High-sugar-resistant saccharomycetes and application thereof

Abstract

The invention relates to the field of microbial fermentation, in particular to high-sugar-resistant saccharomycetes and application thereof. The invention discloses a high-glucose-resistant Kodamaea ohmeri SJY6, the preservation number is GDMCC No: 65816, the high-glucose-resistant Kodamaea ohmeri SJY6 is early in starting period and strong in stress resistance, has better acid resistance and ethanol resistance compared with commercial yeast, particularly has excellent high-glucose resistance, can tolerate 180g / L of NaCl, 800g / L of glucose, 800g / L of fructose, 600g / L of glycerol and 1300g / L of sucrose at most, can still grow and survive in a large scale in an environment of 1800g / L of maltose, and can be used for preparing the high-glucose-resistant Kodamaea ohmeri SJY6. And the method has wide utilization value for industrial production and environmental protection.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and particularly to a high-sugar tolerant yeast strain and its application. Background Art

[0002] Yeast is a common eukaryotic single-celled microorganism in industrial production. In recent years, extreme yeasts have attracted much attention due to their strong tolerance, which is of great help to industrial production applications and scientific research. High-sugar tolerant yeast is one of them. High-sugar tolerant yeast refers to yeast that can still grow normally at a relatively high sugar concentration, generally defined as yeast that can grow at a glucose concentration of 55% - 65% (w / v). Common high-sugar tolerant yeasts include Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Yarrowia lipolytica, Debaryomyces Hansenii, etc. Currently, high-sugar tolerant yeasts have the following applications: (1) Biological fermentation catalysis: Producing erythritol, xylitol, D-arabitol and other polyols used in the production of high-value sugar substitutes such as glycerol, trehalose, etc., and fermenting ethanol in a high-sugar environment of sugar factory wastewater and production waste (lignocellulose, molasses, etc.), which is the main source of ethanol green energy; (2) Food industry: Used for thick mash fermentation, yellow rice wine and ice wine fermentation, and the production of hypertonic fermented foods (soy sauce, balsamic vinegar, sausage, etc.); (3) Environmental protection: Producing green energy, degrading harmful substances such as azo dyes and polycyclic aromatic hydrocarbons in sewage, and reducing the use of fresh water in the fermentation process. It plays a very important role in society and industrial production.

[0003] Currently, the yeast used in the research on the mechanism of high-sugar tolerant yeast has a tolerance range of 30% - 60%. There are few reports on high-sugar tolerant yeast with a sugar content above 60% and its tolerance research. Most of the highest sugar tolerance of the screened yeasts is 700 g / L glucose and below, and the research on yeast high-sugar tolerance is only limited to the tolerance ability of glucose. Therefore, screening yeasts with high tolerance to various sugars has broad application value for industrial production. Summary of the Invention

[0004] In view of this, the present invention provides a high-sugar tolerant yeast strain and its application. The yeast strain has an early start-up period and strong stress resistance. It has better acid and ethanol tolerance than commercial yeast, especially has excellent high-sugar tolerance. It can tolerate up to 180 g / L NaCl, 800 g / L glucose, 800 g / L fructose, 600 g / L glycerol, 1300 g / L sucrose, and can still grow and survive in large quantities in an environment of 1800 g / L maltose, which has broad application value for industrial production and environmental protection.

[0005] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0006] The present invention provides Kodamaea ohmeri with a preservation number of GDMCC No: 65816.

[0007] The present invention also provides the application of the above-mentioned Kodamaea ohmeri in the production of ethanol.

[0008] In some specific embodiments of the present invention, the production of ethanol in the above application can be the fermentation to produce ethanol in a high-sugar environment of sugar mill wastewater and production waste;

[0009] The production waste can be lignocellulose and molasses.

[0010] The present invention also provides the application of the above-mentioned Kodamaea ohmeri in the production of polyols.

[0011] In some specific embodiments of the present invention, the polyols in the above application can be at least one of erythritol, xylitol, D-arabitol, and glycerol.

[0012] In some specific embodiments of the present invention, the polyol in the above application is D(+)-arabitol.

[0013] In some specific embodiments of the present invention, the polyol in the above application is xylitol.

[0014] In some specific embodiments of the present invention, the polyol in the above application is glycerol.

[0015] The present invention also provides the application of the above-mentioned Kodamaea ohmeri in thick mash fermentation.

[0016] In some specific embodiments of the present invention, the thick mash fermentation in the above application can be alcohol thick mash fermentation, beer thick mash fermentation, soy sauce thick mash fermentation, and sake thick mash fermentation.

[0017] The present invention also provides the application of the above-mentioned Kodamaea ohmeri in the production of hypertonic fermented foods.

[0018] In some specific embodiments of the present invention, the hypertonic fermented foods in the above application can be pickles, balsamic vinegar, salted meat, salted fish, candied fruit, preserved fruit, fermented soybeans, fermented bean curd, pickled vegetables, high-salt soy sauce, and pickled olives.

[0019] The present invention also provides the application of the above-mentioned Kodamaea ohmeri in the production of high-added-value sugar substitute foods.

[0020] In some specific embodiments of the present invention, the high-added-value sugar substitute foods in the above application may be allulose beverages, tagatose dairy products, steviol glycoside chewing gums, brazzein chocolate bars, mogroside baked foods, rebaudioside M candies, sugar alcohol ice creams.

[0021] The present invention also provides the application of the above-mentioned Kodamaea ohmeri in the degradation of production waste, sewage degradation or reduction of fresh water usage in the fermentation process.

[0022] In some specific embodiments of the present invention, the sewage degradation in the above application may be the degradation of sugar mill wastewater.

[0023] In some specific embodiments of the present invention, the degradation of production waste and sewage degradation in the above application may be the degradation of azo dyes or polycyclic aromatic hydrocarbons.

[0024] The present invention also provides the fermentation product of the above-mentioned Kodamaea ohmeri.

[0025] The present invention has the following effects:

[0026] 1. The present invention screens out a yeast strain that can grow in a medium containing a high concentration of sugar. It not only has a glucose tolerance concentration not lower than that of other strains (Table 1), but also studies the tolerance of high-sugar-tolerant yeast under different carbon sources based on the fact that most studies are limited to the tolerance of glucose carbon source, and finds that it also has extremely high tolerance under different types of carbon sources.

[0027] 2. The present invention screens out a yeast strain for the first time that can still grow normally under the condition of 1800 g / L maltose and still has a relatively high biomass.

[0028] 3. The present invention screens out a yeast strain with a sucrose tolerance of up to 1300 g / L, a fructose tolerance of up to 800 g / L, and a glycerol tolerance of up to 600 g / L, proving that the growth of yeast is different under different types of sugar conditions.

[0029] 4. The present invention screens out a high-osmotic-tolerant yeast strain that can tolerate a NaCl concentration of 180 g / L. It can replace fresh water with seawater as the fermentation solvent during the fermentation process, saving fresh water resources.

[0030] Biological deposit description

[0031] Biological material: Kodamaea ohmeri SJY6, classified as Kodamaea ohmeri, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on January 16, 2025. The address of the deposit center is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; Institute of Microbiology, Guangdong Academy of Sciences; The deposit number is GDMCC No: 65816. Brief Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0033] Figure 1 Showing the colony morphology and microscopic structure of SJY6;

[0034] Figure 2 Showing the phylogenetic tree of SJY6 yeast;

[0035] Figure 3 Showing the fitting of the growth curve of SJY6 yeast;

[0036] Figure 4 Showing the fitting analysis of the fermentation kinetics of SJY6;

[0037] Figure 5 Showing the change of residual sugar in the fermentation of SJY6;

[0038] Figure 6 Showing the results of the sugar preference experiment of SJY6 yeast;

[0039] Figure 7 Showing the influence of different pH value gradients on the fermentation of SJY6 and commercial yeast;

[0040] Figure 8 Showing the influence of different ethanol concentrations on the fermentation of SJY6 and commercial yeast;

[0041] Figure 9 Showing the influence of different sulfur dioxide concentrations on the fermentation of SJY6 and commercial yeast;

[0042] Figure 10 Showing the influence of different NaCl concentrations on the fermentation of SJY6 yeast;

[0043] Figure 11 Showing the influence of different concentrations of glycerol on the fermentation of SJY6 yeast;

[0044] Figure 12 Showing the influence of different concentrations of glucose on the fermentation of SJY6 yeast;

[0045] Figure 13 Showing the influence of different concentrations of fructose on the fermentation of SJY6 yeast;

[0046] Figure 14 Showing the effects of different concentrations of sucrose on the fermentation of SJY6 yeast;

[0047] Figure 15 Showing the effects of different concentrations of maltose on the fermentation of SJY6 yeast;

[0048] Figure 16 Showing the number of SJY6 yeast cells at high concentration of maltose;

[0049] Figure 17 Showing the LC-MS metabolite classification annotation;

[0050] Figure 18 Showing the LC-MS untargeted metabolite results;

[0051] Figure 19 Showing the LC-MS untargeted metabolite results. Detailed implementation manners

[0052] The present invention discloses a high-sugar tolerant yeast strain and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0053] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0054] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0055] It should be understood that as long as the present application is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0056] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present application.

[0057] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0058] Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in the present invention are all ordinary commercially available products and can be purchased from the market.

[0059] The present invention will be further described below in conjunction with embodiments.

[0060] Embodiment

[0061] (I) Experimental method

[0062] 1. Separation and purification

[0063] 1.1 Yeast enrichment

[0064] Take 10 - 20 g of fruit peels and their leaves, add them to 50 mL of wort medium, and culture in a shaker at 30 °C and 180 r / min for 24 h. Dilute the culture solution to 10 -3 ~10 -7 times and coat it on the Rose Bengal solid medium, and culture in a constant temperature incubator for 1 - 2 d.

[0065] 1.2 Purification and preservation

[0066] Pick single colonies with different morphologies and streak them on WL agar medium plates, culture them inverted at 30 °C for 48 h, repeat the plate streaking more than 3 times, isolate pure single strains and store them in glycerol tubes at -80 °C.

[0067] 2. Yeast morphology observation

[0068] Inoculate and streak the measured yeast on WL medium and Rose Bengal medium, culture them inverted at 30 °C for 48 h, and record the colony morphology. Observe the microscopic morphology using an optical microscope. Dip a little bacterial sludge with an inoculation loop and smear it on a glass slide, stain it with 1% crystal violet and observe it under a 100-fold optical microscope (oil immersion lens).

[0069] 3. Sequence identification

[0070] Inoculate and streak the yeast to be tested on WL medium or Sabouraud dextrose agar with chloramphenicol. Scrape a certain amount of sample from the plate and place it in a 2 mL centrifuge tube. Use a kit to extract deoxyribonucleic acid (DNA) from the yeast genome. Perform genomic PCR amplification, and use the designed primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′, SEQ ID NO: 1) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′, SEQ ID NO: 2) to amplify the 28S rDNA gene sequence. The PCR amplification reaction system is as follows: 1.0 μL of genomic DNA (20 ng / μL), 5.0 μL of 10× Buffer (containing 2.5 mM Mg 2+ ), 1.0 μL of Taq polymerase (5 U / μL), 1.0 μL of dNTP (10 mM), 1.5 μL each of ITS1 primer (10 μM) and ITS4 primer (10 μM), and 39.0 μL of ddH 2 O. The PCR amplification conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, final extension at 72°C for 7 min, for 35 cycles. After the reaction is completed, take 3 μL of the PCR product for 1% agarose gel electrophoresis detection to confirm that the PCR amplification fragment is qualified. Take the purified PCR products of each strain and use the ABI3730-XL sequencer for DNA sequencing. Submit the sequence results to NCBI blast (http: / / www.ncbi.nlm.nih.gov / BLAST / ) for sequence comparison to obtain the species information of the species with the highest sequence similarity to the yeast to be tested. Use the ApE program to reverse-complement the sequences, select several groups of strains with high homology, and use the MEGA11 software to construct a phylogenetic tree.

[0071] 4. Growth curve

[0072] Inoculate the activated yeast in YPD liquid medium and culture it in a shaker at 30°C and 180 r / min for 24 h. Take samples every 3 h and dilute them 20 times. Measure the biomass of the strain at 600 nm using a spectrophotometer, and make a scatter plot with the culture time as the abscissa and the OD value as the ordinate, and use Origin software for analysis - non-linear fitting curve to select the best growth fitting curve.

[0073] 5. pH tolerance experiment

[0074] The selected yeast and Angel commercial yeast were activated and inoculated into YPD liquid medium under different pH conditions. The pH conditions were: 2, 2.5, 3, 3.5, 4. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0075] 6. Ethanol tolerance experiment

[0076] The selected yeast and Angel commercial yeast were activated and inoculated into YPD liquid medium under different ethanol concentrations. The ethanol concentrations were: 6%, 9%, 12%, 15%, 18% (v / v). They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0077] 7. Sulfur dioxide tolerance experiment

[0078] The selected yeast and Angel commercial yeast were activated and inoculated into YPD liquid medium under different sulfur dioxide concentrations. The sulfur dioxide concentrations were: 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0079] 8. Glycerol tolerance experiment

[0080] The selected yeast was activated and inoculated into YPD liquid medium under different glycerol concentrations. The glycerol concentrations were: 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 650 g / L. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0081] 9. NaCl tolerance experiment

[0082] The selected yeast was activated and inoculated into YPD liquid medium under different NaCl concentrations. The NaCl concentrations were: 90 g / L, 120 g / L, 150 g / L, 180 g / L, 210 g / L. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0083] 10. High sugar tolerance experiment

[0084] The selected yeast was inoculated into YPD liquid media with different types of sugars at different concentrations to study the tolerance of the yeast to different types of sugars.

[0085] 10.1 Glucose tolerance

[0086] The selected yeast and Angel commercial yeast were activated and inoculated into YPD liquid media with different concentrations of glucose. The glucose concentrations were: 0 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 850 g / L, 900 g / L. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution 20-fold, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0087] 10.2 Fructose tolerance

[0088] The selected yeast was activated and inoculated into YPD liquid media with different concentrations of fructose. The fructose concentrations were: 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 850 g / L. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution 20-fold, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0089] 10.3 Sucrose tolerance

[0090] The selected yeast was activated and inoculated into YPD liquid media with different concentrations of sucrose. The sucrose concentrations were: 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 900 g / L, 1000 g / L, 1100 g / L, 1200 g / L, 1300 g / L, 1400 g / L. They were cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution 20-fold, the biomass of the strains was measured at 600 nm using a spectrophotometer.

[0091] 10.4 Maltose tolerance

[0092] The selected yeast was activated and inoculated into YPD liquid medium under different concentrations of maltose. The maltose concentrations were: 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 900 g / L, 1000 g / L, 1100 g / L, 1200 g / L, 1300 g / L, 1500 g / L, 1800 g / L. It was cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strain was measured at 600 nm using a spectrophotometer.

[0093] SJY6 yeast was inoculated into high-concentration maltose YPD liquid medium with 0 g / L, 900 g / L, 1000 g / L, 1100 g / L, 1200 g / L, 1300 g / L, 1500 g / L, 1800 g / L. It was cultured in a shaker at 30 °C and 180 r / min for 14 h. The fermentation broth was diluted to 10 -4 ~10 -7 times and spread on Sabouraud dextrose agar medium. It was cultured upside down at 30 °C for 24 h, and the number of colonies was counted.

[0094] 11. Microbial sugar preference experiment

[0095] The selected yeast was inoculated into liquid medium containing 20 g / L peptone, 10 g / L yeast extract powder, and 20 g / L of different types of carbon sources (glycerol, glucose, fructose, maltose, xylose, lactose, sucrose). It was cultured in a shaker at 30 °C and 180 r / min for 14 h. After dilution by 20 times, the biomass of the strain was measured at 600 nm using a spectrophotometer to compare the growth of the strain under different carbon sources.

[0096] 12. Fermentation kinetics and total sugar consumption capacity determination

[0097] Fermentation kinetics determination: Yeast was inoculated into a Durham fermentation tube containing blood orange juice (total sugar content 300 g / L, liquid filling volume 50%). It was anaerobically fermented at 30 °C to test the fermentation ability of the yeast in a high-sugar real system. The weight of the fermentation broth was measured every 1 d until the weight loss was less than 5 g / L, which was regarded as the end of fermentation. A fermentation kinetics scatter plot was made with the culture time as the abscissa and the fermentation weight loss as the ordinate, and the optimal fitting straight line was drawn using Origin software.

[0098] Total sugar consumption capacity determination: Yeast was inoculated into a 400 g / L glucose solution and cultured in a shaker at 30 °C and 180 r / min for 8 d. Samples were taken every 2 d, and the residual sugar concentration of the sample solution was measured using the 3,5-dinitrosalicylic acid method (DNS method) to observe the total sugar consumption of the yeast under high-concentration sugar conditions.

[0099] 13. LC-MS Non-targeted Metabolomics Analysis

[0100] 13.1 Metabolite Extraction

[0101] Transfer 500 μL of the fermentation broth fermented in 400 g / L glucose YPD liquid medium for 8 d into a 1.5 mL centrifuge tube, and centrifuge at 12,000 rpm and 4℃ for 15 min; Transfer 400 μL of the supernatant to a 2 mL centrifuge tube, quickly freeze it in liquid nitrogen and then lyophilize it using a freeze dryer; Add 1 mL of pre-cooled methanol:acetonitrile (1:1, v / v), vortex for 30 s; Place it in a -20℃ refrigerator and freeze for 30 min; Centrifuge at 12,000 rpm and 4℃ for 10 min, take 850 μL of the supernatant and concentrate it to dryness under vacuum; Add 150 μL of 50% methanol (containing 5 ppm 2-chlorophenylalanine) for reconstitution, and vortex for 30 s; Centrifuge at 12,000 rpm and 4℃ for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane, and add the filtrate to the detection bottle.

[0102] 13.2 Chromatographic Method

[0103] Use an ACQUITY UPLC HSS T3 chromatographic column (100 Å, 1.8 µm, 2.1 mm×100 mm), flow rate 0.4 mL / min, column temperature 40℃, autosampler temperature 8℃, injection volume 2 μL.

[0104] Mobile phase for positive and negative modes: Mobile phase A is 0.1% formic acid in water, and mobile phase B is acetonitrile (containing 0.1% formic acid)

[0105] 13.3 Mass Spectrometry Method

[0106] Use a Thermo Orbitrap Exploris 120 mass spectrometer to collect DDA mass spectrometry data in positive and negative ion modes respectively under the control of software Xcalibur (version: 4.7, Thermo). Use an HESI source, spray voltage 3.5 kV / -3.0 kV, sheath gas 40 arb, auxiliary gas 10 arb, capillary temperature 320℃, auxiliary gas temperature 300℃, first-stage resolution 60,000, scanning range 70 - 1000 m / z, AGC Target Standard, Max IT 100 ms, screen the top 4 ions with the highest response for secondary fragmentation, dynamic exclusion time is 4 s, secondary resolution 15,000, HCD collision energy 30%, AGC Target Standard, Max IT Auto.

[0107] 13.4 Metabolite Search

[0108] The off-line data was imported into the software MS-DIAL (version 4.9.221218), and operations such as peak extraction, alignment, filtering, and metabolite identification were performed based on this software. Metabolite comparison was carried out based on the PerSonalbio Next-Generation Metabolomics Database (PSNGM) of Personalbio, which includes a self-built library, the mzCloud library (https: / / www.mzcloud.org / ), LIPID MAPS (https: / / www.lipidmaps.org / ), HMDB (https: / / hmdb.ca / ), MoNA (https: / / mona.fiehnlab.ucdavis.edu / ), NIST_2020_MSMS, and an AI-predicted MSMS spectral library.

[0109] Table 1: Summary of Previous Studies on the Tolerance of High-Sugar Yeasts

[0110]

[0111] (2) Experimental Results

[0112] 1. Results of Bacterial Strain Morphology and Identification

[0113] 1.1 Morphological Results

[0114] The colony characteristics of SJY6 yeast are as Figure 1 shown. On the tiger red medium, the colony surface is relatively smooth, the edge is neat, milky white, round and convex, and it is easy to pick up; on the WL medium, the colony surface is smooth, the edge is neat, white, round and convex, and it is viscous and easy to pick up.

[0115] The microscopic structure of the yeast is as Figure 1 shown. The microscopic morphology of the selected strain is nearly round, medium in size, and reproduces by budding.

[0116] 1.2 Bacterial Strain Identification

[0117] The deoxyribonucleic acid of the SJY6 yeast genome was extracted, PCR amplification was performed using ITS1 and ITS4 primers, the genome was sequenced using a sequencer, and the results were uploaded to NCBI Blast for comparison. The results showed that the SJY6 yeast was Kodamaea ohmeri. A phylogenetic tree was constructed with homologous bacteria as shown in Figure 2 .

[0118] 1.3 Bacterial Strain Preservation

[0119] This strain is preserved in the Institute of Microbiology, Guangdong Academy of Sciences, with the preservation number GDMCC No: 65816. Preservation address: 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences, 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Preservation date: 2025.

[0120] 2. Yeast growth characteristics experiment

[0121] 2.1 Growth curve

[0122] After multi-model fitting, the BiDoseResp model has the best fitting effect. The BiDoseResp model of the SJY6 yeast growth curve is as Figure 3 shown. The curve of the OD 600 value changing with the growth time is approximately S-shaped, and the result of its fitting curve function is:

[0123] , R 2 = 0.99994.

[0124] It can be seen from Figure 3 that the SJY6 yeast has an early start-up period, enters the logarithmic growth period of rapid reproduction within 3 - 6 h, and reaches a steady state after about 10 h.

[0125] 2.2 Fermentation kinetics

[0126] Taking the weight loss of the fermentation broth as the vertical coordinate and the number of fermentation days as the horizontal coordinate, a scatter plot of fermentation kinetics is made, and the optimal fitting straight line is analyzed using Origin as Figure 4 , and the optimal fitting straight line is obtained as y = 1.28204 + 6.11527*x, R 2 = 0.99171. It can be seen from the figure that the SJY6 fermentation starts early and is stable.

[0127] 2.3 Total sugar consumption

[0128] It can be seen from Figure 5 that SJY6 still has high growth vitality in a high-concentration sugar solution. From 0 to 2 d, the glucose content drops from the initial 400 g / L to 130 g / L, and the degradation rate reaches 67.71%. The consumption rate of sugar is fast, and after 4 d, the residual sugar concentration does not change significantly.

[0129] 2.4 Sugar preference experiment

[0130] By inoculating the SJY6 yeast in a medium containing different carbon sources, the utilization preference of this yeast for different carbon sources can be studied. As Figure 6 shown, the SJY6 yeast grows best in sucrose, and also grows well under the conditions of glucose, maltose, and fructose. It can utilize glycerol and lactose, but its growth is restricted.

[0131] 3. Tolerance Experiments

[0132] 3.1 pH Tolerance

[0133] As yeast fermentation progresses, the growth and metabolism of yeast cause the pH value of the fermentation broth to rapidly decrease. The optimal pH value for yeast growth is between 4.0 and 6.0. At lower or higher pH values, it will have a negative impact on yeast growth. As can be seen from Figure 7 it, after 14 h of fermentation, under the conditions of pH values of 2, 2.5, 3, 3.5, and 4, the OD value of SJY6 yeast is significantly higher than that of commercial yeast. It has relatively high growth activity at pH values equal to 2.5 - 4. Among them, it grows best at pH = 4, and the yeast activity rapidly decreases and the growth is limited (OD 600 value < 0.5) at pH = 2.

[0134] 3.2 Ethanol Tolerance

[0135] Yeast ferments anaerobically to produce alcohol. As the fermentation time increases, alcohol accumulates continuously, which is toxic to yeast, inhibits or kills yeast. The tolerance of yeast to alcohol concentration determines the ethanol fermentation yield. As can be seen from Figure 8 it, the OD value of SJY6 is significantly higher than that of commercial yeast under the condition of ethanol concentration of 0 - 6% (v / v). Under the condition of ethanol concentration of 9% (v / v), there is no significant difference in the biomass of the two strains of yeast. The growth of the two strains of yeast is inhibited at ethanol concentrations above 12% (v / v) (OD 600 value < 0.5). This shows that SJY6 has an ethanol tolerance ability not lower than that of commercial yeast, providing a prerequisite for fermenting ethanol green energy under high - sugar conditions.

[0136] 3.3 Sulfur Dioxide Tolerance

[0137] High - sugar yeast can be applied to yellow rice wine fermentation and thick mash fermentation. During the production of fermented wine, SO 2 is added to inhibit microorganisms in fruit wine, prevent the oxidation of components such as phenolic substances in wine, and improve the stability of the wine body. However, the added concentration of SO 2 will affect the growth of yeast. As can be seen from Figure 9 it, SJY6 can grow normally under the condition of 200 - 600 mg / L SO 2 , and its biomass is higher than that of commercial yeast, proving that SO 2 has little impact on the growth of SJY6 yeast.

[0138] 3.4 NaCl Tolerance

[0139] Yeasts that can tolerate a NaCl concentration of 15–25% (w / v) are generally defined as osmotolerant yeasts. In the production process, osmotolerant yeasts can use seawater instead of fresh water as a production solvent, which helps to save fresh water resources. From Figure 10 It can be seen that the yeast grows normally in a medium with a NaCl concentration lower than 120 g / L. When the NaCl concentration reaches 150 g / L, the growth of the yeast is restricted. When the concentration reaches 210 g / L, the growth of the yeast is severely restricted and the yeast stops growing (OD 600 value < 0.5).

[0140] 3.5 Glycerol tolerance

[0141] High-sugar-tolerant yeasts can be used for glycerol fermentation, and certain glycerol tolerance is required for yeast in glycerol production. From Figure 11 it can be known that as the glycerol concentration increases, the growth activity of the yeast is inhibited. The highest tolerance concentration is 600 g / L glycerol. When the concentration is higher than 650 g / L, the yeast stops growing (OD 600 value < 0.5).

[0142] 3.6 Glucose tolerance

[0143] Glucose is the most commonly used sugar for measuring sugar tolerance characteristics. As Figure 12 shown, SJY6 yeast grows best at a glucose concentration of 200 g / L and reaches its highest tolerance concentration at 800 g / L glucose.

[0144] 3.7 Fructose tolerance

[0145] Fructose is the sweetest natural monosaccharide and is commonly used as a sweetener and flavor enhancer. As Figure 13 shown, the growth activity of SJY6 decreases as the fructose concentration increases. The highest fructose tolerance of SJY6 is 800 g / L. When the fructose concentration is higher than 850 g / L, the yeast stops growing (OD 600 value < 0.5).

[0146] 3.8 Sucrose tolerance

[0147] Sucrose is one of the most commonly used sweeteners and is one of the main components of the sewage discharged from sugar factories. High tolerance to sucrose makes it possible for yeast to ferment and utilize the wastewater from sugar factories. From Figure 14 it can be known that at a sucrose concentration of 300 g / L, SJY6 yeast grows best and has the highest growth compared to other types of carbon sources. Its OD 600 value is as high as 24.77. As the sucrose concentration increases, the growth activity of SJY6 yeast slowly decreases. The highest tolerance is 1300 g / L (OD 600 = 0.555). When the sucrose concentration is higher than 1400 g / L, the yeast stops growing (OD 600 value < 0.5).

[0148] 3.9 Maltose Tolerance

[0149] Maltose is a common disaccharide composed of Figure 15 As can be seen, the OD of SJY6 yeast fluctuates at maltose concentrations from 300 to 1000 g / L, indicating that its growth is basically not restricted within this concentration range. At a maltose concentration of 1100 g / L, the growth activity of SJY6 yeast decreases as the maltose concentration increases, and there is still relatively high growth activity (OD 600 = 5.467) even at a maltose concentration of 1800 g / L. 600

[0150] To visually reflect the growth of SJY6 yeast in a high-concentration maltose environment, the fermentation broth cultured for 14 h under high-concentration maltose conditions was spread on plates for counting. The results are as Figure 16 shown. The number of yeast cells stabilizes above 5×10 -8 CFU at maltose concentrations from 900 to 1300 g / L, and there are still 2.02×10 -8 CFU viable cells at a maltose concentration of 1800 g / L, further indicating that SJY6 yeast can still survive and grow well at extremely high concentrations of maltose.

[0151] 4. Analysis of Metabolites

[0152] Using self-built and commercial databases, a total of 770 metabolites were identified from the sample solution of SJY6 fermented in high-concentration glucose for 8 d. According to the metabolites detected in the default mode, metabolite classification was obtained through annotation in the KEGG database ( Figure 17 a in), HMDB database ( Figure 17 b in), and LIPID MAPS database ( Figure 17 c in). The results of the positive and negative ion mode spectra are as Figure 18 , Figure 19 . The LC-MS non-targeted metabolite results show that SJY6 yeast can produce two high-added-value polyols, D(+)-arabitol and xylitol, as well as the common high-sugar-tolerant yeast metabolite glycerol ( Figure 19 , Table 2) under high-concentration glucose.

[0153] Table 2: Results of Liquid Chromatography-Mass Spectrometry Database Search

[0154]

[0155] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.​

Claims

1. Omokoda yeast ( Kodamaea ohmeri ), characterized in that, The deposit number is GDMCC No: 65816.

2. Omokoda yeast as claimed in claim 1 ( Kodamaea ohmeri ) in the production of ethanol.

3. Omokoda yeast as claimed in claim 1 ( Kodamaea ohmeri ) in the production of polyols.

4. The use according to claim 3, characterized in that The polyol is D(+)-arabinitol.

5. The use according to claim 3, characterized in that The polyol is xylitol.

6. The use according to claim 3, characterized in that The polyol is glycerol.

7. Omokoda yeast as claimed in claim 1 ( Kodamaea ohmeri ) in concentrated mash fermentation.

8. Omokoda yeast as claimed in claim 1 ( Kodamaea ohmeri ) in the production of high value-added sugar substitutes or hypertonic fermented foods.

9. Omokoda yeast as claimed in claim 1 ( Kodamaea ohmeri ) in the degradation of production waste, sewage or in reducing the amount of fresh water used in fermentation processes.

10. Omokoda yeast as claimed in claim 1 ( Kodamaea ohmeri ) fermentation products.

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