Wild pichia kluyveri with high methanol reduction capacity and application of wild pichia kluyveri in navel orange wine brewing

Abstract

The invention discloses a Pichia kluyveri strain and an application of the Pichia kluyveri strain in navel orange wine brewing. The pichia kluyveri is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation name is UT002, and the preservation number is CGMCC No.39116. In the navel orange wine brewing process, the UT002 shows excellent methanol reducing capacity, the methanol content is lower than that of commercially available commercial yeast, the safety is high, meanwhile, the fruit wine aroma can be improved, the navel orange variety aroma is highlighted, the complexity and typicality of the navel orange wine aroma are increased, and a more balanced and rich aroma profile is formed, so that the highest acceptability is obtained in the sense organ, and the navel orange wine has good market prospects. The navel orange wine has commercial application potential, and industrial development of navel orange wine in the whole production area is promoted.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and specifically to a strain of Kluyveromyces yeast and its application in reducing methanol content during the fermentation of navel orange wine. Background Technology

[0002] Navel orange wine is an important processed product of navel orange fruit, and its sensory flavor and safety are the core factors determining its quality. During fermentation, methanol, produced by the degradation of pectin in the fruit raw material by pectin methyl esterase (PME), is a key risk indicator for the safety of fruit wine products. Excessive methanol is clearly toxic to the human visual and nervous systems, causing various symptoms such as loss of consciousness, difficulty breathing, blurred vision, and even blindness, abdominal pain, diarrhea, and vomiting; in severe cases, it can be life-threatening. Current research on controlling methanol in fruit wine mainly focuses on two aspects: fermentation process optimization and brewing yeast improvement. In terms of process, selecting brewing fruit raw materials with lower pectin content, lowering fermentation temperature, selecting appropriate types and amounts of pectinase, and adding bentonite have been proven to effectively reduce the methanol content in fruit wine. Yeast improvement mainly involves inducing yeast mutations to reduce methanol produced by yeast through glycine metabolism. However, these methods may introduce exogenous substances, limit the development of flavor diversity to some extent, or face the uncontrollability of mutation direction, thus having certain limitations in the modern brewing concept that emphasizes naturalness and microbial diversity. Meanwhile, research shows that methanol levels are mainly regulated by pectin degradation, with the contribution of yeast strains themselves being relatively weak. Therefore, utilizing yeast to degrade methanol produced during fermentation offers another novel perspective. Among these methods, compared to engineering modification, screening natural yeast strains with methanol-degrading potential from abundant natural environmental microbial resources is a simpler and safer approach, providing a more promising biological solution for reducing methanol in fruit wines; however, current research in this area is still quite lacking.

[0003] In fruit wine production, using locally selected non-saccharifying yeasts in mixed fermentation with brewing yeast has become an effective strategy to enhance the complexity and regional characteristics of the product's flavor. Some local non-saccharifying yeast strains possess excellent properties such as degrading harmful substances and increasing the content of glycerol and esters, thereby significantly improving the safety of fruit wine and enhancing its flavor profile. However, most non-saccharifying yeasts have poor fermentation performance and low alcohol production efficiency, making them unable to complete fermentation independently. Therefore, using brewing yeast in mixed fermentation with non-saccharifying yeasts possessing functional properties is a feasible method to improve the safety and flavor of fruit wine.

[0004] Hunan Province is a renowned navel orange producing area in my country, with the navel oranges from Langshan (located in Xinning County, Shaoyang City, Hunan Province) being hailed as one of the "Four Great Navel Oranges of China." They are highly regarded for their bright orange-red color, delicate flesh, and rich aroma, making them a shining example of my country's citrus industry. This region features typical Danxia landforms, a warm and humid climate, abundant rainfall, and a long frost-free period. The unique red soil and mountain microclimate provide excellent ecological conditions for the accumulation of sugar and the formation of flavor compounds in the navel oranges. After years of industrial development, Langshan navel oranges have achieved remarkable results in terms of planting scale, quality control, and brand building, with fresh fruit sales becoming one of the pillar industries of the local agricultural economy. However, the current Langshan navel orange industry mainly focuses on fresh consumption, with a relatively low rate of deep processing. Particularly in the fruit wine brewing sector, research and application of local fermentation microbial resources specific to Langshan navel oranges are almost nonexistent. Currently, when developing navel orange wine, enterprises and farmers often tend to use general commercial yeasts. This not only fails to fully release the complex citrus aromas of Langshan navel oranges but also leads to homogenization of the wine products, making it difficult to reflect the unique flavor of Langshan navel oranges and to create a distinctive Langshan regional flavor. Therefore, in-depth exploration and development of the local wild navel orange yeast resources in Langshan, and screening for local yeast strains with excellent brewing characteristics, especially those with methanol-reducing abilities, can not only improve the safety, taste harmony, and flavor complexity of navel orange wine but also help to create navel orange wine products with the terroir of Langshan. This will play a crucial role in extending the Langshan navel orange industrial chain and increasing the added value of agricultural products. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a strain of wild Kluwer-Pichia pastoris selected from Langshan area of ​​Hunan Province, which has good fermentation performance, high methanol reduction ability, and improves the quality of navel orange wine.

[0006] Another objective of this invention is to provide the application of a strain of Kluwerpić yeast with good methanol-reducing ability in the brewing of navel orange wine.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] The *Pichia kluyveri* strain provided by this invention, named UT002, belongs to the genus *Pichia* and was isolated from soil in a mature navel orange orchard of the 'Newhall' variety in Langshan Town, Xinning County, Shaoyang City, Hunan Province. This strain was deposited on December 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39116. The suggested classification name is *Pichia kluyveri*.

[0009] Unless otherwise specified, Pichia kluyveri UT002 CGMCC No.39116 is referred to as Pichia kluyveri UT002 in this article.

[0010] The Kluwer Pichia pastoris UT002 provided by this invention can be used to brew navel orange wine. The advantages of using Kluwer Pichia pastoris UT002 to brew navel orange wine are: during the screening process, it exhibits excellent methanol reduction ability, with a degradation rate of 34.63% compared to the blank control group. In real navel orange juice fermentation, when mixed with another strain of Maggi-Megaki Yeast U405 screened in the laboratory and commercial brewing yeast QA23, the methanol content was 137.54 mg / L. Compared to the control group (commercial brewing yeast QA23 single-strain fermentation), it can reduce methanol by 10.11 mg / L, which is lower than the methanol content of commercially available yeasts. The resulting navel orange wine has low methanol content and high safety, while also enhancing the aroma of the fruit wine and highlighting the aroma of the navel orange variety. Therefore, inoculating local navel orange fruit from Langshan, Hunan Province, with Kluyveromyces UT002 can effectively reduce methanol content and increase the complexity and typicality of the aroma of navel orange wine without affecting its aroma, resulting in a more balanced and richer aroma profile and thus achieving the highest sensory acceptance. Simultaneously, the use of mixed fermentation with local wild yeast strains and commercial brewing yeast helps to overcome the homogenization of navel orange wine quality caused by the use of imported commercial brewing yeast, better showcasing the characteristics of navel orange wine from Langshan, Hunan Province. This helps the navel orange wine industry in Langshan develop its own local characteristics and promotes the development of the entire navel orange wine industry in the region. Attached Figure Description

[0011] Figure 1 The colony morphology of strain UT002 on WL medium;

[0012] Figure 2 The methanol content in the simulated system after group fermentation in Example 2 is given by: F: strain from Fukumoto navel orange, I: strain from Nevelina navel orange, L: strain from Langfeng navel orange, U: strain from Newhall navel orange; Ct: Candida tropicalis, Cf: Corynebacterium tumefaciens, Dh: Hansenula d'Barry, Ho: Hansenula spores of cactus, Ht: Hansenula spores of Thailand, Hu: Hansenula spores of grape juice, Kq: Kurzmania citrinum, Mp: Maggimycium magna, Mc: Meyer's fruit yeast, Pkl: Pichia kluvii, Pku: Pichia kudrica, Po: Pichia westernis, Sb: Candida astrosa, Si: Matsutani spp., Td: Saccharomyces delleoides, Wa: Wickham anomala.

[0013] Figure 3 The methanol content in the simulated system after fermentation with a single strain in Example 2 is given by F: strain from Fukumoto navel orange, I: strain from Nevelina navel orange, L: strain from Langfeng navel orange, U: strain from Newhall navel orange; Mp: Maggi Mitchell yeast, Pkl: Kluyveromyces kluyveromyces, and Hu: Hansenula spores of grape juice.

[0014] Figure 4 The methanol content in the navel orange juice and each navel orange wine fermentation group in Example 3;

[0015] Figure 5 This is a radar chart for the aroma evaluation of the navel orange wine in Example 3. Detailed Implementation

[0016] To better understand and implement this invention, the present invention will be described in detail below with reference to the embodiments and accompanying drawings; the embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0017] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0018] Unless otherwise specified, all reagents used in the following examples are analytical grade reagents and are available from legitimate commercial sources.

[0019] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are taken as the statistical average.

[0020] The culture medium formulations involved in the examples are as follows:

[0021] WL solid medium: WL nutrient agar medium 80.25 g / L.

[0022] Yeast Extract Peptone Dextrose (YPD) Liquid Culture Medium: Glucose 20 g / L, Peptone 20 g / L, Yeast Extract 10 g / L.

[0023] YPD slant culture medium: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, agar 20 g / L.

[0024] Simulated fermentation medium: YNB medium 6.7 g / L, glucose 50 g / L, fructose 50 g / L, add 0.5 mol / L NaOH solution to adjust pH to 5.8, autoclave at 121℃ for 20 min, cool to room temperature and add methanol filtered through a 0.22 μm polyethersulfone (PES) filter membrane to sterilize until the methanol concentration of the medium is 400 mg / L.

[0025] Example 1: Isolation, purification and identification of Kluyveromyces UT002

[0026] This invention screened a strain of Kluwer-Pichia pastoris with strong methanol-reducing ability, obtained from the soil of a mature navel orange orchard of the "Newhall" variety in Langshan Town, Xinning County, Shaoyang City, Hunan Province. The colony morphology of strain UT002 on WL medium is as follows: Figure 1 As shown, the filtering process is as follows:

[0027] Soil samples were collected from the base of navel orange trees after removing the top 3 cm of topsoil, and from areas where fallen or rotten fruit had fallen to the ground. Three replicates were prepared, with each soil sample weighing approximately 5 g, and all samples were stored in sterile bags. Soil samples collected on the same day were processed on the same day. The samples were mixed thoroughly in sterile bags, and under sterile conditions, 2 g of the soil sample was added to 500 mL of YPD liquid culture medium and incubated at 28°C and 160 r / min for 12 h.

[0028] After culturing in a constant temperature shaking incubator at 28℃ and 160 r / min for 12 h, samples were taken and serially diluted with sterile water. Three appropriate dilutions of 100 μL each were selected and spread on sterilized WL solid medium. After incubation in an inverted incubator at 28℃ for 72 h, plates with colony counts of 30-300 were selected. According to the method of yeast classification and identification on WL solid medium, colonies with different characteristics were classified, numbered, and the characteristics and number of each type of colony were recorded and photographed for archiving.

[0029] From each type of yeast with different colony morphology and emergence time, 2-3 strains were selected and preserved in YPD slant medium at 4°C. After the initial screening of yeast, to prevent impurity, the strains were purified. Single colonies were picked and purified on WL solid medium plates using the streak plate method. The plates were incubated upside down at 28°C for 72 hours. The characteristics of the colonies after each purification were recorded and photographed for archiving. Each strain was purified 2-3 times. The purified yeast was stored in 50% glycerol cryovials at -80°C for molecular identification.

[0030] The yeast to be tested was pre-activated and inoculated into YPD liquid medium, and cultured at 28℃ with constant temperature shaking at 160 r / min for 12-16 h until the logarithmic growth phase. After treatment with the M5 high-speed mix (catalog number MF848-10) from Beijing Polymer Biotechnology Co., Ltd., PCR amplification was performed. Primers for the 26S rDNA D1 / D2 region amplification were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are as follows: Forward primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3' (SEQ ID NO: 1); Reverse primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3' (SEQ ID NO: 2). The PCR reaction system was 20 μL: 2 μL template DNA, 1.5 μL 10 μM NL1 primer, 1.5 μL 10 μM NL4 primer, 10 μL 2x M5 Hiper high-AT high-speed mix, and 5 μL double-distilled water. PCR reaction conditions: 95 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 1 min, 55 ℃ annealing for 1 min, 72 ℃ extension for 1 min, 35 cycles; 72 ℃ final extension for 5 min. 3 μL of amplification product was analyzed by 1% agarose gel electrophoresis.

[0031] PCR product sequencing was performed by Shanghai Sangon Biotech Co., Ltd.

[0032] The target sequence of the test strain was searched using the BLAST tool on the website of the National Center for Biotechnology Information (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Similarity sequence searches were performed, and the species position of strain UT002 was initially determined based on the similarity of homologous sequences. At the same time, the sequencing map was corrected. The similarity between UT002 and the corresponding sequences of known yeasts was compared. The similarity exceeded 99%. The species position of the test strain was finally identified by the colony morphology of UT002 on WL solid medium.

[0033] The sequencing results of the 26S rDNA D1 / D2 region of *Pichia pastoris* UT002 (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 39116) are as follows:

[0034] (SEQ ID NO: 3).

[0035] Similarity to the related type strain Pichia kluyveri CBS 188: 99.18%, identified as Pichia kluyveri.

[0036] Example 2 Screening of methanol-degrading yeast

[0037] (1) Preliminary screening of bacterial strains

[0038] Remove the strain frozen in glycerol tubes, thaw it, and use an inoculation loop to streak a small amount of bacterial solution onto WL solid medium in four zones. Incubate the streaked plates at 28°C for 48 h, and purify twice. Add commercial dry yeast powder to 5% glucose water at a ratio of 1:10 (w / v), and incubate at 35°C for 20-25 min until dense bubbles appear, indicating yeast activation. Use an inoculation loop to take one loopful of bacterial solution and purify it on WL solid medium using the four-zone streak method, incubating at 28°C for 48 h, and purifying twice.

[0039] After purifying the yeast twice on WL solid medium, single colonies of the target strain were picked from WL plates and inoculated into YPD liquid medium for 12–16 h. Then, 1% of the activated bacterial culture was inoculated into 10 mL of YPD liquid medium for further culture. The absorbance of the pre-cultured bacterial solution at 600 nm was measured using a UV spectrophotometer, and the OD value of the bacterial solution was adjusted using YPD liquid medium. 600 Value up to 1.

[0040] Yeasts of the same genus selected from the same navel orange variety were grouped into one group, totaling 41 groups. Simultaneously, 60 wild *Saccharomyces cerevisiae* strains from our laboratory's existing strain resource bank were grouped according to their different origins (HJZ-W, HW-W, ZHYS-W, HJZ-R, JX-R, ZHYS-R), totaling 6 groups. Six commercial *Saccharomyces cerevisiae* strains (F15, L13, B2, D254, QA23, and Angel Yeast) were each grouped into a separate group, totaling 6 groups. A total of 53 fermentation groups were formed, as shown in Table 1. Fermentation was carried out in simulated fermentation media. 2% initial inoculum (OD) was inoculated into 50 mL of simulated fermentation media. 600 The value is 1). For groups with only one strain, 2% OD is directly inoculated. 600 Bacterial suspension with an OD value of 1; for groups with more than one strain, pre-culture each strain in the group to an OD value of 1. 600 The value is 1. Then, take 2% of the bacterial suspension from each culture, mix them in equal volumes, and inoculate them into fresh YPD liquid medium. Culture again until OD reaches 1. 600 A value of 1 was set, and 2% of the bacterial culture was used as the initial inoculum. A blank control group, with all other conditions identical and without any inoculation, was also set up. The main purpose of this was to eliminate the influence of abiotic factors (such as physical volatilization and chemical degradation) on the methanol content in the culture medium, so as to accurately and reliably attribute the change in methanol content after fermentation to the biological metabolism of the yeast strain. After fermentation in a constant temperature incubator at 25℃ for 14 days, the residual methanol content in the simulated fermentation medium was measured for initial screening to evaluate the methanol degradation capacity of each group of yeast strains.

[0041] Table 1 Grouping in the initial screening experiment

[0042] (2) Secondary screening of strains

[0043] Fermentation groups with lower remaining methanol content from the initial screening were selected, and the yeast in each group was fermented in a simulated fermentation medium with a single strain. 2% pre-cultured bacterial solution (OD200) was inoculated into 50 mL of the simulated fermentation medium. 600The value was set to 1), and a blank control group with the same conditions but without any inoculation was set up. After fermentation in a constant temperature incubator at 25℃ for 14 days, the residual methanol content in the simulated fermentation medium was measured, and the strains with strong methanol degradation ability were obtained through secondary screening.

[0044] (3) Determination of methanol content

[0045] After 14 days of simulated fermentation, the bacterial culture was centrifuged at 4℃ and 5000 r / min for 10 min. The supernatant was collected, and the methanol was qualitatively identified by gas chromatography based on the peak time of the methanol standard. The methanol was quantified by plotting a standard curve of the standard, thereby evaluating the methanol reduction ability of different strains.

[0046] The results are as follows:

[0047] (1) Preliminary screening of bacterial strains

[0048] Initial screening results by Figure 2 It is known that most wild non-brewing yeasts have the ability to degrade methanol, and some yeast strains can degrade methanol very efficiently. Among them, the most outstanding are *Umbrella Magnix* (UMp, degradation rate 34.57%) from Newhall, *Hansenula polysaccharide* (UHu, degradation rate 32.04%), and *Pichia pastoris* (UPkl, degradation rate 28.95%), as well as *Pichia pastoris* (LPkl, degradation rate 27.98%) from Langfeng. These yeast strains can effectively reduce the methanol content in the fermentation system while completing alcoholic fermentation, providing key functional strains for the development of low-methanol navel orange wine.

[0049] (2) Secondary screening of strains

[0050] Based on the initial screening results, and considering alcoholic fermentation performance, it was found that *Megmecium mihanovichii* (Mp), *Hansenula polysaccharide* (Hu), and *Pichia kluyveromyces* (Pkl) from various navel orange varieties all exhibited strong methanol degradation capabilities. Therefore, all yeasts from these three genera underwent secondary screening to evaluate the methanol degradation capabilities of specific strains and to select the optimal single strain. The results are as follows: Figure 3 As shown, this study further clarifies the advantages of *Kluyveromyces* (Pkl) as a highly efficient methanol-degrading strain. It not only exhibits high overall degradation efficiency but also relatively stable performance among strains. Among them, strain UT002 demonstrated the strongest degradation ability, with a residual methanol content of 153.24 mg / L and a degradation rate of 34.63%, showing great application potential. Highly efficient strains also exist in *Megrichia coli* (Mp), with strain U405 exhibiting the best methanol degradation ability, a methanol content of 192.81 mg / L, and a degradation rate of 17.74%. It can be used in mixed fermentation with *Kluyveromyces* (Pkl) to improve and enrich the flavor of navel orange wine.

[0051] Example 3: Comparative small-scale fermentation test of Kluyveromyces UT002 and commercial Saccharomyces cerevisiae.

[0052] Navel orange juice from Langshan, Hunan Province, stored at -20℃, was thawed and filtered through four layers of gauze to separate the clear juice from the pulp. The clear juice was adjusted to °Brix 20.0 with added white sugar and then pasteurized (65℃, 30 min). After cooling to room temperature, pectinase was added at a ratio of 50 mg / L and activated in a 45℃ water bath for 2 h. After cooling to room temperature, 400 mL portions were added in batches to a 500 mL fermenter, and the mixture was then adjusted to OD 20.0. 600 The bacterial culture with a value of 1 was added to different fermentation tanks according to the fermentation experimental group settings shown in Table 2. Each fermentation experimental group was set up in triplicate, and the fermentation process was started. The ambient temperature was set at 25℃ during fermentation, and the Brix value was monitored daily. When the °Brix value did not decrease for 3 consecutive days, the alcoholic fermentation was considered to be completed. After fermentation, samples were taken and frozen at -20℃ for methanol content determination. The remaining samples were added with 120 mg / L potassium metabisulfite, sealed, and placed in a 4℃ refrigerator for aroma evaluation.

[0053] Table 2 Fermentation Experiment Group Setup

[0054] Note: QA23: Commercial brewing yeast, U405: Maggi Mickey yeast, UT002: Kluyveromyces kluyveromyces.

[0055] Aroma Evaluation: The aroma evaluation team consisted of 12 well-trained food professionals who used a five-point intensity method to evaluate the aroma of the navel orange wine in six aspects (alcoholic aroma, sweet aroma, sour aroma, fruity aroma, floral aroma, and herbal aroma) and ranked the samples according to their preference.

[0056] The results are as follows:

[0057] (1) Methanol content in navel orange juice and various navel orange wine fermentation groups

[0058] The methanol content produced at the end of alcoholic fermentation under different mixed-culture fermentation methods is as follows: Figure 4 As shown in the figure. The results showed that the methanol degradation rate of the S+M+P(X) group was 6.85%, and the methanol content was 137.54 mg / L, which was 10.11 mg / L lower than that of the control group (commercial brewer's yeast QA23 single-strain fermentation).

[0059] (2) Aroma evaluation of navel orange juice and each navel orange wine fermentation group

[0060] Scent evaluation results as follows Figure 5As shown, the groups fermented with different non-brewing yeasts exhibited diverse aroma characteristics, with the S+M+P(X) group showing the most prominent sweet, fruity, and herbal aromas. Each panel of tasters selected their three favorite navel orange wines. Among the top-ranked navel orange wines, the S+M+P(X) group received the highest approval rating, reaching 50%. This demonstrates that the S+M+P(X) group was most favored by the tasters due to its rich, sweet aroma, prominent floral and fruity notes, and harmonious aroma.

[0061] In conclusion, the selected wild Kluwer Pichia pastoris UT002 exhibits excellent methanol reduction capabilities during the brewing of navel orange wine, increases the complexity and typicality of the aroma, and forms a more balanced and rich aroma profile, thus achieving the highest sensory acceptance and possessing the potential for commercial application.

Claims

1. A strain of Kluyveromyces yeast, characterized in that, The Kluyveromyces yeast is deposited at the China General Microbiological Culture Collection Center (CGMCC) under the name UT002 and accession number CGMCC No. 39116.

2. The application of Kluyveromyces yeast as described in claim 1 in the production of navel orange wine.

3. The application of Kluyveromyces yeast as described in claim 1 in reducing methanol content during the fermentation of navel orange wine.

4. A method for producing navel orange wine, comprising fermenting navel orange raw materials with Kluyveromyces yeast as described in claim 1 to obtain navel orange wine.

5. The method for isolating, purifying, and identifying *Kluyveromyces* as described in claim 1, characterized in that, Includes the following steps: (1) Soil samples were taken from the root of the navel orange tree after removing the top 3 cm of loose soil and from the ground where the fruit had fallen and rotted. Three replicates were set up for each sample, with each soil sample weighing about 5 g. The samples were stored in sterile bags. Soil samples collected on the same day were processed on the same day. They were mixed evenly in sterile bags, and 2 g of soil sample was added to 500 mL of YPD liquid culture medium under sterile conditions. The samples were then incubated in a constant temperature shaking incubator at 28℃ and 160 r / min for 12 h. (2) After culturing in a constant temperature shaking incubator at 28℃ and 160 r / min for 12 h, samples were taken and serially diluted with sterile water. Three appropriate dilution concentrations of 100 μL each were selected and spread on sterilized WL solid medium. After incubating in an inverted incubator at 28℃ for 72 h, plates with 30-300 colonies were selected. According to the method of classifying and identifying yeast on WL solid medium, colonies with different characteristics were classified and numbered. The characteristics and number of each type of colony were recorded and photographed for archiving. (3) Select 2-3 strains from each type of yeast with different colony morphology and different emergence time and preserve them in YPD slant medium at 4℃; After the initial screening of yeast was completed, the strain was purified to prevent impurity of the colonies. Single colonies were picked and purified on WL solid medium plates using the streak plate method. The plates were incubated upside down at 28°C for 72 hours. The characteristics of the colonies after each purification were recorded and photographed for archiving. Each strain was purified 2-3 times. The purified yeast was stored in 50% glycerol cryovials at -80°C for molecular identification. (4) Activate the yeast to be tested in advance, inoculate it into YPD liquid medium, and culture it at 28℃ and 160 r / min for 12-16 h until the logarithmic growth phase; after treatment with M5 high-speed mix (catalog number MF848-10) from Beijing Polymer Biotechnology Co., Ltd., perform PCR amplification; the primers for the 26S rDNA D1 / D2 region amplification were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are as follows: forward primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3'; reverse primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3'; the PCR reaction system is 20 μL: 2 μL template DNA, 1.5 μL of 10 μM NL1 primer, 1.5 μL of 10 μM NL4 primer, 10 μL of 2x M5 Hiper high-AT high-speed mix, and 5 μL of double-distilled water; PCR reaction conditions: 95 ℃ pre-denaturation 3 denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min, extension at 72 ℃ for 1 min, repeated 35 times; supplementary extension at 72 ℃ for 5 min; 3 μL of amplification product was detected by 1% agarose gel electrophoresis. (5) Sequencing of PCR products was performed by Shanghai Sangon Biotech Co., Ltd. (6) The target sequence of the test strain was searched using the BLAST tool in the website of the National Center for Biotechnology Information (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Similar sequence search was performed, and the species status of the UT002 strain was initially determined based on the similarity of homologous sequences. At the same time, the sequencing map was corrected. The similarity between UT002 and the corresponding sequences of known yeasts was compared. The similarity was more than 99%. The species status of the test strain was finally identified by the colony morphology of UT002 on WL solid medium.

6. A method for screening yeasts with characteristics of reducing methanol content using a simulated fermentation medium, characterized in that, The Kluyveromycin yeast as described in claim 1 can be obtained by the following steps: (1) Take out the strain frozen in the glycerol tube, thaw it, use an inoculation loop to dip a small amount of bacterial solution and streak it on the four zones of WL solid medium. After streaking, incubate the plate at 28℃ for 48 h and purify it twice. (2) After the yeast was purified twice on WL solid medium, a single target colony was picked from the WL plate and inoculated into YPD liquid medium and cultured for 12-16 h. Then, 1% of the activated bacterial solution was inoculated into 10 mL of YPD liquid medium for expansion culture. The absorbance of the pre-cultured bacterial solution at 600 nm was measured with a UV spectrophotometer, and the OD of the bacterial solution was adjusted with YPD liquid medium. 600 Value up to 1; (3) Prepare simulated fermentation medium (preparation method: YNB medium 6.7 g / L, glucose 50 g / L, fructose 50 g / L, add 0.5 mol / L NaOH solution to adjust pH to 5.8, autoclave at 121℃ for 20 min, cool to room temperature and add methanol filtered through a 0.22 μm polyethersulfone (PES) filter membrane to sterilize until the methanol concentration of the medium is 400 mg / L); inoculate 2% of the OD obtained in (2) into 50 mL of simulated fermentation medium. 600 The bacterial culture with a value of 1 was prepared, and a blank control group with the same conditions but without any inoculation of any strains was set up. Each group was set up in triplicate. After fermentation in a constant temperature incubator at 25℃ for 14 days, the residual methanol content in the simulated fermentation medium was measured to evaluate the methanol degradation ability of each yeast strain. (4) After 14 days of simulated fermentation, the bacterial culture was centrifuged at 4℃ and 5000 r / min for 10 min. The supernatant was collected and the methanol was qualitatively determined by gas chromatography based on the peak time of the methanol standard. The methanol was quantified by plotting the standard curve of the standard, thereby evaluating the methanol reduction ability of different strains.

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