A 3-methylthiopropanol-producing strain of williopsis saturnus and culture method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and provides a high-yield 3-methylthiopropanol Torulopsis pullulans, with a preservation number of CGMCC No.23667.The Torulopsis pullulans can be used for producing 3-methylthiopropanol, and the yield can reach 4.07 g / L, and the ethanol tolerance is high, so that the Torulopsis pullulans can be applied in the industries of brewing, tobacco fermentation, condiment and food additive.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a yeast strain that produces 3-methylthiopropanol, its culture method, and its application. Background Technology

[0002] Baijiu is China's national liquor, and currently has twelve major aroma types. Sesame-flavored baijiu is an innovative aroma type, characterized by "prominent sesame aroma, harmonious flavors, full-bodied and delicate taste, and a long aftertaste." The uniqueness of its flavor compounds creates the differences in the style of the liquor. GB / T 20824-2007 "Sesame-flavored Baijiu" identifies 3-methylthiopropanol as a characteristic flavor compound of sesame-flavored baijiu, and stipulates that the mass concentration of 3-methylthiopropanol in high-proof baijiu must be greater than 0.5 mg / L, and the mass concentration in low-proof baijiu must be greater than 0.4 mg / L.

[0003] 3-Methylthiopropanol (molecular formula C4H10OS), also known as pineapple alcohol, has a typical sulfurous odor and is a major flavor compound in fermented foods. It is widely used in the food, cosmetics, and tobacco industries and is a very important sulfur-containing aroma compound. As a key flavor compound that provides a caramelized flavor, it is found in many fruits and various fermented foods such as sesame-based liquor, cheese, and soy sauce. Currently, 3-methylthiopropanol is usually synthesized chemically in industrial production. Although this method is relatively inexpensive, the raw materials and products inevitably pose health risks. Furthermore, the increasing consumer demand for "natural flavorings" has spurred the development of green and safe microbial transformation methods.

[0004] With the advancement of technology and time, people's demand for natural products is increasing. Although the chemical synthesis of 3-methylthiopropanol has gradually matured, there is growing resistance to chemically synthesized substances. Therefore, the production of 3-methylthiopropanol using microorganisms has broad application prospects. Current research on the production of 3-methylthiopropanol using microbial metabolism shows that high-yield strains are mainly engineered strains, while natural strains have relatively low production capacity. This paper selects a natural yeast strain obtained from brewing yeast to synthesize 3-methylthiopropanol for screening purposes. The aim is to obtain a natural yeast strain capable of producing 3-methylthiopropanol, and optimize its fermentation conditions, providing a theoretical basis for the microbial synthesis of 3-methylthiopropanol. Summary of the Invention

[0005] In response to the problem that natural green 3-methylthiopropanol cannot meet market demand, and in order to improve the content of 3-methylthiopropanol in traditional brewed food sesame-flavored baijiu and enhance the quality of baijiu, the present invention aims to provide a strain of 3-methylthiopropanol newly isolated from baijiu yeast, named Y1402, and to provide the culture medium, culture method and application of Y1402 for producing 3-methylthiopropanol.

[0006] Another object of the present invention is to provide a method for producing 3-methylthiopropanol using Y14O2.

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

[0008] A strain of cladocyst-coated yeast ( Saccharomycopsis fibuligera Y1402, with accession number CGMCC No.23667.

[0009] The colony characteristics and biochemical properties of the Y1402 cladomycin are as follows: On solid YPD medium, the colonies of this strain are milky white, small, with regular edges, a slightly raised center, a smooth and moist surface, and are relatively viscous. The cells are oval, with budding at the tip, and no mycelium. It produces a strong aroma during fermentation, can tolerate 9% (v / v) ethanol, can tolerate 15% (w / v) NaCl, has a pH growth range of 2-13, and a growth temperature range of 20-40℃.

[0010] The described *Saccharomyces cerevisiae* Y1402 can be used to produce 3-methylthiopropanol, for applications in brewing, tobacco fermentation, condiments, and food additives industries. *Saccharomyces cerevisiae* Y1402 is mainly used for microbial fermentation to prepare 3-methylthiopropanol, with a yield of 2.0~4.07 g / L. It can be used as a functional microbial strain to increase the 3-methylthiopropanol content in baijiu (Chinese liquor).

[0011] A method for producing 3-methylthiopropanol using *Saccharomyces cerevisiae* Y1402 includes the following steps:

[0012] (1) Inoculate the Y1402 capsule-coated yeast into the liquid seed activation medium to activate it and obtain the seed activation solution;

[0013] (2) The seed activation solution obtained in step (1) is inoculated into a liquid fermentation medium for fermentation to obtain a culture medium containing 3-methylthiopropanol.

[0014] Preferably, the liquid seed activation culture medium in step (1) includes: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, and natural pH.

[0015] Preferably, in step (1), the activation conditions are: 24 h of incubation at 28℃ and 180 r / min.

[0016] Preferably, in step (2), the inoculum amount is 0.1~6.4%, and the fermentation conditions are: 0~270 r / min, cultured at 20~40℃ for 12~96 h.

[0017] More preferably, in step (2), the inoculum amount is 0.8%, and the fermentation conditions are: 90~270 r / min, cultured at 24~36℃ for 36~96 h.

[0018] Preferably, in step (2), the liquid fermentation culture medium comprises: glucose 10~60 g / L, yeast extract 0~2 g / L, L-methionine 2~10 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, and pH 3.0~7.0.

[0019] More preferably, in step (2), the liquid fermentation medium comprises: 40-50 g / L glucose, 0.8-1.2 g / L yeast extract, 2-10 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 4.0-6.0.

[0020] More preferably, in step (2), the liquid fermentation medium comprises: 40-50 g / L glucose, 0.9 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, and pH 5.0.

[0021] Preferably, in step (2), the liquid fermentation medium further includes a surfactant selected from at least one of glycerol, Tween-20, Tween 40, Tween 60, Tween 80 and X-100; preferably Tween 80.

[0022] More preferably, the concentration of Tween 80 in the fermentation medium is 2~64 g / L; preferably 2 g / L.

[0023] A composition containing the above-mentioned *Saccharomyces cerevisiae* Y1402.

[0024] Preferably, depending on the intended use, the composition may also contain other microorganisms, substrates of *Saccharomyces cerevisiae* Y1402, protectants, fillers, or adsorbents.

[0025] The present invention has the following advantages:

[0026] (1) The strain of *Saccharomyces cerevisiae* screened in this invention (… Saccharomycopsis fibuligera Y1402 originated from the environment of Baijiu brewing and has the characteristic of 3-methylthiopropanol. The test results showed that the 3-methylthiopropanol content of this strain can reach 4.07 g / L.

[0027] (2) This invention utilizes the method of treating cyst-forming yeast ( Saccharomycopsis fibuligera The study on the fermentation characteristics of Y1402 optimized the culture medium composition and culture conditions, thereby increasing the yield of 3-methylthiopropanol by this bacterium.

[0028] (3) The capsule-forming yeast screened in this invention ( Saccharomycopsis fibuligera The Y1402 strain has a high yield of 3-methylthiopropanol and high tolerance to ethanol, which is beneficial for the production of 3-methylthiopropanol and its application in baijiu brewing.

[0029] Biological Preservation Information

[0030] Capsule-forming yeast ( Saccharomycopsis fibuligera Y1402 was deposited on October 26, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 23667. Attached Figure Description

[0031] Figure 1 The colony morphology (a) and cell morphology (b) of *Saccharomyces cerevisiae* strain Y1402 on WL differential medium are shown.

[0032] Figure 2 This is the phylogenetic tree of the Y1402 strain of *Saccharomyces cerevisiae*.

[0033] Figure 3 The results show the temperature (a), pH (b), NaCl (c), ethanol (d), and nicotine (e) tolerance of the Y1402 strain of *Saccharomyces cerevisiae*.

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

[0035] Figure 5 The effects of glucose (a), yeast extract (b), L-methionine (c) concentrations and initial pH (d) on the synthesis of 3-methylthiopropanol by *Saccharomyces cerevisiae* Y1402 were investigated.

[0036] Figure 6 The effects of temperature (a), rotation speed (b), liquid volume (c), and inoculum size (d) on the synthesis of 3-methylthiopropanol by *Saccharomyces cerevisiae* Y1402 were investigated.

[0037] Figure 7 The effects of surfactant type (a) and Tween-80 concentration (b) on the synthesis of 3-methylthiopropanol by *Saccharomyces cerevisiae* Y1402;

[0038] Figure 8 The effects of L-methionine addition time (a) and culture time (b) on the synthesis of 3-methylthiopropanol by *Saccharomyces cerevisiae* Y1402;

[0039] Figure 9 These are 3D surface plots showing the effects of the pairwise interactions of various factors on the concentration of 3-methylthiopropanol: (a) Surface plot showing the effects of glucose concentration and methionine addition time on the concentration of 3-methylthiopropanol; (b) Surface plot showing the effects of yeast extract concentration and methionine addition time on the concentration of 3-methylthiopropanol; (c) Surface plot showing the effects of glucose concentration and yeast extract concentration on the production of 3-methylthiopropanol by Yeast Y1402. Detailed Implementation

[0040] 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.

[0041] The culture medium formulations used in the following examples are as follows:

[0042] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar powder, and distilled water to a final volume.

[0043] WL medium (g / L): glucose 50, yeast extract 5, peptone 5, potassium dihydrogen phosphate 0.55, potassium chloride 0.425, calcium chloride 0.125, ferric chloride 0.0025, magnesium sulfate 0.125, manganese sulfate 0.0025, bromocresol green 0.022, agar 20, pH 6.5, sterilized at 121℃ for 20 min.

[0044] Example 1: Isolation of Y1402, a type of yeast with a capsid membrane

[0045] Yeast strains were isolated from Daqu powder using traditional microbial isolation methods, as follows: 1 g of pulverized and well-mixed Daqu powder was diluted 10-fold with sterile water. Under sterile conditions, the bacterial culture was serially diluted to 10⁻⁵, 10⁻⁶, and 10⁻⁷. 0.1 mL of each serial dilution was plated onto YPD plates and incubated at 30°C for 2 days. Single colonies with white, raised, opaque structures exhibiting yeast morphology were picked and streaked onto YPD plates to obtain pure yeast culture. These were then stored in glycerol tubes for later use.

[0046] Yeast strains screened from Daqu (a type of yeast) were inoculated into YPD medium and activated for 24 h at 28℃ and 180 r / min. Three activated yeast seed cultures were inoculated into sterilized 3-methylthiopropanol conversion medium at an inoculation rate of 0.2% and cultured for 48 h in a shaker at 30℃ and 200 r / min. After culture, the yield of 3-methylthiopropanol synthesized by the three yeast strains was determined by UV-HPLC. Finally, a strain with good conversion ability and capable of accumulating a high concentration of 3-methylthiopropanol was obtained, namely *Saccharomyces cerevisiae*. Saccharomycopsis fibuligera Y1402.

[0047] The above-mentioned capsule-forming yeast ( Saccharomycopsis fibuligera Y1402 is saved using the following method:

[0048] (1) Slant preservation: The purified strain was inoculated onto YPD slant, placed in an incubator and cultured for 48-72 h, and then stored in a refrigerator at 4℃;

[0049] (2) Glycerin tube preservation: Take 1 mL of 50% glycerol and mix it with 1 mL of activated bacterial solution, then store it in a 1.5 mL sterile PE tube at -80℃.

[0050] Example 2 Identification of Y1402, a type of yeast with a cladostomata-like membrane

[0051] The obtained capsule-coated yeast ( Saccharomycopsis fibuligera The identification of Y1402 includes the following steps:

[0052] Step 1: Morphological characteristics

[0053] To identify the aforementioned yeast strains, the following morphological characteristics were observed:

[0054] Yeast colony morphology observation: Yeast was inoculated into YPD liquid medium and cultured for 24-48 h. The activated strain was then streaked into WL differential medium and incubated at 30℃ for 48 h. Colony morphology was observed after the incubation period. The colony morphology of strain Y1402 on WL differential medium is shown below. Figure 1 As shown in Figure a, the colonies are white, round and protruding, and relatively small. The color of the culture medium at the site of bacterial growth changes from blue-green to yellow, and the surface is smooth, moist, and easy to pick up.

[0055] Yeast cell morphology observation: Place a drop of sterile water in the center of a sterilized glass slide. Using an inoculation loop, pick a small number of yeast cells from a single colony on WL differential medium and spread them evenly on the sterile water on the slide. Fix the cells by heating over an alcohol lamp flame. Add methylene blue staining solution, cover with a coverslip, and blot away excess staining solution with absorbent paper. Observe the cell morphology under an electron microscope. Cell morphology is as follows: Figure 1 As shown in b, it is oval in shape with a bud at the tip.

[0056] Step 2: Physiological and biochemical identification

[0057] To identify the aforementioned yeast strains, the following physiological and biochemical assays were performed:

[0058] (1) Sugar fermentation experiment

[0059] The sugars tested in this invention include the following sugar sources: D-galactose, L-sorbose, D-arabinose, D-(+)-xylose, sucrose, glucose, lactose, maltose, and D-fructose.

[0060] The sugar fermentation experiment conducted in this invention consists of the following steps: Prepare the sugar fermentation medium, dispense it into test tubes, add Durham tubes, and sterilize at 121°C for 20 min; prepare the sugar concentration to 50 mmol / L, filter (0.22 μm) for sterilization, and add it and 1.6% bromocresol purple ethanol to the medium containing Durham tubes; The activated bacterial culture was inoculated into the above fermentation tubes. Three parallel experiments were performed for each sugar. Fermentation tubes without test sugar and without inoculation were used as blank controls. The tubes were placed at 25°C for static incubation. The amount of bubbles and color changes in the Durham tubes were observed daily for two weeks. The presence of bubbles in the Durham tube was recorded as positive (+), indicating that the yeast could ferment the sugar. The absence of bubbles was recorded as negative (-), indicating that the yeast could not ferment the sugar. When the color changed from purple to yellow, it indicated that the yeast could produce acid using the sugar.

[0061] (2) Carbon assimilation experiment

[0062] The present invention identifies the required assimilation carbon sources including glycerol, ethanol, mannitol, soluble starch, α-lactose, D-sorbose, cellobiose, inulin, D-(+)-ribose, and D-(+)-raffinose.

[0063] The carbon assimilation experiment conducted in this invention includes the following steps: The above substances were prepared into a 20% solution, filtered (0.22 μm) for sterilization, and then added to a carbon source assimilation medium containing Durham tubes, along with bromothymol blue at a final concentration of 0.0024%. Add the activated yeast to the above culture medium, and perform three replicates for each carbon source. Use a test tube without carbon source as a blank control. Incubate at 25°C for two weeks. After two weeks, observe the test tubes and shake them thoroughly. If the culture medium in the test tube becomes turbid, it is recorded as positive (+), otherwise it is recorded as negative (-).

[0064] (3) Nitrogen assimilation experiment

[0065] The nitrogen source assimilation substances identified in this invention include urea, potassium nitrite, ammonium sulfate, potassium nitrate, L-phenylalanine, ammonium nitrate, sodium nitrate, sodium nitrite, and aluminum nitrate.

[0066] The nitrogen assimilation experiment conducted in this invention includes the following steps: The above substances were prepared into solutions, filtered (0.22 μm) for sterilization, and then added to nitrogen source assimilation culture medium to achieve a final concentration of 0.1 mol / L. The activated yeast was inoculated into the test tubes described above, with three replicates for each nitrogen source. A culture medium without nitrogen source was used as a blank control. The tubes were incubated at 25°C for one week, and the results were observed after one week. If the culture medium in the test tubes became turbid, it was recorded as positive (+), and otherwise as negative (-).

[0067] (4) Physiological and biochemical experiments of the strain

[0068] ① Hydrogen sulfide experimental medium (g / L): pH 7.0, NaCl 5, tryptone 25, gelatin 120, beef extract 7.5. Sterilize at 115℃ for 20 min. Add 5 mL of 10% FeSO4·7H2O (sterilized by 0.22 μm membrane filtration), inoculate with seed culture by puncture, and incubate at 37℃ for 48 h.

[0069] ② Indole test medium (g / L): pH 7.8, NaCl 5, tryptone 10. Sterilize at 121℃ for 20 min. Inoculate 50 μL of seed culture and place in a 37℃ incubator. After two days, remove the medium, add 3-4 drops of ether, shake vigorously, let stand for a few minutes, and then add 3-4 drops of indole reagent along the tube wall.

[0070] ③ Methyl red test medium (g / L): K2HPO4 2, glucose 5, tryptone 5, pH 7.0~7.2. Sterilize for 30 min at 112 ℃. Inoculate 50 μL of seed culture and place in a 37 ℃ incubator. After two days, remove the medium, add 2~3 drops of methyl red reagent, and then observe.

[0071] ④ Volta-Putschland experimental medium (g / L): NaCl 5, tryptone 10, pH 7.8, sterilized for 20 min at 121 ℃. Inoculate with 50 μL of seed culture and incubate at 37 ℃ for 48 h. Add 7-8 drops of 40% KOH solution, then add 5% α-naphthol. Shake well, let stand, and observe the phenomenon.

[0072] ⑤ Citrate culture medium (g / L): NH4H2PO4 1, NaCl 5, K2HPO4 1, sodium citrate 2, agar 20, MgSO4 0.2, pH 6.8. Sterilize at 121℃ for 20 min. Add 10 mL of 1% bromothymol blue ethanol solution. Inoculate the culture medium and incubate at 37℃ for 48 h. Observe after 48 h.

[0073] ⑥ Starch hydrolysis experimental medium (g / L): NaCl 5, beef extract 5, agar 20, tryptone 10, soluble starch 2. Sterilize at 121℃ for 20 min. Inoculate the seed culture onto plates and place them in a 37℃ incubator. After 24 h, remove the plates, drop iodine solution onto the surface, and gently rotate the plates to ensure complete contact of the iodine solution with the medium surface. Observe.

[0074] ⑦ Urea test medium (g / L): Urea medium slant: tryptone 1, agar 15, NaCl 5, KH2PO4 2, sterilized at 121℃ for 20 min. While the medium is still liquid, add 20 g of urea (sterilized by membrane filtration), then add 0.012 g of phenol red. Streak the seed culture slant and incubate at 35℃ for 48 h.

[0075] ⑧ Gelatin hydrolysis experimental culture medium (g / L): NaCl 5, tryptone 10, gelatin 120, beef extract 3, pH 7.2~7.4, sterilized at 121℃ for 30 min. Inoculate the seed culture by puncture, and observe after 2~5 days at 20℃.

[0076] The physiological and biochemical results of the above-mentioned *Saccharomyces cerevisiae* strain Y1402 are shown in Tables 1-4. The sugar fermentation test results showed that Y1402 can utilize D-maltose, D-xylose, glucose, maltose, and sucrose for growth. It can produce acid and gas using glucose, but only acid without gas using D-galactose, D-maltose, maltose, and sucrose. It cannot utilize L-rhamnose, D-xylose, D-arabinose, or lactose for acid and gas production. In the carbon source assimilation test, yeast Y1402 can utilize ethanol and D-ribose as the sole carbon source for growth, and its growth is good. Although it can utilize inulin and glycerol as carbon sources, its growth is slightly worse than with ethanol and D-ribose. It cannot utilize D-trehalose, D-raffinose, mannitol, or D-sorbose as the sole carbon source for growth. Nitrogen source assimilation tests showed that yeast Y1402 could grow using sodium nitrite and potassium nitrate as the sole nitrogen source, and it could also grow well using urea, ammonium nitrate, L-lysine, and L-phenylalanine as the sole nitrogen source. The indole test, methyl red test, citrate test, and starch hydrolysis test were positive, indicating that yeast Y1402 can produce indole, possess tryptophanase, produce amylase to decompose starch, and can use sodium citrate as a carbon source for growth. The hydrogen sulfide test, voltaic test, urea hydrolysis test, and gelatin liquefaction test were all negative.

[0077] Table 1. Capsule-forming yeast ( Saccharomycopsis fibuligera Results of sugar fermentation experiment of strain Y1402

[0078]

[0079] Table 2. Capsule-forming yeast ( Saccharomycopsis fibuligera Results of carbon source assimilation experiment of strain Y1402

[0080]

[0081] Table 3. Capsule-forming yeast ( Saccharomycopsis fibuligera Results of nitrogen source assimilation experiment of strain Y1402G

[0082]

[0083] Table 4. Capsule-covered yeast ( Saccharomycopsis fibuligera Physiological and biochemical experimental results of strain Y1402

[0084] .

[0085] Step 3: Molecular biological identification

[0086] To identify the aforementioned yeast strains, the molecular biological assays performed included the following steps:

[0087] (1) Bacterial culture and collection

[0088] The activated strain was inoculated into YPD liquid medium and cultured at 28℃ and 180 r / min for 48 h. The bacterial cells were then collected by centrifugation at 10000 r / min for 10 min.

[0089] (2) Extraction of yeast strain genomic DNA and amplification of 26S rDNA

[0090] The extraction method for yeast genomic DNA followed the method described in the fungal DNA extraction kit. Amplification primers were used: using the extracted total yeast DNA as a template, the 26S universal primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') were used to amplify the 26S rDNA D1 / D2 region sequence. The PCR reaction mixture consisted of 2.5 μL LA PCR Buffer, 1 μL each of primers, 2 μL dNTPs, 0.2 μL LAtaq enzyme, and 2 μL DNA, adjusted to a final volume of 25 μL with ddH2O.

[0091] PCR amplification program: 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 30 s, 58 ℃ annealing for 30 s, 72 ℃ extension for 1 min, for a total of 30 cycles, with a final extension at 72 ℃ for 10 min. The PCR amplification products of yeast strain DNA were examined by electrophoresis on a 1% agarose gel.

[0092] (3) Sequencing and phylogenetic tree construction

[0093] The 26S amplification product was purified and sent for sequencing to obtain the original sequence of the PCR amplified fragment of the strain. The sequence was proofread using BioEdit sequence mapping software, referring to the forward sequence map. The proofread 26S rDNA D1 / D2 region sequence was then used to perform a BLAST search in the GenBank nucleic acid sequence database to compare the similarity between the tested strain and the corresponding sequences of known yeasts.

[0094] Based on 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 ).

[0095] After BLAST sequence alignment of the sequencing results of yeast Y1402 in NCBI, MEGA7 software was used to perform sequence alignment of strains with high homology with strain Y1402, and a phylogenetic tree was constructed using Neighbor-Joining. Figure 4 As shown), yeast Y14012 and cladomycin (as shown) Saccharomycopsis fibuligera KU956952.1 is the most closely related species. Based on morphological observation, physiological and biochemical characteristics, and molecular biology, yeast Y1402 was identified as *Saccharomyces cerevisiae*. Saccharomycopsis fibuligera This strain was deposited with the China General Microbiological Culture Collection Center (CGMCC) on October 26, 2021, with accession number CGMCC No. 23667.

[0096] Example 4: Growth characteristics of *Saccharomyces cerevisiae* Y1402

[0097] The growth characteristics of cladocyst-coated yeast involved in this invention are characterized by the following steps:

[0098] Step 1: Temperature Tolerance

[0099] Using YPD medium as the basal medium, activated bacterial culture was inoculated into YPD medium at an inoculum volume of 0.2%. The culture was then incubated for 48 hours at temperatures of 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, with a rotation speed of 180 r / min. The OD560 value was then measured using a 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°C, and its optimal growth temperature is 25°C.

[0100] Step 2: pH tolerance

[0101] 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 4.

[0102] Step 3: NaCl tolerance

[0103] 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 48 h of incubation in a shaker at 28℃ and 180 r / min, their OD560 was determined using a turbidimetric method. The results are as follows: Figure 3As shown in c, this bacterium can grow at NaCl concentrations below 15%, indicating its strong salt tolerance.

[0104] Step 4: Ethanol Tolerance

[0105] Activated yeast strains were inoculated at a rate of 0.2% into sterilized YPD medium with ethanol concentrations (v / v) of 0%, 3%, 6%, 9%, 12%, 15%, 18%, and 21%, respectively. The cultures were incubated for 48 h in a shaker at 28 °C and 180 r / min, and then the 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 9%, and is a moderately ethanol-tolerant strain.

[0106] Step 5: Nicotine Tolerance

[0107] Activated yeast strains were inoculated at a rate of 5% into nicotine-containing 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 using a turbidimetric method. The results are as follows: Figure 3 As shown in e, the nicotine tolerance concentration of this strain reaches 1.2 g / L.

[0108] Example 5: Fermentation of 3-methylthiopropanol using capsule-forming yeast Y1402

[0109] The present invention relates to the capsule-forming yeast ( Saccharomycopsis fibuligera The steps for preparing 3-methylthiopropanol are as follows:

[0110] (1) Activation of strain

[0111] Capsule-coated yeast ( Saccharomycopsis fibuligera Y1402 was inoculated into YPD culture and activated for 24 h at a temperature of 28 ℃ and a rotation speed of 180 r / min.

[0112] (2) Basic conditions for the production of 3-methylthiopropanol by the strain

[0113] The seed culture obtained in (1) was inoculated into sterilized 3-methylthiopropanol conversion medium at an inoculation rate of 0.2%, and cultured in a shaker at 30°C and 200 r / min for 48 h to obtain the seed culture.

[0114] The liquid seed culture medium in step (1) consists of: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, natural pH, and distilled water to a final volume.

[0115] The liquid fermentation medium in step (2) consists of: 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, and 0.01 g / L magnesium chloride. The pH is adjusted to 5, and the medium is sterilized at 121 °C for 20 min. 0.02 g of ferrous chloride is then sterilized by passing it through a 0.22 μm filter membrane.

[0116] (3) Determination of product and product concentration

[0117] The determination of *Saccharomyces cerevisiae* (a type of yeast) using ultraviolet-high performance liquid chromatography (Agilent 1260 Infinity) was performed. Saccharomycopsis fibuligera The yield of 3-methylthiopropanol synthesized from Y1402 was determined. The high-performance liquid chromatography (HPLC) parameters were as follows: C-18 reversed-phase column (ZORBAX Eclipse Plus C-18, 4.6 × 250 mm, 5 μm); mobile phase: methanol:water = 30:70; 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.

[0118] (4) Construction of the standard curve of 3-methylthiopropanol

[0119] Accurately pipette 0.2 mL of 3-methylthiopropanol standard and dilute to 10 mL with deionized water to prepare a stock solution with a concentration of 20 g / L. Pipe 0.0, 0.5, 1.0, 1.5, 2, 2.5 and 3.0 mL of the stock solution and dilute to 10 mL with deionized water to obtain 3-methylthiopropanol standard solutions with concentrations of 0.0, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 g / L.

[0120] The above-mentioned 3-methylthiopropanol standard solutions with different gradients were measured under the above conditions. A standard curve was plotted with concentration as the x-axis and peak area as the y-axis. The peak elution time of the 3-methylthiopropanol standard was between 9 and 10 min. Each concentration was measured three times, and the average values ​​were calculated to plot the 3-methylthiopropanol standard curve. Figure 4 As shown, the equation obtained from this curve is y = 3123.5x + 245.62 (R² = 0.9929).

[0121] (5) Sample preparation

[0122] 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;

[0123] The high-performance liquid chromatography (Agilent 1260 Infinity) method parameters were as follows: C-18 reversed-phase column (ZORBAX Eclipse Plus C-18, 4.6 × 250 mm, 5 μm); mobile phase: methanol:water = 30:70; 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.

[0124] The peak area of ​​the sample was determined by high performance liquid chromatography and substituted into the standard curve equation y=3123.5x+245.62 (R2=0.9929). The peak area was the value of y, and the concentration of 3-methylthiopropanol x was calculated.

[0125] Step 1: Optimization of glucose concentration

[0126] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the glucose concentration in the liquid fermentation medium is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively, with an inoculum of 0.2% (v / v), and cultured at 30℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 5 a. It can be seen that when the glucose concentration is 40 g / L, the *Saccharomyces cerevisiae* (Saccharomyces cerevisiae)... Saccharomycopsis fibuligera Y1402 synthesized the highest amount of 3-methylthiopropanol from glucose, at 3.07 g / L; therefore, the optimal glucose concentration was selected as 40 g / L. The yield of 3-methylthiopropanol did not differ significantly between glucose concentrations of 40 g / L and 50 g / L.

[0127] Step 2: Optimization of yeast extract concentration

[0128] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the yeast extract concentrations in the liquid fermentation medium are 0 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L, and 2.0 g / L, respectively, with an inoculum of 0.2%, and cultured at 30 ℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 5 b shows that when the yeast extract concentration is 0.4 g / L, the endocarp-coated yeast ( Saccharomycopsis fibuligeraY1402 synthesized the highest content of 3-methylthiopropanol, at 3.28 g / L, therefore the optimal yeast extract concentration was selected as 0.4 g / L. There was no significant difference in the yield of 3-methylthiopropanol between yeast extract concentrations of 0.8 g / L and 1.2 g / L.

[0129] Step 3: Optimization of L-methionine concentration

[0130] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the methionine concentrations in the liquid fermentation medium were 0 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, and 10 g / L, respectively, with an inoculum of 0.2%, and cultured at 30 ℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 5 c shows that when the yeast extract concentration is 4 g / L, the capsule-coated yeast ( Saccharomycopsis fibuligera Y1402 synthesized the highest content of 3-methylthiopropanol, which was 2.73 g / L. Therefore, the optimal L-methionine concentration of 4 g / L was selected.

[0131] Step 4: Optimization of initial pH

[0132] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the initial pH of the liquid fermentation medium was 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, with an inoculum of 0.2%, and cultured at 30℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 5 As can be seen from d, when the initial pH is 5.0, the capsid-forming yeast ( Saccharomycopsis brooch Y1402 synthesized the highest content of 3-methylthiopropanol, which was 3.75 g / L. Therefore, the initial pH of the culture medium was selected to be 5.0.

[0133] Step 5: Optimization of incubation temperature

[0134] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the liquid fermentation medium was cultured at temperatures of 20℃, 24℃, 28℃, 32℃, 36℃, and 40℃, with an inoculum of 0.2%, at the corresponding temperature and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 6 a. It can be seen that when the temperature is 32℃, the cyst-forming yeast (…) Saccharomycopsis brooch Y1402 synthesized the highest content of 3-methylthiopropanol, which was 3.31 g / L, so the culture temperature was selected as 32℃.

[0135] Step 6: Optimization of culture rotation speed

[0136] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the liquid fermentation medium was cultured at rotation speeds of 0 r / min, 45 r / min, 90 r / min, 135 r / min, 180 r / min, 225 r / min, and 270 r / min, with an inoculum of 0.2%, at 30℃ for 48 h at the corresponding rotation speeds. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 6 b shows that when the rotation speed is 225 r / min, the capsule-forming yeast ( Saccharomycopsis fibuligera Y1402 synthesized the highest content of 3-methylthiopropanol, which was 3.99 g / L, so the culture speed was selected as 225 r / min.

[0137] Step 7: Optimization of liquid volume

[0138] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the liquid fermentation medium volumes were 25 mL, 50 mL, 75 mL, 100 mL, and 125 mL, respectively, with an inoculum of 0.2%, and cultured at 30℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 6 c shows that when the liquid volume is 25 mL, the capsule-covered yeast ( Saccharomycopsis brooch Y1402 synthesized the highest content of 3-methylthiopropanol, which was 3.52 g / L, so the optimal liquid volume was 25 mL.

[0139] Step 8: Optimization of Inoculation Dosage

[0140] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the inoculum amounts in the liquid fermentation medium were 0.1%, 0.2%, 0.4%, 0.8%, 1.6%, 3.2%, and 6.4%, respectively, and the cultures were incubated at 30℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 6 d shows that when the inoculum size is 0.8%, the *Saccharomyces cerevisiae* (Saccharomyces cerevisiae)... Saccharomycopsis fibuligeraY1402 synthesized the highest content of 3-methylthiopropanol, at 3.18 g / L, so the inoculum size of strain Y1402 was selected as 0.8%.

[0141] Step 9: Selection of Surfactant Types

[0142] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that 0.2% of different surfactants (blank, glycerol, Tween-20, Tween-40, Tween-60, Tween-80, and Triton-100) were added to the liquid fermentation medium, with an inoculum of 0.2%, and cultured at 30℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 7 a. It can be seen that when the surfactant is Tween-80, the capsule-forming yeast ( Saccharomycopsis fibuligera Y1402 synthesized the highest content of 3-methylthiopropanol, at 2.53 g / L. Therefore, *Saccharomyces cerevisiae* (Y1402) is the most likely to synthesize this product. Saccharomycopsis fibuligera The preferred surfactant for the synthesis of 3-methylthiopropanol using Y1402 is Tween-80.

[0143] Step 10: Optimization of Tween-80 concentration

[0144] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the liquid fermentation medium Tween-80 concentrations were 0 g / L, 2 g / L, 4 g / L, 8 g / L, 16 g / L, 32 g / L, and 64 g / L, and cultured at 30 ℃ and 200 r / min for 48 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 7 b shows that when the Tween-80 concentration is 2 g / L, the cyst-coated yeast ( Saccharomycopsis fibuligera Y1402 synthesized the highest content of 3-methylthiopropanol, at 2.53 g / L. Different concentrations of Tween-80 promoted the yield of 3-methylthiopropanol without significant differences. Therefore, *Saccharomyces cerevisiae* (Y1402) is the most effective synthesizer. Saccharomycopsis fibuligera The optimal Tween-80 concentration for the synthesis of 3-methylthiopropanol using Y1402 is 2 g / L.

[0145] Step 11: Optimization of the timing of L-methionine addition

[0146] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligeraY1402 differs in that L-methionine was added to the liquid fermentation medium at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and cultured at 30℃ and 200 r / min for 96 h. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 8 As can be seen from a, when L-methionine is added at 0 h, i.e. at the beginning of culture, the cyst-forming yeast ( Saccharomycopsis fibuligera Y1402 synthesized the highest content of 3-methylthiopropanol, at 3.73 g / L. Therefore, L-methionine was added to the culture medium at the beginning of the culture.

[0147] Step 12: Optimization of incubation time

[0148] Cultivate cyst-covered yeast according to the basic conditions in (2). Saccharomycopsis fibuligera Y1402 differs in that the liquid fermentation medium was cultured for 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h at 30℃ and 200 r / min. The concentration of 3-methylthiopropanol was determined by high-performance liquid chromatography (HPLC). Figure 8 b shows that when the culture time is 60 h, the cyst-covered yeast ( Saccharomycopsis brooch Y1402 synthesized the highest content of 3-methylthiopropanol, which was 2.66 g / L, so the culture time was selected as 60 h.

[0149] Example 6: Response Surface Design Optimization of Fermentation Conditions

[0150] Step 1: Plackett–Burman Trial

[0151] The results of the single-factor experiments showed that nine factors, including glucose concentration, rotation speed, and temperature, were selected. A Plackett-Burman experimental design was conducted using Mintab 17, with three replicates for each factor. The response value was the concentration of 3-methylthiopropanol. The factors and levels of the Plackett-Burman experimental design are shown in Table 5, and the experimental results are shown in Table 6. The yield of 3-methylthiopropanol ranged from 0.5 to 3.81 g / L. The regression equation was: Y = -6.13 + 0.00291 A + 0.712 B + 0.1210 C + 0.0548 D + 0.631 E + 0.02493 F + 0.632 G - 0.00459 H + 0.00494 I

[0152] Table 5. Factor Levels in the Plackett-Burman Experimental Design

[0153]

[0154] Table 6 Plackett-Burman Experimental Design and Results

[0155]

[0156] Table 7. Analysis of variance of the regression equation for 3-methylthiopropanol production

[0157]

[0158] Table 8. Results of Plackett-Burman significance analysis

[0159]

[0160] Based on the ANOVA results in Table 7 and the significance test results in Table 8, it can be seen that each factor has a certain degree of influence on the yield of 3-methylthiopropanol. The liquid volume has a negative effect on the experimental results, while the other eight factors all have positive effects. Table 7 shows that the model corresponds to... P The value is 0.006. P <0.01) indicates that the model is significant. As shown in Table 8, seven factors, namely pH, culture time, yeast extract concentration, inoculum size, L-methionine concentration, rotation speed, and culture temperature, have a significant impact on the yield of 3-methylthiopropanol. Therefore, these seven factors were selected as the subjects of the steepest climbing experiment, while the optimal level among the single-factor results was selected for the climbing experiment of the two factors, namely liquid volume and glucose concentration.

[0161] Step 2: Optimization of the steepest climb test

[0162] Based on the Plackett-Burman test results, and combined with the regression coefficients in Table 8, the step size and direction of the climbing test were determined. The results of the steepest climbing test are shown in Table 9:

[0163] Table 9. Experimental Design and Results for the Steepest Climb

[0164]

[0165] As shown in Table 9, the highest yield of 3-methylthiopropanol was observed in the third group of experiments. Specifically, when the pH was 5, the culture time was 52 h, the yeast extract concentration was 0.6 g / L, the inoculum size was 0.8%, and the L-methionine concentration was 5 g / L, the yield of 3-methylthiopropanol was 3.48 g / L. Therefore, the three fermentation conditions in the third group of experiments—pH, culture time, and yeast extract concentration—were used as the central values ​​for response surface methodology in subsequent experiments.

[0166] Example 8: Optimization of Response Surface Experiments

[0167] Based on the Plackett-Burman experiment results and the steepest ascent experiment, the center point of the values ​​for the three factors—pH, culture time, and yeast extract concentration—was determined, and this center point was used as the zero level. Following the principles of the Box-Benhnken central composite design, the response surface methodology was designed using Design-Expert.V8.0.6.1 software. A total of 15 experiments with 3 factors and 3 levels were determined for the Box-Benhnken central composite design. The coded levels obtained with pH, ​​culture time, and yeast extract concentration as independent variables are shown in Table 10. Table 11 shows the Box-Benhnken experimental design and results.

[0168] Table 10 Response Surface Experiment Design Factor Levels

[0169]

[0170] Table 11 Box-Behnken Design and Results

[0171]

[0172] The results in Table 11 show that the highest yield of 3-methylthiopropanol was achieved in the fifth group of experiments, when the initial pH of the culture medium was 5, the yeast extract concentration was 0.9 g / L, and the culture time was 60 h, with a yield of 3.97 g / L.

[0173] Using Design Expert 8.0.6 software, multiple regression analysis and multiple quadratic equation regression fitting were performed on the comprehensive value Y and each factor, resulting in the regression equation: Y = 2.46 + 0.41A + 0.30B + 0.089C + 0.17AB - 0.032AC - 0.073BC + 0.10A² + 0.34B² + 0.69C².

[0174] Table 12 Analysis of Variance of Regression Model

[0175]

[0176] Note: p < 0.01 indicates a highly significant difference; p<0.05, the difference is significant.

[0177] The model can be seen from Table 12. P <0.05 indicates that the model is significant, and the quadratic regression equation is valid. R2=0.9266, 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 variable, and the influence of the independent variable on the dependent variable can be reflected by the significance in the analysis of variance. As shown in the correlation coefficients in Table 12, the linear terms A (pH) and B (time) are significant (…). P <0.05, none of the interaction terms were significant, and only term C2 among the quadratic terms was significant ( P <0.05).

[0178] The effects of pairwise interactions of various factors on the concentration of 3-methylthiopropanol can be presented by a 3D response surface plot. Figure 9 The regression equation shows that the optimal fermentation conditions are pH=5, fermentation time 60 h, and yeast extract concentration 0.9 g / L. To verify the accuracy of the model prediction, the experiment was repeated three times under the above optimal fermentation conditions. The yield of 3-methylthiopropanol was 4.07 g / L, which is close to the predicted value of 3.81 g / L, with a deviation of 6.5%, proving the reliability of the model.

Claims

1. A strain of cladocyst-coated yeast ( Saccharomycopsis fibuligera Y1402, with accession number CGMCC No.23667.

2. The application of the capsule-forming yeast Y1402 as described in claim 1 in the production of 3-methylthiopropanol, characterized in that, Y1402, a type of yeast with a capillary membrane, can be used in brewing or food additive production.

3. The application of the capsule-forming yeast Y1402 as described in claim 1 in the production of 3-methylthiopropanol, characterized in that, Y1402, a type of yeast with a vesicle covering, was used in the production of condiments.

4. A method for producing 3-methylthiopropanol using the capsule-forming yeast Y1402 as described in claim 1, characterized in that, Includes the following steps: (1) Inoculate the Y1402 capsule-coated yeast into the liquid seed activation medium to activate it and obtain the seed activation solution; (2) The seed activation solution was inoculated into a liquid fermentation medium for fermentation to obtain a culture medium containing 3-methylthiopropanol.

5. The method according to claim 4, characterized in that, In step (1), the liquid seed activation culture medium comprises: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, and natural pH. The activation conditions were: incubation at 28℃ and 180 r / min for 24 h. In step (2), the inoculum size is 0.1-6.4%; the fermentation conditions are: 90-270 r / min, cultured at 24-36℃ for 12-96 h; The liquid fermentation medium comprises: glucose 15-60 g / L, yeast extract 0.4-2 g / L, L-methionine 2-10 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, and pH 3.5-6.

0.

6. The method according to claim 4, characterized in that, In step (2), the inoculum amount is 0.8%, and the fermentation conditions are: 90~270 r / min, cultured at 24~36℃ for 36~96 h; The liquid fermentation medium comprises: glucose 40-50 g / L, yeast extract 0.8-1.2 g / L, L-methionine 2-10 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, and pH 4.0-6.

0.

7. The method according to claim 4, characterized in that, In step (2), the liquid fermentation medium contains: 40-50 g / L glucose, 0.9 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, and pH 5.

0.

8. The method according to any one of claims 4-7, characterized in that, In step (2), the liquid fermentation medium further includes a surfactant selected from at least one of glycerol, Tween-20, Tween 40, Tween 60, Tween 80 and X-100.

9. The method according to claim 8, characterized in that, In step (2), the surfactant is Tween 80; the concentration of Tween 80 in the fermentation medium is 2~64 g / L.

10. A composition containing the capsule-forming yeast Y1402 as described in claim 1.

11. The composition according to claim 9, characterized in that, The composition also contains a substrate, protectant, filler or adsorbent of Yeast Y1402.

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