producing Saccharomyces cerevisiae in the preparation of fruit wine.

[0011] Preferably, the raw material for the fruit wine is citrus or grapes. More preferably, the brewing yeast is used in the preparation of citrus fruit wine with an inoculation amount of 1%-1.5% for Saccharomyces cerevisiae GJY6-9.

[0012] More preferably, the method for using the brewing yeast to prepare citrus fruit wine is as follows: glucose is added to citrus juice, sterilized, and brewing yeast Saccharomyces cerevisiae GJY6-9 is inoculated, and fermentation is carried out to prepare citrus fruit wine.

[0013] More preferably, the method for using the brewing yeast in the preparation of citrus fruit wine is as follows: add 10%-14% glucose to citrus juice, sterilize at 75-85℃ for 15-25 minutes, inoculate with brewing yeast Saccharomyces cerevisiae GJY6-9, and ferment at 28-32℃ for 45-50 hours.

[0014] More preferably, the method for using the brewing yeast in the preparation of citrus fruit wine is as follows: add 12% glucose to citrus juice, sterilize at 80℃ for 20 min, inoculate with 1% of mutagenized brewing yeast Saccharomyces cerevisiae GJY6-9, and ferment at 30℃ for 48 h. Beneficial effects of this invention: 1. When using the strains screened by this invention to produce fruit wine, the flavor substances produced are only ethyl acetate, resulting in a pure flavor and a high sugar alcohol conversion rate.

[0015] 2. The typical ester of citrus is ethyl acetate. When fermented with the above-mentioned strains, a product with a purer and richer flavor can be obtained.

[0016] 3. Using the strain of the present invention, the fermentation flavor is pure and singular, which can make full use of the raw materials to obtain the target product. In addition, the sugar alcohol conversion rate is high, which ensures the full utilization of the sugar in the raw materials and improves the utilization rate of the raw materials. Attached Figure Description

[0017] Figure 1 Growth capacity OD of cells grown in YPD medium for 12 hours 600 ; Figure 2 Glucose consumption capacity of the strain with optimal flavor after six mutagenesis; Figure 3 Ethanol production capacity of the strain with optimal flavor after six mutagenesis; Figure 4 Sugar alcohol conversion rate of the strain with the best flavor after six mutagenesis. Detailed Implementation

[0018] The present invention will be better understood through specific examples below. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0019] Example 1 Microbial strain preservation: Submit the mutagenic strain to a preservation institution recognized by the State Intellectual Property Office for preservation. Source of the starting strain: Angel high-temperature resistant brewing yeast with high activity, designated as CK, is the starting strain.

[0020] Mutagenesis method: ARTP mutagenesis, power 120W, time 60s; 1. Strain Activation and Preparation: Inoculate the starting strain, Angel high-activity thermostable brewing yeast, into 100 ml of YPD liquid medium at a 10% inoculum and activate for 12 h. Take 2 mL of the bacterial suspension, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant. Resuspend the precipitated cells in 1 mL of physiological saline, mix thoroughly by pipetting, and centrifuge at 3000 rpm for 8 minutes, discarding the supernatant. Repeat this washing step once to ensure cell purity. After the final centrifugation, discard the supernatant, add 1 mL of physiological saline to resuspend the cells, and mix thoroughly by pipetting.

[0021] 2. ARTP mutagenesis treatment: 10 μL of bacterial suspension was evenly spread onto a sterile iron plate, which was then placed in a sterile petri dish. The bacterial cells were treated using an ARTP mutagen. The parameters for each mutagenesis were set as follows: power 120 W, gas flow rate 10 SLM, and treatment time 60 seconds. 3. Preparation and Fermentation of Mutagenic Bacterial Broth: After mutagenesis, the small iron discs were transferred to centrifuge tubes containing 1 mL of physiological saline. The bacteria on the iron discs were eluted into the saline solution by vortexing. The eluted bacterial broth was diluted to 3 mL. 100 μL portions of the diluted broth were then inoculated into 30 bottles of fruit juice (5 mL volume per bottle, with 12% glucose added). The inoculated fermentation broth was placed in a 30℃ incubator and allowed to ferment statically for 48 hours until the fermentation endpoint was reached.

[0022] 4. Strain Screening and Preservation: After fermentation, 1 mL of the fermentation broth was taken from each bottle for preservation. The flavor of the fermentation broth was evaluated for similarity to the original fruit juice using olfactory assessment. Glucose residue and ethanol production were detected using a biosensor, and the sugar-to-alcohol conversion rate was calculated. After comprehensive evaluation, five bottles of strains with excellent flavor and high sugar-to-alcohol conversion rate were selected for glycerol tube preservation. The strain with the best overall evaluation was selected for the next round of ARTP mutagenesis screening. Six rounds of mutagenesis were performed using the above method.

[0023] Identification characteristics: Morphologically, the cells are round and reproduce through polygonal budding, forming milky white, smooth colonies on YPD medium; its physiological and biochemical characteristics are significant, it can ferment glucose, sucrose, maltose and galactose, but not lactose, nor does it ferment nitrate, and under specific conditions, ascocells can form 1-4 smooth ascospores; as a facultative anaerobe, it can efficiently convert sugars into ethanol.

[0024] Strain selection criteria: For the first four mutagenesis trials, five strains with high sugar alcohol conversion rates were selected from those with good odor and flavor and preserved in glycerol tubes. For the fifth and sixth trials, all strains with good flavor and high sugar alcohol conversion rates were preserved. The strains with good flavor in the fifth trial were U5-5, U5-7, U5-17, U5-21, and U5-28; those with high sugar alcohol conversion rates were U5-6, U5-9, U5-10, U5-11, U5-12, U5-15, U5-22, U5-24, U5-25, and U5-27. The sixth mutagenesis trial was conducted based on U5-21. The following flavors were rated 6th: U6-8, U6-10, U6-12, U6-20, and U6-21; the following had high sugar alcohol conversion rates: U6-6, U6-7, U6-9, U6-11, U6-13, U6-14, U6-15, U6-16, U6-26, and U6-28.

[0025] Tables 1 and 2 show the dominant strains selected in the fifth and sixth mutagenesis screenings. The relevant data on sugar alcohol conversion rate are as follows: Table 1. Data corresponding to the fifth mutation

[0026] Table 2 Data corresponding to the sixth mutation

[0027] The specific fermentation verification method is as follows: Preparation of citrus juice: Peel fresh citrus fruits, press the juice manually, and filter it through 12 layers of gauze to obtain citrus juice.

[0028] Citrus fruit wine fermentation method: Add 12% glucose to citrus juice, sterilize at 80℃ for 20 minutes, inoculate with 1% mutagenized brewing yeast, shake well and dispense into 30ml vials, 5ml per vial, and place in a 30℃ constant temperature incubator for static fermentation for 48 hours.

[0029] Flavor analysis was performed using headspace solid-phase microextraction-gas chromatography (HS-GC). The specific method was as follows: 5.0 mL of citrus wine sample was accurately transferred to a 20 mL headspace vial. 1.5 g NaCl and 10 μL of internal standard solution (1.98 g / L 4-methyl-2-pentanol) were added, and the vial was immediately sealed with a cap fitted with a PTFE septum. The vial was placed in a 50°C constant-temperature magnetic stirrer and equilibrated at 600 rpm for 30 min. Then, an aged 50 / 30 μm DVB / CAR / PDMS SPME fiber tip was inserted into the headspace vial, and the fiber tip was removed. Extraction was performed at 50°C for 30 min. After extraction, the fiber tip was immediately inserted into the GC inlet, and desorption was performed at 250°C for 5 min. The target analyte was introduced into the chromatographic column with the carrier gas, and separation was performed using a temperature-programmed method. The chromatogram was detected and recorded, and the target analyte was quantitatively analyzed using the internal standard method. Specific conditions were as follows: Gas chromatography conditions: An Agilent 7890B gas chromatograph was used, equipped with a DB-WAX fused silica capillary column (60m × 0.32mm × 0.50μm, catalog number 123-7063, Agilent J&W, USA), and a flame ionization detector (FID). High-purity nitrogen was used as the carrier gas. The column flow program was: initial flow rate 3.1945 mL / min, decreasing to 2.1209 mL / min during operation; the injection port was in splitless mode, with an injection port temperature of 250℃ and a septum purge flow rate of 3 mL / min. The column oven temperature program was: initial temperature 40℃, held for 5 min; increased to 240℃ at a rate of 5℃ / min, held at that temperature for 8 min; the column's maximum tolerance temperature was 250℃, with an equilibration time of 0.5 min. The FID detector temperature was 300℃, hydrogen flow rate was 30 mL / min, air flow rate was 400 mL / min, and make-up gas (nitrogen) flow rate was 25 mL / min; the data acquisition frequency was 20 Hz. The results are shown in Table 3. Table 3 shows the optimal strain from each of the six mutagenesis trials, which underwent gas phase flavor analysis. The selection of the optimal strain was determined by a combination of sugar alcohol conversion rate and flavor.

[0030] Table 3

[0031] Note: Independent samples t-tests were used to compare each mutagenized strain group with the CK group for each aroma substance, and the results showed that there were significant differences.

[0032] As shown in Table 3, the U6-20 strain obtained by six rounds of mutagenesis in this invention produces only ethyl acetate and no other esters. The ethyl acetate yield is more than 17 times higher than that of the starting strain. The target strain for screening is a strain with high sugar alcohol conversion, a clean flavor background, and high ethyl acetate production.

[0033] After six rounds of mutagenesis screening, single-colony isolation and purification were performed. Based on U6-20, 30 single colonies were selected through plate dilution and fermentation verification, and the optimal strain was chosen. The resulting *Saccharomyces cerevisiae* was named *Saccharomyces cerevisiae* GJY6-9. It is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2026897, deposit date May 8, 2026, at Wuhan University, Wuhan, China.

[0034] Characteristic evaluation of the strain: The detection of sugar consumption, alcohol production, and sugar alcohol conversion rate all involved centrifuging the fruit wine after fermentation for 48 hours, taking the supernatant, diluting it by a certain factor until the detection concentration was within the detection range of the biosensor, detecting the content of glucose and ethanol using the biosensor, and calculating the sugar alcohol conversion rate according to the formula.

[0035] The calculation method is as follows: Sugar consumption capacity =

[0036] Sugar alcohol conversion rate (%) = *100% Growth capacity (OD) 600 The optimal flavor strain after six mutagenesis experiments and the growth of *Saccharomyces cerevisiae* GJY6-9 in YPD medium were compared. The mutagenic strains showed superior growth compared to the original strain. Experimental conditions: 1% *Saccharomyces cerevisiae* seed culture was added to 100 ml of YPD medium and cultured at 30℃ and 200 rpm for 12 h. 1 ml of the culture was taken, centrifuged to obtain the cells, and the absorbance at 600 nm was measured using a UV spectrophotometer. See below for detailed results. Figures 1-4 .

[0037] The pH change of the purified Saccharomyces cerevisiae GJY6-9 strain after fermentation was then measured, and the results are shown in Table 4 below. There were no significant pH changes (change < 0.1) before and after fermentation for either the original strain or the mutant strain.

[0038] Table 4

[0039] Based on all the data above, it can be seen that the mutagenic strain Saccharomyces cerevisiae GJY6-9 exhibits superior characteristics compared to the original strain.

[0040] Citrus wine and grape wine were prepared using the *Saccharomyces cerevisiae* strain GJY6-9, with grapes as the raw material, and their ethyl acetate content was compared. The citrus wine and grape wine were analyzed by biosensor and gas chromatography after 48 hours of fermentation.

[0041] Table 5 Sugar alcohol conversion rate, glucose consumption, and ethanol production.

[0042] Table 6 Comparison of flavors between the two groups of fermentation broths

[0043] According to Table 6, the independent samples t-test results show that, among the three flavor compounds, the content of isobutanol and phenylethanol in the citrus juice group was significantly higher than that in the grape juice group, while there was no significant difference in the content of ethyl acetate between the two groups, both of which produced high levels of ethyl acetate.

[0044] Comparison of sugar alcohol conversion rate data shows that when using citrus as raw material to make fruit wine, the corresponding strain exhibits a significantly superior sugar alcohol conversion rate compared to wine. Specifically, the strain Saccharomyces cerevisiae GJY6-9 screened in this invention demonstrates a high sugar alcohol conversion rate and produces a pure flavor in the fermentation process using citrus as raw material. The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations; therefore, any obvious variations or modifications derived therefrom remain within the scope of protection of this invention.

Claims

1. A brewing yeast that produces high levels of ethyl acetate, characterized in that, The brewing yeast is named Saccharomyces cerevisiae GJY6-9, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026897 and deposit date of May 8, 2026. When the brewing yeast is used to ferment fruit wine, it produces only ethyl acetate among the esters, with an ethyl acetate yield of over 250 mg / L and a sugar alcohol conversion rate of over 95%.

2. The application of the high ethyl acetate-producing Saccharomyces cerevisiae of claim 1 in the preparation of fruit wine.

3. The application according to claim 2, characterized in that, The raw material for the fruit wine is citrus or grapes.

4. The application according to claim 3, characterized in that, The brewing yeast is used in the preparation of citrus fruit wine: the inoculation amount of brewing yeast Saccharomyces cerevisiae GJY6-9 is 1%-1.5%.

5. The application according to claim 4, characterized in that, The method for using the brewing yeast to prepare citrus fruit wine is as follows: glucose is added to citrus juice, sterilized, and brewing yeast Saccharomyces cerevisiae GJY6-9 is inoculated, and fermentation is carried out to prepare citrus fruit wine.

6. The application according to claim 5, characterized in that, The method for using the brewing yeast in the preparation of citrus fruit wine is as follows: add 10%-14% glucose to citrus juice, sterilize at 75-85℃ for 15-25 minutes, inoculate with brewing yeast Saccharomyces cerevisiae GJY6-9, and ferment at 28-32℃ for 45-50 hours.

7. The application according to claim 6, characterized in that, The method for using the brewing yeast to prepare citrus wine is as follows: 12% glucose is added to citrus juice, sterilized at 80℃ for 20 minutes, 1% of the mutagenized brewing yeast Saccharomyces cerevisiae GJY6-9 is inoculated, and fermented at 30℃ for 48 hours.

Images