Candida parapsilosis producing 3-methylthiopropanol and application thereof

Abstract

The application belongs to the field of microorganism and fermentation engineering, and provides a Candida magnoliae with high yield of 3-methylthiopropanol, and the preservation number is CGMCC NO. 23668. The Candida magnoliae can be used for producing 3-methylthiopropanol, the yield can reach 3.16 g / L, and the ethanol tolerance is high, and can be applied in the wine making, condiment and food additive industries.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and fermentation engineering, and relates to a strain of Candida albicans, particularly a strain of Candida albicans that produces high levels of 3-methylthiopropanol. Background Technology

[0002] 3-Methylthiopropanol possesses a strong sweet, soupy, or meaty aroma. As early as 1973, the Flavor and Extract Manufacturers Association (FEMA) in the United States classified it as a generally considered safe flavoring substance, recommending a usage of 0.1 mg / kg-50 mg / kg in food. In 1986, relevant national food standards in my country also listed it as a temporarily permitted food flavoring. Although there is debate about whether 3-methylthiopropanol is a characteristic flavoring substance in sesame-flavored baijiu, it plays an important role in the style formation of some brands of sesame-flavored baijiu and also makes significant contributions to the flavor of soy sauce-flavored baijiu and fruit wines, exhibiting aromas of cauliflower and cooked vegetables, thus significantly contributing to flavor quality.

[0003] 3-Methylthiopropanol is an important food flavoring agent, widely used in meat, fish, and soy sauce flavorings. It can be prepared through chemical synthesis and biotransformation. Chemical synthesis offers advantages such as low cost, rapid synthesis, and high product concentration; however, it also presents challenges such as high toxicity of raw materials, difficulty in removing toxic byproducts, and significant pollution, limiting its long-term industrial use and necessitating the development of new production methods. Microorganisms can convert methionine into 3-methylthiopropanol. Although the current rate of microbial synthesis of 3-methylthiopropanol is lower than that of chemical methods, its advantages, including mild reaction conditions, a single product configuration, environmental friendliness, and safe and pollution-free raw materials, have made it an important development direction for the preparation of "green" and "natural" 3-methylthiopropanol in recent years.

[0004] With the development of technology and time, people's demand for natural products is increasing, and they are becoming more resistant to chemically synthesized substances. Although the chemical synthesis of 3-methylthiopropanol is gradually maturing, the production of 3-methylthiopropanol by microbial metabolism has broad prospects. Summary of the Invention

[0005] To address the issue that the demand for natural green 3-methylthiopropanol cannot be met, this invention provides a high-yield natural strain YHM-G isolated from Daqu (a type of starter culture), which exhibits high ethanol tolerance and can utilize methionine to produce 3-methylthiopropanol.

[0006] Another object of the present invention is to provide a method for producing 3-methylthiopropanol using YHM-G.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A strain of Candida albicans ( Candida naeodendra YHM-G, with accession number CGMCC NO. 23668.

[0009] The aforementioned Candida albicans YHM-G can be used to produce 3-methylthiopropanol, such as in the brewing, condiment, and food additive industries.

[0010] A method for producing 3-methylthiopropanol using Candida albicans YHM-G includes the following steps:

[0011] (1) Inoculate Candida albicans YHM-G into seed culture medium to activate it and obtain seed activation solution;

[0012] (2) The seed activation solution was inoculated into the fermentation medium for fermentation to obtain a culture medium containing 3-methylthiopropanol.

[0013] Preferably, the seed culture medium contains: 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract.

[0014] Preferably, the activation step is: activation at 28 ℃ and 180 r / min for 24 h.

[0015] Preferably, the fermentation medium contains: glucose 10-70 g / L, yeast extract 0.4-2.8 g / L, L-methionine 2-14 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, pH 3-7, and ferrous chloride 0.02 g / L.

[0016] More preferably, the fermentation medium contains: 30-60 g / L glucose, 0.8-1.2 g / L yeast extract, 4-6 g / L L-methionine, 8 g / L potassium dihydrogen phosphate, 6 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 0.03 g / L zinc sulfate, 0.01 g / L magnesium chloride, 0.02 g / L ferrous chloride, and pH 4-6.

[0017] More preferably, the fermentation medium further comprises a surfactant selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Triton-100. The concentration of the surfactant in the fermentation medium is 2-64 g / L; preferably 2-16 g / L.

[0018] Preferably, the inoculum amount in step (2) is 0.1%~6.4% (v / v); the fermentation conditions are: 0~270 r / min, cultured at 20~36℃ for 24~96 h.

[0019] More preferably, in step (2), the inoculum amount is 0.2%~3.2% (v / v); the fermentation conditions are: 135~210 r / min, 28~30 ℃ for 48~60 h.

[0020] A method for producing 3-methylthiopropanol using Candida albicans YHM-G includes the following steps:

[0021] (1) Inoculate Candida albicans YHM-G into seed culture medium and activate it at 28 ℃ and 180 r / min for 24 h to obtain seed activation solution;

[0022] (2) The seed activation solution was inoculated into the fermentation medium at 28 °C and 210 r / min at an inoculation rate of 0.5% and fermented for 48 h to obtain a culture medium containing 3-methylthiopropanol;

[0023] The seed culture medium contains 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract.

[0024] The fermentation medium contains: glucose 50 g / L, yeast extract 1.2 g / L, L-methionine 6 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, Tween-80 2.5 g / L, ferrous chloride 0.02 g / L, pH 6.

[0025] A composition containing the aforementioned Candida albicans YHM-G.

[0026] Depending on the intended use, the composition may also contain other microorganisms, substrates of Candida albicans YHM-G, protectants, fillers or adsorbents, etc.

[0027] The present invention has the following advantages:

[0028] The Candida albicans provided by this invention ( Candida naeodendraYHM-G, derived from Baijiu Daqu (a type of starter culture), exhibits a high yield of 3-methylthiopropanol compared to known strains. Testing showed that this strain can produce up to 3.16 g / L of 3-methylthiopropanol. This strain demonstrates high ethanol tolerance, capable of growing at 11% ethanol concentration, classifying it as a highly ethanol-tolerant strain. Furthermore, it exhibits a wide pH tolerance range (growing within pH 2-12), making it suitable for application in various fields for 3-methylthiopropanol production. Further, this invention utilizes *Candida albicans* (… Candida naeodendra The study of the fermentation characteristics of YHM-G optimized the culture medium composition and culture conditions, thereby increasing the yield of 3-methylthiopropanol by this bacterium.

[0029] Biological Preservation Information

[0030] Candida albicans ( Candida naeodendra YHM-G was deposited on October 26, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC NO.23668. Attached Figure Description

[0031] Figure 1 The colony morphology (a) and cell morphology at 10×100 times magnification (b) of yeast strain YHM-G on WL differential medium;

[0032] Figure 2 It is the phylogenetic tree of yeast strain YHM-G;

[0033] Figure 3 The tolerance of yeast strain YHM-G to temperature (a), pH (b), NaCl (c), ethanol (d), and nicotine (e);

[0034] Figure 4 It is a standard curve made from 3-methylthiopropanol standard;

[0035] Figure 5 The effects of glucose concentration (a), yeast extract concentration (b), and L-methionine concentration (c) on the synthesis of 3-methylthiopropanol by Candida albicans YHM-G.

[0036] Figure 6 The effects of initial pH (a), temperature (b), rotation speed (c), liquid volume (d), and inoculum size (e) on the synthesis of 3-methylthiopropanol by Candida albicans YHM-G;

[0037] Figure 7The effects of surfactant type (a), Tween-80 concentration (b), L-methionine addition time (c), and culture time (d) on the synthesis of 3-methylthiopropanol by Candida albicans YHM-G;

[0038] Figure 8 These are 3D surface plots showing the effects of the pairwise interactions of various factors on the concentration of 3-methylthiopropanol. Among them, (a) is a surface plot showing the effects of glucose concentration and methionine addition time on the concentration of 3-methylthiopropanol; (b) is a surface plot showing the effects of yeast extract concentration and methionine addition time on the concentration of 3-methylthiopropanol; and (c) is a surface plot showing the effects of glucose concentration and yeast extract concentration on the concentration of 3-methylthiopropanol. Detailed Implementation

[0039] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0040] Example 1: Isolation and Identification of YHM-G Strains

[0041] Prepare the culture medium according to the following formula, dispense it into containers, and sterilize it at 121 ℃ for 20 min.

[0042] YPD medium (g / L): yeast extract 10, peptone 20, glucose 20, agar powder 20;

[0043] 3-Methylthiopropanol conversion medium formula (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, and 0.02 g / L ferrous chloride (sterilized by passing through a 0.22 μm filter membrane) was added after sterilization.

[0044] 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.

[0045] 1. Isolation of the target strain

[0046] Take 1 g of pulverized and well-mixed Daqu powder and dilute it 10 times with sterile water. Under sterile conditions, serially dilute the bacterial culture to 10⁻⁵, 10⁻⁶, and 10⁻⁷. Spread 0.1 mL of each serial dilution onto YPD plates and incubate at 30 ℃ for 2 days. Select single colonies with white, raised, opaque yeast morphology and streak them onto YPD plates to obtain pure yeast culture. Store the pure yeast culture in glycerol tubes for later use. Inoculate the yeast strains selected from Daqu into YPD medium and activate them at 28 ℃ and 180 r / min for 24 h. Inoculate the activated yeast seed culture at a rate of 0.2% into sterilized 3-methylthiopropanol conversion medium and incubate at 30 ℃ and 200 r / min for 48 h. After incubation, determine the yield of 3-methylthiopropanol synthesized by the yeast using high-performance liquid chromatography (HPLC). Using the yield of *Saccharomyces cerevisiae* strains as a reference, select strains with higher 3-methylthiopropanol yields. A strain with the code YHM-G was obtained from Daqu (a type of Chinese liquor) and showed good transformation ability, accumulating a high concentration of 3-methylthiopropanol. This strain was preserved in slant agar and glycerol tubes.

[0047] 2. Identification of YHM-G strain

[0048] (1) Colony morphology: Activated yeast cells were inoculated into WL differential medium and cultured at 30 °C for 48 h. Colony morphology was then observed. The results are as follows: Figure 1 As shown in Figure a, the colonies are large, white, round, and raised with regular edges. The surface of the colonies is dry and rough, making them difficult to pick up, and the culture medium at the colony growth site changes from green to yellow. Cell morphology: The yeast cells from step (1) were fixed and methylene blue staining solution was added. The cell morphology was observed under an electron microscope. The cell morphology observed under the microscope is as follows: Figure 1 As shown in b, it is rod-shaped, with a bud at one end but no spores and no mycelium.

[0049] (2) The yeast was subjected to physiological and biochemical identification by sugar fermentation, carbon assimilation, nitrogen assimilation and hydrogen sulfide, indole, methyl red, Volta-P. test, citrate test, starch hydrolysis, urea test and gelatin hydrolysis. The sugars tested in the sugar fermentation test included: D-galactose, D-arabinose, D-xylose, sucrose, glucose, lactose, maltose, D-fructose and L-rhamnose. The carbon sources tested in the carbon assimilation test included: glycerol, ethanol, mannitol, D-sorbose, inulin, D-ribose, D-raffinose and D-trehalose. The nitrogen sources tested in the nitrogen assimilation test included: urea, sodium nitrite, ammonium sulfate, potassium nitrate, L-phenylalanine and L-lysine. The results of physiological and biochemical experiments are shown in Table 1. The YHM-G strain can utilize most sugars, and can use ethanol, glycerol and inulin as the sole carbon source for growth. It can use urea, ammonium nitrate, L-lysine, L-phenylalanine and sodium nitrite and potassium nitrate as the sole nitrogen source for growth. It can produce indole and tryptophanase, can produce amylase to decompose starch, and can use sodium citrate as a carbon source for growth.

[0050] Morphological and physiological-biochemical characteristics of yeast YHM-G and Candida albicans ( Candida naeodendra The yeast strain YHM-G is highly similar to Candida albicans and is preliminarily considered to be Candida albicans. Candida naeodendra ).

[0051] Table 1. Physiological and biochemical identification results of YHM-G strain

[0052]

[0053] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0054] (3) Molecular biological identification: Yeast cells were collected, and DNA was extracted as a template. PCR amplification was performed using the universal 26S rDNA primers: NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'). The amplified products were examined by electrophoresis and sequenced. The sequence was proofread using the BioEdit software, referring to the forward sequence map. The proofread 26S rDNA D1 / D2 region sequence was used to perform a homology search (BLASTsearch) in the GenBank nucleic acid sequence database to compare the similarity between the tested strain and the corresponding sequences of known yeasts. Based on the homology search results, MEGA 6.0 biological software was used to perform comparative analysis on multiple sequences of the test strain and related strains, and the Neighbour-Joining method was used to construct a phylogenetic tree (…). Figure 2 Blast sequence alignment and phylogenetic tree construction revealed that yeast YHM-G is related to Candida albicans (…). Candida naeodendraKC798430.1 is the most closely related.

[0055] Based on morphological observation, physiological and biochemical characteristics, and molecular biology, yeast YHM-G was identified as *Candida nebsiella*. Candida naeodendra This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 26, 2021, with accession number CGMCC NO.23668.

[0056] Example 2: Growth characteristics of strain YHM-G

[0057] 1. Temperature tolerance of the strain

[0058] Using YPD medium as the basal medium, activated bacterial suspensions were inoculated into YPD medium and cultured for 48 h at temperatures of 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, and 50 ℃, with a rotation speed of 180 r / min. The OD560 was then measured by turbidimetric method. Turbidimetric method: The fermentation broth was placed in a cuvette, and the optical density of the fermentation broth was measured at a wavelength of 560 nm using a spectrophotometer. The measured OD value can be used to reflect the bacterial concentration. Results are as follows... Figure 3 As shown in a, the strain involved in this invention cannot grow at temperatures above 40 ℃, and its optimal growth temperature is 25 ℃.

[0059] 2. pH tolerance of the strain

[0060] Activated yeast cells were inoculated at a rate of 0.2% into YPD basal medium with initial pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14. After incubation at 28 °C and 180 r / min for 48 h, the OD560 was determined using a turbidimetric method. The results are as follows: Figure 3 As shown in b, the strain involved in this invention has a wide growth pH range, and can grow between pH 2 and 12, with its optimal growth pH being 6.

[0061] 3. NaCl tolerance of the strain

[0062] Activated yeast strains were inoculated at a rate of 0.2% into YPD medium with NaCl concentrations (w / v) of 0%, 3%, 6%, 9%, 12%, 15%, 18%, and 21%, respectively. After incubation at 28 °C and 180 r / min for 48 h, the OD560 was determined using a turbidimetric method. The results are as follows: Figure 3 As shown in c, this bacterium can grow at NaCl concentrations below 21%, indicating its strong salt tolerance.

[0063] 4. Ethanol tolerance of the strain

[0064] Activated yeast strains were inoculated at a rate of 0.2% into sterilized YPD medium containing 0%, 3%, 6%, 9%, 12%, 15%, 18%, and 21% ethanol (v / v). The cultures were incubated for 48 h in a shaker at 28 °C and 180 r / min, and then their OD560 was determined using a turbidimetric method. The results are as follows: Figure 3 As shown in d, this bacterium can grow at ethanol concentrations below 12%, and is a highly ethanol-tolerant strain.

[0065] 5. Nicotine tolerance of the strain

[0066] Activated yeast strains were inoculated at a 5% inoculation rate into nicotine media with initial nicotine concentrations of 0.3 g / L, 0.4 g / L, 0.6 g / L, 0.7 g / L, 1.0 g / L, and 1.2 g / L. After incubation at 28 ℃ and 180 r / min for 48 h, the OD560 was determined by turbidimetric method. The nicotine liquid culture medium consisted of: 13.3 g / L dipotassium hydrogen phosphate, 4 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, and 0.5 mL of trace element solution (0.008 g manganese sulfate, 0.05 g calcium chloride, 0.05 g copper chloride, 0.004 g ferrous sulfate, 0.1 g zinc sulfate, 0.1 g sodium molybdate, diluted to 1 L with 0.1 mol / L hydrochloric acid). The medium was sterilized at 121 ℃ for 20 min. Nicotine was mixed 1:1 with sterile enrichment medium (initial nicotine concentration in the medium was 0.05 g / L). Results are as follows: Figure 3 As shown in e, the nicotine tolerance concentration of this strain reaches 1.2 g / L.

[0067] Example 3: Single-factor screening of fermentation conditions for strain YHM-G

[0068] 1. Detection method for 3-methylthiopropanol

[0069] The content of 3-methylthiopropanol in the fermentation broth of YHM-G was determined using high performance liquid chromatography (Agilent 1260 Infinity). Chromatographic conditions: C-18 reversed-phase column (ZORBAX Eclipse Plus C-18, 4.6 × 250 mm, 5 μm); mobile phase: methanol:water = 30:70 (v / v); flow rate: 0.7 mL / min; detection wavelength: 215 nm; column temperature: 30 ℃; injection volume: 10 μL; retention time of 3-methylthiopropanol: 9.3 min. A standard curve was plotted using 0.0–6.0 g / L 3-methylthiopropanol standard solutions under the above conditions, with concentration as the x-axis and peak area as the y-axis. Figure 4As shown, the regression equation is y = 3123.5x + 245.62 (R² = 0.9929). Substituting the peak area of ​​the sample into the regression equation, with the peak area as the y-value, the concentration x (g / L) of 3-methylthiopropanol is calculated.

[0070] 2. YHM-G basic fermentation conditions

[0071] (1) Yeast YHM-G was inoculated into YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and activated for 24 h at 28 ℃ and 180 r / min to obtain seed culture;

[0072] (2) The seed culture was inoculated into the fermentation medium (30 g / L glucose, 0.8 g / L yeast extract, 4 g / L L-methionine, 8 g / L potassium dihydrogen phosphate, 6 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 0.03 g / L zinc sulfate, 0.01 g / L magnesium chloride, pH adjusted to 5, sterilized and then 0.02 g / L ferrous chloride (sterilized by passing through a 0.22 μm filter membrane)) at 0.2% inoculation rate and cultured at 30 ℃ and 200 r / min for 48 h to obtain the fermentation broth;

[0073] (3) Take 2 mL of fermentation broth into a 2 mL centrifuge tube, centrifuge at 10000 r / min for 10 min, take 1.5 mL of supernatant, and filter it through a 0.22 μm aqueous filter membrane to obtain the sample to be tested.

[0074] 3. Optimal selection of glucose concentration

[0075] Yeast YHM-G was cultured under basal fermentation conditions, the difference being that the glucose concentration in the fermentation medium was 1.0% (10 g / L), 2.0% (20 g / L), 3.0% (30 g / L), 4.0% (40 g / L), 5.0% (50 g / L), 6.0% (60 g / L), and 7.0% (70 g / L), respectively. The results are as follows: Figure 5 As shown in figure a: when the glucose concentration was 6.0% (60 g / L), yeast YHM-G synthesized the highest amount of 3-methylthiopropanol from glucose, at 1.82 g / L. The yield of 3-methylthiopropanol did not differ significantly between glucose concentrations of 3.0% (30 g / L) and 6.0% (60 g / L).

[0076] 4. Optimal concentration of yeast extract

[0077] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the yeast extract concentrations in the fermentation medium were 0% (0 g / L), 0.04% (0.4 g / L), 0.08% (0.8 g / L), 0.16% (1.6 g / L), 0.2% (2.0 g / L), 0.24% (2.4 g / L), and 0.28% (2.8 g / L), respectively. The results are as follows: Figure 5 As shown in b, the highest 3-methylthiopropanol content (2.02 g / L) was achieved by yeast YHM-G when the yeast extract concentration was 0.2% (2.0 g / L). There was no significant difference in 3-methylthiopropanol yield between yeast extract concentrations of 0.2% (2.0 g / L) and 0.28% (2.8 g / L).

[0078] 5. Optimal L-methionine concentration

[0079] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the methionine concentration in the fermentation medium was 0 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L, and 14 g / L, respectively. The results are as follows: Figure 5 As shown in c: when the L-methionine concentration was 4 g / L, the yeast YHM-G synthesized the highest content of 3-methylthiopropanol, which was 2.03 g / L.

[0080] 6. Optimal initial pH

[0081] Yeast YHM-G was cultured under basal fermentation conditions, the difference being that the initial pH of the fermentation medium was 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. The results are as follows: Figure 6 As shown in figure a: when the initial pH was 4.5, the yeast YHM-G synthesized the highest amount of 3-methylthiopropanol, which was 1.69 g / L. The yield of 3-methylthiopropanol did not differ significantly between the initial pH values ​​of 4.0 and 5.5.

[0082] 7. Optimal Temperature

[0083] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the fermentation temperatures in step (2) were 20℃, 24℃, 28℃, 32℃, 36℃, and 40℃, respectively. The results are as follows: Figure 6 As shown in b: when the temperature is 32 ℃, the yeast YHM-G synthesizes the highest content of 3-methylthiopropanol, which is 1.87 g / L.

[0084] 8. Optimal Rotation Speed

[0085] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the fermentation speed in step (2) was 0 r / min, 45 r / min, 90 r / min, 135 r / min, 180 r / min, 225 r / min, and 270 r / min, respectively. The results are as follows: Figure 6 As shown in c: when the rotation speed is 135 r / min, the yeast YHM-G synthesizes the highest content of 3-methylthiopropanol, which is 2.49 g / L.

[0086] 9. Optimal liquid volume

[0087] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the liquid volume in step (2) was 25 mL, 50 mL, 75 mL, 100 mL, and 125 mL / 250 mL, respectively. The results are as follows: Figure 6 As shown in d: when the liquid volume is 75 mL, the yeast YHM-G synthesizes the highest content of 3-methylthiopropanol, which is 1.92 g / L.

[0088] 10. Optimal Inoculation Dosage

[0089] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the inoculum amounts in step (2) were 0.1%, 0.2%, 0.4%, 0.8%, 1.6%, 3.2%, and 6.4%, respectively. The results are as follows: Figure 6 As shown in e, the highest 3-methylthiopropanol content (1.89 g / L) was achieved by yeast YHM-G when the inoculum size was 0.4%. There was no significant difference in 3-methylthiopropanol yield between inoculum sizes of 0.2% and 3.2%.

[0090] 11. Preferred surfactants

[0091] Yeast YHM-G was cultured under basal fermentation conditions, the difference being the addition of 0.2% of different surfactants (blank, glycerol, Tween-20, Tween-40, Tween-60, Tween-80, Triton-100, and Triton-114) to the fermentation medium. The results are as follows: Figure 7 As shown in Figure a, Tween-20, Tween-40, Tween-60, Tween-80, and Triton-100 all promoted the production of 3-methylthiopropanol without significant differences. When Tween-80 was used as the surfactant, the yeast YHM-G synthesized the highest content of 3-methylthiopropanol, which was 2.31 g / L.

[0092] 12. Preferred concentration of Tween-80

[0093] Yeast YHM-G was cultured under basal fermentation conditions, the difference being the addition of Tween-80 at concentrations of 0 g / L, 2 g / L, 4 g / L, 8 g / L, 16 g / L, 32 g / L, and 64 g / L to the fermentation medium. The results are as follows: Figure 7 As shown in b, when the Tween-80 concentration was 16 g / L, the yeast YHM-G synthesized the highest amount of 3-methylthiopropanol, at 2.08 g / L. Different concentrations of Tween-80 promoted the yield of 3-methylthiopropanol without significant differences.

[0094] 13. Optimal timing of L-methionine addition

[0095] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that L-methionine was added to the fermentation medium at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The results are as follows: Figure 7 As shown in c: when L-methionine was added for 24 h, the yeast YHM-G synthesized the highest content of 3-methylthiopropanol, which was 1.47 g / L.

[0096] 14. Optimal selection of incubation time

[0097] Yeast YHM-G was cultured under basic fermentation conditions, the difference being that the culture time in step (2) was 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h, respectively. The results are as follows: Figure 7 As shown in c: when the culture time was 60 h, the yeast YHM-G synthesized the highest content of 3-methylthiopropanol, which was 1.72 g / L. There was no significant difference in the yield of 3-methylthiopropanol between the culture times of 48 h and 96 h.

[0098] Example 4: Response surface methodology for fermentation conditions of YHM-G strain

[0099] 1. Plackett-Burman test

[0100] The Plackett-Burman experimental design was used to test 11 factors in Example 3, excluding the type of surfactant, using Mintab 17 (Minitab, Inc. State College, PA, USA). Each factor had three replicates, as shown in Table 2. The response value was the concentration of 3-methylthiopropanol.

[0101] Table 2. Factors and levels in the Plackett–Burman experimental design

[0102]

[0103] The experimental results are shown in Table 3. The variation range of 3-methylthiopropanol was 0.00~2.00 g / L, and the regression equation was: 3-methylthiopropanol concentration = 2.1126 + 0.002173 Rotation speed (rpm) - 0.04118 Temperature (°C) + 0.018272 Glucose concentration (g / L) + 0.18997 Initial pH - 0.005191 Liquid volume (mL) + 0.3529 Inoculum size (%) - 0.029632 Methionine addition time (h) - 0.008589 Culture time (h) - 0.4476 Yeast extract concentration (g / L) + 0.05511 Methionine concentration (g / L) + 0.05673 Tween-80 concentration (g / L).

[0104] Table 3. Plackett–Burman Experimental Design and Results

[0105]

[0106] Table 4. Significance analysis results of the Plackett-Burman test.

[0107]

[0108] The adequacy of the model was tested using analysis of variance (ANOVA). ANOVA showed that the model was significant. The p-value was significant (0.001), and a p-value less than 0.05 is generally considered acceptable. Table 4 shows that all 11 factors had a significant impact on the concentration of 3-methylthiopropanol; therefore, further research was conducted on these 11 factors.

[0109] 2. Steepest Climb Test

[0110] Based on the Plackett-Burman experiment results, and combined with the regression coefficients in Table 4, the steepest climbing experiment was designed with step length and direction. Among them, rotation speed, glucose concentration, initial pH, inoculum size, methionine concentration, and Tween-80 concentration had positive effects on the yield of 3-methylthiopropanol, while temperature, liquid volume, methionine addition time, culture time, and yeast extract concentration had negative effects.

[0111] Table 5. Experimental Design and Results for the Steepest Climb

[0112]

[0113] The design and results of the steepest climb test are shown in Table 5. Among them, the fifth group had the highest yield of 3-methylthiopropanol, which was 2.03 g / L. Therefore, the experimental conditions of the fifth group were used as the center point for subsequent response surface methodology experiments.

[0114] 3. Response surface methodology

[0115] After identifying the region with the highest 3-methylthiopropanol yield through the steepest ramp-up experiment, a Box-Behnken experimental design from the response surface methodology was employed. Based on the results of the PB experiment and the steepest ramp-up experiment, further investigation was conducted on the three most critical factors (methionine addition time (A), glucose concentration (B), and yeast extract concentration (C)) to enhance 3-methylthiopropanol yield. Each factor was assigned three levels, coded as -1, 0, and 1, with the 3-methylthiopropanol concentration as the response value Y. Table 6 shows the experimental design and results. Table 6 indicates that the 13th group of experiments yielded the highest 3-methylthiopropanol yield, at 2.74 g / L.

[0116] Table 6 Box-Behnken Experimental Design and Results

[0117]

[0118] Multiple regression analysis was conducted on 15 sets of experimental data. After fitting the regression equation, the influence of each factor on the response value can be represented by the following function: Y = 1.39 - 0.36 A + 0.43 B + 0.38 C - 0.055 A² + 0.45 B². The analysis of variance and model reliability of the regression equation are shown in Table 7.

[0119] Table 7 Analysis of Variance of Regression Model

[0120]

[0121] Table 7 shows that the model P < 0.05, indicating that the model is significant. The R² of the quadratic regression equation is 0.7699, indicating that the equation fits the experiment well with a small error. Furthermore, the lack of significance in the missing-fit term suggests a good correlation between the actual and predicted values. The yield of 3-methylthiopropanol can be reflected and predicted using this model. Changes in the dependent variable depend on changes in the independent variables, and the influence of the independent variables on the dependent variable can be reflected by the significance in the analysis of variance.

[0122] The effects of pairwise interactions of various factors on the concentration of 3-methylthiopropanol can be presented by a 3D response surface plot. Figure 8 The regression equation shows that the optimal fermentation conditions are: methionine addition time 0 h, glucose concentration 50 g / L, yeast extract concentration 1.2 g / L, temperature 28 ℃, rotation speed 210 r / min, initial pH 6, liquid volume 50 mL, inoculum size 0.5%, culture time 48 h, methionine concentration 6 g / L, and Tween-80 concentration 2.5 g / L.

[0123] To verify the accuracy of the model's predictions, the experiment was repeated three times under the above-mentioned optimal fermentation conditions. The average yield of 3-methylthiopropanol was 3.16 g / L, which was close to the predicted value of 2.96 g / L, proving that the model is reliable.

[0124] Application Example 1: Application of YHM-G yeast in brewing

[0125] (1) Yeast YHM-G was inoculated into YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and activated at 28℃ and 180 r / min for 24 h to obtain seed culture;

[0126] (2) Take 800 g of red sorghum with husks, crush the sorghum grains into 4-5 pieces using a pulverizer, add 1.6 L of 80℃ hot water, mix thoroughly and soak for 24 h, add 200 g of rice husks, mix again, and spread evenly on a gauze-lined rack (not too thick), place in a boiling water steamer, cover and steam for 1.5 h. Immediately remove the grain, mix well, and spread out to cool. When the grain temperature is about 30℃, add 100 g of Daqu powder (20-40 mesh), and inoculate the experimental group with 10% (w / w) of the yeast cell suspension prepared above, while the control group uses brewer's yeast instead. After mixing well, divide into 2 L ceramic jars and carry out solid-state fermentation at room temperature;

[0127] (3) Accurately weigh 1 g of mixed mash, place it in a 15 mL gas chromatography-mass spectrometry bottle, add 5 mL of saturated saline and 1 µL of 0.5 g / L 2-octanol, equilibrate at 60℃ for 30 min, adsorb at 60℃ for 30 min, and then perform gas chromatography-mass spectrometry to detect volatile flavor substances.

[0128] (4) The results showed that the experimental group inoculated with 10% YHM-G had a richer content of flavor substances than the control group. 3-methylthiopropanol was detected in the experimental group, while it was not detected in the control group. This indicates that the strain has certain application potential in increasing the content of 3-methylthiopropanol in Baijiu.

Claims

1. A strain of Candida albicans ( Candida naeodendra YHM-G, with accession number CGMCC NO. 23668.

2. The application of *Candida albicans* YHM-G as described in claim 1 in the production of 3-methylthiopropanol, characterized in that, The applications are in brewing and food additive preparation.

3. The application of *Candida albicans* YHM-G as described in claim 1 in the production of 3-methylthiopropanol, characterized in that, The application is in the preparation of condiments.

4. A method for producing 3-methylthiopropanol using *Candida albicans* YHM-G as described in claim 1, characterized in that, Includes the following steps: (1) Inoculate Candida albicans YHM-G into seed culture medium to activate it and obtain seed activation solution; (2) The seed activation solution was inoculated into the fermentation medium for fermentation to obtain a culture medium containing 3-methylthiopropanol.

5. The method according to claim 4, characterized in that, The seed culture medium contains: 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract. The activation step is as follows: activation at 28 ℃ and 180 r / min for 24 h; The fermentation medium contains: glucose 10-70 g / L, yeast extract 0.4-2.8 g / L, L-methionine 2-14 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, pH 3.5-6.0, and ferrous chloride 0.02 g / L. In step (2), the inoculum amount is 0.1%~6.4% (v / v); the fermentation conditions are: 90~270 r / min, 24~36 ℃ for 24~96 h.

6. The method according to claim 4, characterized in that, The fermentation medium contains: glucose 30-60 g / L, yeast extract 0.8-1.2 g / L, L-methionine 4-6 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, ferrous chloride 0.02 g / L, pH 4-6; In step (2), the inoculum amount is 0.2%~3.2% (v / v); the fermentation conditions are: 135~210 r / min, 28~30 ℃ for 48~60 h.

7. The method according to claim 4, characterized in that, The fermentation medium also contains a surfactant; selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Triton-100; the concentration of the surfactant in the fermentation medium is 2~64 g / L.

8. The method according to claim 7, characterized in that, The concentration of the surfactant in the fermentation medium is 2~16 g / L.

9. A method for producing 3-methylthiopropanol using *Candida albicans* YHM-G as described in claim 1, characterized in that, Includes the following steps: (1) Inoculate Candida albicans YHM-G into seed culture medium and activate it at 28 ℃ and 180 r / min for 24 h to obtain seed activation solution; (2) The seed activation solution was inoculated into the fermentation medium at 28 °C and 210 r / min at an inoculation rate of 0.5% and fermented for 48 h to obtain a culture medium containing 3-methylthiopropanol; The seed culture medium contains 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract. The fermentation medium contains: glucose 50 g / L, yeast extract 1.2 g / L, L-methionine 6 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, Tween-80 2.5 g / L, ferrous chloride 0.02 g / L, pH 6.

10. A composition containing the Candida albicans YHM-G of claim 1.

11. The composition according to claim 10, characterized in that, It also contains substrates, protectants, fillers, and adsorbents of Candida albicans YHM-G.

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