Geotrichum candidum strain capable of resisting high sugar, producing aroma and producing wine and application of geotrichum candidum strain

Abstract

The invention belongs to the technical field of microorganisms, and relates to a high-sugar-resistant, aroma-producing and wine-producing geotrichum candidum strain and application thereof. The high-sugar-resistant, aroma-producing and wine-producing geotrichum candidum strain BS-2 is preserved in the China Center for Type Culture Collection on January 8, 2025, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 2025067. The invention further discloses application of the geotrichum candidum in production of acid, ester, alcohol and aldehyde ketone substances. The bacterial colony is flat, milky white and flock-shaped, and the cell is cylindrical. The strain has excellent salt (6%, g / L) and acid (pH 3.0) tolerance, good ethanol tolerance (4%, V / V), sugar tolerance (400g / L), high ester yield (7.11 g / L) and certain wine production capacity (2.9% vol). GC-MS (Gas Chromatography-Mass Spectrometer) analysis shows that the relative content of total ester is up to 33.31 The hemolysis effect is avoided, and the hereditary stability is good. Therefore, a theoretical basis is provided for unique flavor application research of the strain leavening agent in fermentation of foods, beverages, spices, cosmetics and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a high-sugar-tolerant aroma-producing and wine-producing strain of Geotrichum candida and its applications. Background Technology

[0002] *Geotrichum candidum* is a fungus widely found in nature, attracting significant attention due to its applications in agriculture, environmental remediation, and biopharmaceuticals. As a microorganism with diverse biological activities, *Geotrichum candidum* not only effectively promotes plant growth and inhibits plant diseases but also plays a crucial role in environmental protection. In recent years, with the development of biotechnology, the application of *Geotrichum candidum* strains in metabolic engineering has been continuously expanding, particularly in the synthesis of natural products, enzyme production, and organic acid synthesis, demonstrating great potential.

[0003] Esters, as a class of natural products with significant commercial value, are widely used in food, fragrances, cosmetics, and pharmaceuticals. There are various methods for producing esters, including chemical synthesis, enzymatic catalysis, and microbial fermentation. Compared with traditional chemical synthesis, microbial fermentation offers advantages such as being environmentally friendly, low-cost, and highly efficient. Therefore, utilizing microorganisms to produce esters has become an important research direction.

[0004] Geotrichum candida strains show potential in metabolizing ester compounds, but most existing Geotrichum candida strains produce only a single type of ester compound, and their production capacity and efficiency are low. Although some Geotrichum candida strains can synthesize a certain amount of esters under specific conditions, their ester production capacity and the variety of esters still cannot meet the high-demand standards of industrial production.

[0005] Current research on *Galactomyces geotrichum* strains is a key research direction. For example, patent application CN202411261441.7 (A lactic acid-tolerant ester-producing *Galactomyces geotrichum* strain and its application) discloses a *Galactomyces geotrichum* strain with high lactic acid tolerance and high ester production capacity, and its application. The ester-producing *Galactomyces geotrichum* strain J-A2-85 was deposited at the China Center for Type Culture Collection on July 8, 2024, with the accession number CCTCCM20241517XJ-YDG001-1. This strain exhibits high lactic acid (8%, V / V) tolerance, good ethanol tolerance (6%, V / V), and high ester production capacity.

[0006] Therefore, continuing to search for and develop *Geotrichum candida* strains with high ester production capacity and tolerance to acids, alkalis, salts, sugars, and ethanol, and studying their ester production mechanisms, is of great significance for improving the yield and diversity of ester products. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a novel *Geotrichum candida* strain with superior aroma and alcohol production characteristics. This strain can produce unique aroma compounds during fermentation and exhibits good alcohol production capacity. The application of this strain can not only improve the production efficiency of aroma and alcohol but also provide a new biotechnological platform for the development of aroma and alcoholic beverage products. Details are as follows: A high-sugar-tolerant, aroma- and alcohol-producing strain of *Galactomyces candidum*, strain BS-2, was deposited at the China Center for Type Culture Collection (CCTCC) on January 8, 2025, with accession number CCTCC NO: M 2025067. The strain's applications in the production of acids, esters, alcohols, and aldehydes / ketones are also discussed. The colonies are flat, milky white, and short-fibered, with cylindrical cells. This strain exhibits excellent salt (6%, g / L) and acid (pH 3.0) tolerance, good ethanol tolerance (4%, V / V), sugar tolerance (400 g / L), high ester production (7.11 g / L), and a certain alcohol production capacity (2.9% vol). GC-MS analysis shows that the relative total ester content is over 30%. It exhibits no hemolytic activity and good genetic stability. This provides a theoretical basis for the research on the unique flavor application of this strain of starter culture in fermented foods, beverages, spices, and cosmetics.

[0008] This strain can produce at least one ester compound with strong aromatic characteristics under certain fermentation conditions, and the total relative content of its ester compounds is as high as 30% or more.

[0009] This strain exhibits good tolerance to acid, alkali, salt, sugar, and ethanol, and can grow in media with pH 3.0-7.0, 6% salinity, 400 g / L glucose, or 4% ethanol concentration.

[0010] Furthermore, the strain exhibits excellent salt (6%, g / L) and acid (pH 3.0) tolerance, good ethanol tolerance (4%, V / V), sugar tolerance (400 g / L), high ester production (7.11 g / L), and a certain alcohol production capacity (2.9% vol).

[0011] Furthermore, this strain can efficiently produce compounds with distinctive aromas, including esters, alcohols, aldehydes, ketones, and their derivatives.

[0012] The application of the aforementioned high-sugar-tolerant, aroma-producing, and alcohol-producing *Geotrichum candida* strain in alcoholic beverage production. Preferably, the alcoholic beverage production refers to brewed alcoholic beverages, particularly including high-proof rice wine from southeastern Guizhou, ordinary fermented rice wine, and fruit rice wine.

[0013] The application of the high-sugar-tolerant, aroma-producing, and alcohol-producing *Geotrichum candida* strain in the production of aroma compounds. Preferably, the aroma compounds include esters, alcohols, aldehydes, ketones, and their derivatives.

[0014] The application of the aforementioned high-sugar-tolerant, aroma- and alcohol-producing *Geotrichum candida* strain in the production of dairy products and dairy beverages. Preferably, the production of dairy products and dairy beverages includes yogurt, cheese, fermented rice milk, and other dairy beverages.

[0015] The application of the aforementioned high-sugar-tolerant aroma-producing and alcohol-producing *Geotrichum candida* strain in the fermentation of alcohols, acids, and esters. Further, the ester compounds are isobutyraldehyde, ethyl acetate, 2-methylbutyraldehyde, isovaleraldehyde, ethyl propionate, ethyl isobutyrate, isobutyl acetate, ethyl butyrate, 2-methylbutyrate, ethyl isovalerate, isobutyl propionate, isobutanol, isoamyl acetate, ethyl crotonate, isoamyl propionate, isoamyl isobutyrate, methyl isocyanate, 3-isopropyloxetane, eucalyptol, ethyl 3-methyl-2-butenoate, ethyl thiocyanate, styrene, amyl butyrate, isoamyl isovalerate, 3... 2-Methyl-2-butenoic acid-2-methylpentyl ester, 2-methyl-2-butenoic acid-2-methylpropyl ester, ethyl 3-hydroxy-3-methylbutanoate, 2-methyl-2-butenoic acid-2-methylbutyl ester, (E)-2-methylcrotonic acid isoamyl ester, 3-hydroxy-2-butanone, 2-methylbutyric acid, ethyl phenylacetate, phenethyl acetate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, phenethyl alcohol, 2-phenylethyl docosanoate, dimethyl phthalate, or mixtures thereof.

[0016] A fermentation agent comprising live cells, liquid inoculum, and solid inoculum (including lyophilized powder) of the said Geotrichum candida strain BS-2.

[0017] The high-sugar-tolerant, aroma- and alcohol-producing *Galactomyces candidum* strain BS-2 and related fermentation agents can be used in the food, beverage, spice, cosmetic, or pharmaceutical fields, as well as in the fermentation of alcohols, acids, and esters.

[0018] The beneficial effects of this invention are: This invention provides a *Galactomyces candidum* strain with high aroma and alcohol production capabilities. The aroma- and alcohol-producing *Galactomyces candidum* strain BS-2 was deposited at the China Center for Type Culture Collection (CCTCC) on January 8, 2025, with accession number CCTCC NO: M 2025067. The invention also describes the uses of this *Galactomyces candidum* in the production of acids, esters, alcohols, and aldehydes and ketones. The colonies are flat, milky white, and short-fibered, with cylindrical cells. This strain exhibits excellent salt (6%, g / L) and acid (pH 3.0) tolerance, good ethanol tolerance (4%, V / V), sugar tolerance (400 g / L), high ester production (7.11 g / L), and a certain alcohol production capacity (2.9% vol). GC-MS analysis shows that the relative total ester content is over 30%. It has no hemolytic activity and good genetic stability. This provides a theoretical basis for research on the unique flavor applications of this strain of starter culture in fermented foods, beverages, spices, and cosmetics. This strain can produce high yields of various ester compounds during fermentation, including but not limited to ethyl butyrate, ethyl isovalerate, isoamyl acetate, ethyl valerate, propyl isovalerate, isoamyl propionate, and other ester compounds with specific functions. The application of this strain can provide a new, green, and environmentally friendly technological pathway for the industrial production of ester compounds, with broad application prospects. Attached Figure Description

[0019] Figure 1 Strain isolation, identification, and characteristics: (a) BS-2 colonies; (b) BS-2 cell morphology (10 × 40); (c) sugar fermentation, carbon source assimilation, and nitrogen source assimilation; (d) growth curves; (e) phylogenetic tree; (f) pH tolerance; (g) salt tolerance; (h) ethanol tolerance; (i) sugar tolerance; (j) total ester content and alcohol content; (k) TTC alcohol production. Note: "+" indicates a positive reaction; "-" indicates a negative reaction. OD600 ≤ 0.200 indicates no growth.

[0020] Figure 2 Temperature optimization for strains.

[0021] Figure 3 Growth chart of strain BS-2 on hemolytic plate.

[0022] Figure 4 GC-MS total ion spectrum of fermentation broth for strain BS-2 PDB.

[0023] Figure 5 BS-2 26S rDNA sequence. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0025] Example 1 Isolation, identification, physiological and biochemical characteristics of strain BS-2 1. Isolation and screening of Geotrichum candida a. Preparation of selective culture medium b. Initial screening of aroma- and wine-producing strains c. Secondary screening for aroma and alcohol production, i.e., verification of the aroma and alcohol production capabilities of aroma- and alcohol-producing strains. Step a may further include: preparing PDB solid culture medium. Step b may further include: weighing 1 mL of fresh sample into a 50 mL sterile centrifuge tube, adding 9 mL of sterile water, and incubating in a shaker at 30 ℃ and 160 r / min for 30 min to prepare a suspension. Take 100 μL and inoculate it into 10 mL of PDB medium, and incubate with shaking at 30 ℃ and 160 r / min for 2 days. Dilute the culture to 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶, and take 100 μL of each dilution, spread it on PDB plates, and incubate the plates in a 30 ℃ incubator for 72 h. Select and preserve single colony strains with different morphological characteristics.

[0026] A single colony of the bacterial strain was streaked onto the lower layer of 2,3,5-triphenyltetrazolium chloride (TTC) solid medium. The plates were then incubated at 30 °C for 24 h. The cooled upper layer of TTC solid medium was then slowly poured into the growth plates, and the plates were incubated at 30 °C (in the dark) for 20 min. The plates were then observed to see if they turned red. The deeper the red color, the stronger the ethanol production capacity.

[0027] Potato Dextrose Broth (PDB): 5.0 g / L potato extract, 20.0 g / L glucose, 0.1 g / L chloramphenicol, pH 5.6-6.2. Add 20 g / L agar to obtain Potato Dextrose Agar (PDA) medium.

[0028] 2,3,5-Triphenyltetrazolium chloride (TTC) lower medium: Dissolve 10 g glucose, 1.5 g yeast extract, 1 g potassium dihydrogen phosphate, 20 g agar, 0.4 g magnesium sulfate, and 2 g peptone in 1 L of purified water, adjust the pH to 5.5, and sterilize at 115 °C for 20 min. TTC upper medium: Dissolve 5 g glucose, 0.5 g red tetrazolium, and 15 g agar in 1 L of purified water, and sterilize at 115 °C for 20 min.

[0029] Step c may further include: inoculating the screened strains into PDB liquid medium, culturing at 30℃ and 160r / min for 24h, and taking 5mL of fermentation broth for GC-MS analysis.

[0030] 2. Colony morphology observation After culturing on PDB medium for 48 hours, the colonies spread in a planar pattern, grew rapidly, were flat, milky white, and had a short, fluffy texture. Figure 1 As shown in Figure a. Further observation under a microscope reveals that the vegetative cells are single-celled, cylindrical in shape, as... Figure 1 As shown in b, the bacteria can grow in large quantities when cultured at 30°C for 48 hours in the above culture medium.

[0031] 3. Physiological and biochemical characteristics of the strain Experiments on sugar fermentation, carbon source assimilation, and nitrogen source assimilation of bacterial strains are used to examine the strains' utilization of various sugar carbon and nitrogen sources, and the results are recorded. "+" indicates a positive reaction; "-" indicates a negative reaction. Carbon source assimilation, nitrogen source assimilation, and gas production from sugar fermentation are shown in the figures. Figure 1 As shown in c.

[0032] 4. Strain identification The target bacteria were identified using 26S rDNA gene sequence analysis. Yeast DNA was extracted from the target bacteria using a yeast DNA extraction kit, and PCR amplification was performed using the forward primer NL-1: 5'-GCATATCAATAAGCGGAAAAG-3' and the reverse primer NL-4: 5'-GGTCCGTGTTTCAAGACGG-3'. The PCR amplification program was: initial denaturation at 95℃ for 10 min, denaturation at 94℃ for 30 s, annealing at 55℃ for 45 s, extension at 72℃ for 90 s, extension at 72℃ for 10 min, and storage at 16℃ for 59 min. The PCR products were separated by electrophoresis, and the PCR products with clear bands were sent to Shanghai Sangon Biotech for first-generation sequencing. The resulting gene sequences were compared against the GenBank database using BLAST, and finally, sequences with higher homology were selected to construct a phylogenetic tree using the nearest neighbor method in MEGA 11.0.

[0033] Most of the 26S rDNA sequence was determined, such as Figure 5 As shown. The sequence was aligned using BLAST from the NCBI website, and a phylogenetic tree based on the 26S rDNA full sequence was constructed. The results showed that the strain was 98% homologous to *Galactomyces candidum*. Therefore, it was determined that the strain used in this invention is *Galactomyces candidum*, specifically strain BS-2, which was deposited at the China Center for Type Culture Collection on January 8, 2025, with accession number CCTCC NO: M 2025067.

[0034] Most of the 26S rRNA sequence was determined. As shown in 1e, the determined sequence was aligned using BLAST from the NCBI website, and a phylogenetic tree was constructed. The results showed that the strain was 98% homologous to *Galactomyces candidum*. Therefore, it was determined that the strain used in this invention is *Galactomyces candidum* BS-2.

[0035] 5. Growth characteristics of the strain 100 μL of BS-2 bacterial culture was added to 30 mL of MRS liquid medium. Growth characteristics were measured using an automated growth curve analyzer (RTS-1C) at 30°C and 160 rpm. The results are as follows: Figure 1 As shown in d.

[0036] 6. Salt and alkali tolerance of the strain (1) Evaluation of acid tolerance. Activated yeast seed culture was inoculated at a 2% inoculum into PDB liquid medium with pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, respectively. After incubation at 30 ℃ for 24 h, growth was observed. Each group was repeated three times, with uninoculated PDB liquid medium serving as a control. Figure 1 As shown in f, strain BS-2 can grow at pH 3.0.

[0037] (2) Salt tolerance evaluation. Activated yeast seed culture was inoculated at a 2% inoculum into PDB liquid medium with sodium chloride concentrations of 2.0%, 4.0%, 6.0%, 8.0%, 10.0%, and 12.0%, respectively. After incubation at 30 °C for 24 h, growth was observed. Each group was repeated three times, with uninoculated PDB liquid medium serving as a control. Figure 1 As can be seen from g, strain BS-2 can grow under conditions of 8% salt content, and has strong salt tolerance.

[0038] 7. Ethanol tolerance of the strain Ethanol tolerance evaluation. Activated yeast seed culture was inoculated at a 2% inoculum into PDB liquid medium with anhydrous ethanol concentrations of 2.0%, 4.0%, 6.0%, 8.0%, 10.0%, and 12.0%, respectively. After incubation at 30 °C for 24 h, growth was observed. Each group was repeated three times. Uninoculated PDB liquid medium served as a control. Figure 1 As can be seen from h, strain BS-2 can grow under conditions of 6% ethanol content, and has strong ethanol tolerance.

[0039] 8. Glucose tolerance Evaluation of high sugar tolerance. Activated yeast seed culture was inoculated at a 2% inoculum into PDB liquid medium with glucose additions of 10%, 15%, 20%, 25%, 30%, 35%, and 40%, respectively. After incubation at 30 °C for 24 h, growth was observed. Each group was repeated three times. Uninoculated PDB liquid medium served as a control. Figure 1 As can be seen, strain BS-2 can grow under conditions with a glucose content of 300 g / L, and has strong sugar tolerance.

[0040] 9. Determination of total esters and alcohol content The seed culture was inoculated into PDB liquid medium and cultured at 30℃ and 160 rpm for 24 h before analysis. Total esters were determined according to GB / T 10345-2022 "Analytical Methods for Baijiu". 50 mL of fermentation broth was transferred to a 250 mL reflux flask, 2 drops of phenolphthalein indicator were added, and titrated with 0.1 mol / L NaOH standard solution until a faint red color appeared. Then, 50 mL of 0.1 mol / L NaOH standard solution was accurately added. A reflux condenser was attached, and the mixture was refluxed in a boiling water bath for 30 min for saponification. After cooling, it was immediately titrated with 0.1 mol / L sulfuric acid standard solution until the red color just disappeared. The volume of sulfuric acid standard solution consumed was recorded. The blank test group used ester-free ethanol instead of the sample. Calculation formula: In the formula: X represents the total ester content in the sample (calculated as ethyl acetate), g / L; c represents the concentration of sulfuric acid standard solution, mol / L; V0 represents the volume of sulfuric acid consumed by the blank test group, mL; V1 represents the volume of sulfuric acid consumed by the sample group, mL; 50 represents the sample aspiration volume.

[0041] Qualitative determination of alcohol production capacity: If the strain has the ability to produce ethanol, the TTC plate will turn red. The deeper the red, the stronger the alcohol production capacity. The alcohol content was determined according to GB 5009.225-2016 "National Food Safety Standard - Determination of Ethanol Concentration in Alcohol". The results are shown in Figures j and k.

[0042] 10. Temperature screening Activated yeast seed culture was inoculated into PDB liquid medium at a 2% inoculum rate. After incubation at 24℃, 26℃, 28℃, 30℃, 32℃, and 34℃ for 24 h, growth was observed. Each group was repeated three times. Uninoculated PDB liquid medium served as a control. Figure 2 It can be seen that strain BS-2 grows best at 26℃.

[0043] 11. Hemolysis test The activated strain BS-2 was streaked onto blood agar medium and incubated at 30°C for 2 days. The presence of hemolytic zones around the colonies in the culture dish was observed and photographed. Figure 3 As shown, the strain has no hemolytic activity.

[0044] 12. Determination of volatile components in the fermentation broth of the strain Take 5g of fermentation broth or distillate, add 1.5g of sodium chloride, mix well, and test the sample using an instrument.

[0045] GC-MS conditions: Determination was performed using an Agilent 8890-7000E GC-MS system. Column: DB-WAX 122-7032 capillary column (30 m × 0.25 mm, 0.25 μm); Chromatographic conditions: 40 ℃ for 2 min, ramped to 100 ℃ at 4 ℃ / min, ramped to 120 ℃ at 5 ℃ / min and held for 5 min, then ramped to 230 ℃ at 5 ℃ / min and held at this temperature for 5 min; Carrier gas: helium, flow rate 0.8 mL / min, injector temperature 250 ℃, split ratio: 20:1; MS conditions: Electron impact ionization (EI) energy 70 eV, ion source temperature 230 ℃, quadrupole temperature 150 ℃, EI mass spectra recorded in the range of 45–500 AMU.

[0046] The volatile components of the fermentation broth of strain BS-2 are shown in Table 1 (pre-existing components in the blank culture medium have been removed), with a total ester content as high as 33.31%. Fermentation can produce aroma compounds, including but not limited to isobutyraldehyde, ethyl acetate, 2-methylbutyraldehyde, isovaleraldehyde, ethyl propionate, ethyl isobutyrate, isobutyl acetate, ethyl butyrate, 2-methylbutyrate, ethyl isovalerate, isobutyl propionate, isobutanol, isoamyl acetate, ethyl crotonate, isoamyl propionate, isoamyl isobutyrate, methyl isocyanate, 3-isopropyloxetane, eucalyptol, ethyl 3-methyl-2-butenoate, ethyl ethyl terephthalate, styrene, amyl butyrate, isoamyl isovalerate, and 3-methyl... 2-Methylpentyl 2-butenoate, 2-methyl-2-butenoate-2-methylpropyl ester, ethyl 3-hydroxy-3-methylbutyrate, 2-methyl-2-butenoate-2-methylbutyl ester, isoamyl (E)-2-methylcrotonate, 3-hydroxy-2-butanone, 2-methylbutyric acid, ethyl phenylacetate, phenethyl acetate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, phenethyl alcohol, 2-phenylethyl docosanoate, and dimethyl phthalate, or mixtures thereof, GC-MS total ion chromatogram as shown in the figure. Figure 4 As shown.

[0047] Table 1 Volatile components of PDB fermentation broth

[0048] Example 2 1. Analysis of volatile components in different culture media Take 5g of YEPD and PDB culture medium fermentation broth, add 1.5g of sodium chloride, mix well, and analyze the sample using an GC-MS instrument. GC-MS conditions: Analyze using an Agilent 8890-7000E GC-MS instrument. Chromatographic column: DB-WAX 122-7032 capillary column (30 m × 0.25 mm, 0.25 μm); Chromatographic conditions: 40 ℃ for 2 min, ramped to 100 ℃ at 4 ℃ / min, ramped to 120 ℃ at 5 ℃ / min and held for 5 min, then ramped to 230 ℃ at 5 ℃ / min and held at this temperature for 5 min; Carrier gas: helium, flow rate: 0.8 mL / min, injection port temperature: 250 ℃, split ratio: 20:1; MS conditions: electron impact ionization (EI) energy: 70 eV, ion source temperature: 230 ℃, quadrupole temperature: 150 ℃, EI mass spectra recorded in the range of 45–500 AMU.

[0049] The volatile components of the fermentation broth of strain BS-2 are shown in Table 2 (the components of the blank medium have been removed). The results show that the relative content of total esters of the strain is different in different fermentation media. The total ester content of strain BS-2 is as high as 30% or more in PDB fermentation broth, while it is only 9.58% in YEPD fermentation broth.

[0050] Table 2 Volatile components of YEPD and PDB liquid culture media

[0051] "-" indicates that it was not detected.

[0052] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A high-sugar, aroma-producing, and alcohol-producing strain of *Geotrichum candida*, characterized in that, It is deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 2025067, deposit date: January 8, 2025.

2. The *Geotrichum candida* strain tolerant to high sugar content and producing aroma and alcohol according to claim 1, characterized in that, The strain exhibits excellent salt and acid tolerance, good ethanol tolerance, sugar tolerance, high ester production, and a certain alcohol production capacity.

3. The application of the high-sugar-tolerant, aroma-producing, and wine-producing Geotrichum candida strain according to claim 1 in alcoholic beverage production.

4. The application according to claim 3, characterized in that, The alcohol production mentioned refers to the brewing of alcoholic beverages.

5. The application of the high-sugar-tolerant, aroma-producing, and wine-producing Geotrichum candida strain according to claim 1 in the production of aroma substances.

6. The application according to claim 5, characterized in that, The aroma compounds include esters, alcohols, aldehydes, ketones and their derivatives.

7. The application of the high-sugar-tolerant, aroma-producing, and alcohol-producing Geotrichum candida strain according to claim 1 in the production of dairy products and dairy beverages.

8. The application of the high-sugar-tolerant aroma-producing and alcohol-producing *Geotrichum candida* strain according to claim 1 in the fermentation of alcohols, acids, and esters.

9. The application according to claim 8, characterized in that, The ester compounds mentioned are isobutyraldehyde, ethyl acetate, 2-methylbutyraldehyde, isovaleraldehyde, ethyl propionate, ethyl isobutyrate, isobutyl acetate, ethyl butyrate, 2-methylbutyrate, ethyl isovalerate, isobutyl propionate, isobutanol, isoamyl acetate, ethyl crotonate, isoamyl propionate, isoamyl isobutyrate, methyl isocyanate, 3-isopropyloxetane, eucalyptol, ethyl 3-methyl-2-butenoate, ethyl ethyl phthalate, styrene, amyl butyrate, isoamyl isovalerate, 3-methyl... 2-Methylpentyl 2-butenoate, 2-methyl-2-butenoate-2-methylpropyl ester, ethyl 3-hydroxy-3-methylbutanoate, 2-methyl-2-butenoate-2-methylbutyl ester, isoamyl (E)-2-methylcrotonate, 3-hydroxy-2-butanone, 2-methylbutyric acid, ethyl phenylacetate, phenethyl acetate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, phenethyl alcohol, 2-phenylethyl docosanoate, dimethyl phthalate, or mixtures thereof.

10. A fermenting agent, characterized in that, It contains the live cells of Geotrichum candida that are resistant to high sugar, produce aroma and alcohol as described in claim 1.

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