Pichia kluyveri and application thereof

Abstract

The invention discloses a Pichia kluyveri yeast and its application. The Pichia kluyveri yeast is classified and named as Pichia kluyveri JLG30201, which has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242295 and a deposit date of October 22, 2024. The Pichia kluyveri JLG30201 has strong killer affinity and high sugar tolerance, unique aroma production, high production of extracellular superoxide dismutase (SOD), and low alcohol production; the strain is applied to the processing of fruit wine or edible plant enzymes, and has the effects of increasing ester and flavor, improving juice yield and product antioxidant activity.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to an excellent high-sugar-tolerant Pichia kluyveri yeast with good aroma production, strong killing ability, high extracellular SOD production and low alcohol production, and an application thereof. [Background technology]

[0002] Fruit wine is a beverage with a certain alcohol content made from fresh fruit or juice through microbial fermentation. It is popular among consumers for its high nutritional value and good taste, and can also increase the added value of fruits. The native grapes in southern China (including wild grapes, thorny grapes and hairy grapes, hereinafter referred to as native grapes) are the more distinctive germplasm resources in the East Asian population and are a grape variety with strong regional typicality. Compared with Eurasian grapes, native grapes have strong disease resistance, high nutritional value, and contain a variety of bioactive substances, especially resveratrol, which is significantly higher than the Eurasian population. It is a high-quality raw material for brewing fruit wine. However, native wines generally have problems such as weak flavor and insufficient aroma. Yeast is an important microorganism in fruit wine brewing, and plays a decisive role in alcohol production and wine flavor. Therefore, screening yeasts with good aroma production and excellent comprehensive performance will help improve the quality of native wines.

[0003] Edible plant enzymes are edible enzyme products containing specific biologically active ingredients, which are made from fruits, vegetables and grains that can be used for food processing, with or without auxiliary materials, and are fermented by microorganisms. They contain a variety of active ingredients such as functional enzymes, polyphenols, organic acids, amino acids and vitamins, and have physiological functions such as laxative, free radical scavenging, anti-oxidation, hangover and liver protection. With the increase of public health awareness, enzyme products are highly respected. In the diversified deep processing of native grapes, in addition to fruit wine, the development of new enzyme products is also an effective way to increase its added value. Antioxidant activity is one of the typical functional characteristics of enzymes, but at present, in the process of enzyme brewing, concentration treatment is often required to achieve a certain antioxidant activity. Studies have shown that the antioxidant activity of grape enzymes is related to the synergistic effect of polyphenols, superoxide dismutase (SOD) and organic acids; it is also found that the compound fermentation of yeast and lactic acid bacteria can enhance the antioxidant capacity of enzymes. Therefore, based on the functional characteristics of enzymes and the requirements of the enzyme fermentation environment for microorganisms, screening out yeasts suitable for the physical properties of native grapes can provide a high-quality bacterial source for the research and development of high-quality native grape enzymes. [Summary of the invention]

[0004] The technical problem to be solved by the present invention is to provide a Pichia kluyveri yeast and an application thereof. The Pichia kluyveri yeast has good aroma production, strong killing ability, high extracellular SOD production, low alcohol production and excellent sugar tolerance.

[0005] The present invention is achieved in that:

[0006] A Pichia kluyveri yeast, wherein the Pichia kluyveri yeast is Pichia kluyveri (Pichiakluyveri) JLG30201, which was deposited in the China Center for Type Culture Collection in Wuhan on October 22, 2024, with a deposit number of CCTCC NO: M20242295.

[0007] Furthermore, the application of the Pichia kluyveri JLG30201 in the processing of edible plant enzymes.

[0008] Furthermore, the application of the Pichia kluyveri JLG30201 in fruit wine processing.

[0009] The present invention has the following advantages:

[0010] The Pichia kluyveri JLG30201 of the present invention has strong killer instinct and high sugar tolerance, unique aroma production, high extracellular SOD production and low alcohol production. The strain is used for processing fruit wine or edible plant enzymes, and has the effects of increasing ester and aroma, improving juice yield and product antioxidant activity.

Brief Description of the Drawings

[0011] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0012] Figure 1 Schematic diagram of the killer properties of the five tested strains in the examples of the present invention.

[0013] Figure 2 Schematic diagram of the colony morphology of strain JLG30201 in the examples of the present invention.

[0014] Figure 3 Schematic diagram of the cell morphology (light microscope, ×1000) of the strain JLG30201 in the example of the present invention.

[0015] Figure 4 Schematic diagram of the phylogenetic tree of strain JLG30201 in the examples of the present invention.

[0016] Figure 5 Schematic diagram of the growth curve of strain JLG30201 in the embodiment of the present invention.

[0017] Figure 6 Schematic diagram of the growth of strain JLG30201 in the embodiment of the present invention at different temperatures.

[0018] Figure 7 This is a schematic diagram of the growth of strain JLG30201 in the example of the present invention under different sugar concentrations.

[0019] Figure 8 Schematic diagram of the sugar consumption capacity of strain JLG30201 in the examples of the present invention.

[0020] Fig. 9 Schematic diagram of aroma components of samples from fermentation groups treated with different enzymes in an embodiment of the present invention. [Specific implementation method]

[0021] The present invention relates to a Pichia kluyveri yeast, wherein the Pichia kluyveri yeast is Pichia kluyveri (Pichia kluyveri) JLG30201, which was preserved in the China Center for Type Culture Collection in Wuhan on October 22, 2024, and the preservation number is CCTCC NO: M 20242295.

[0022] The present invention also relates to one application of Pichia kluyveri, namely, application of Pichia kluyveri JLG30201 in processing of edible plant enzymes.

[0023] The present invention further relates to a second application of Pichia kluyveri, which is the application of Pichia kluyveri JLG30201 in fruit wine processing.

[0024] The following will be combined with the attached Figure 1-9 The technical scheme of the present invention is clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0025] Example 1. Isolation, screening and identification of strain JLG30201

[0026] 1 Materials and methods

[0027] 1.1 Materials

[0028] 1.1.1 Sample and source: Naturally fermented plum pickle liquid was collected by laboratory staff from a food processing factory in Ningde City, Fujian Province.

[0029] 1.1.2 Strain and source: Saccharomyces cerevisiae CICC 1296, a sensitive strain, was purchased from the China Industrial Microbiological Culture Collection Center.

[0030] 1.1.3 Culture medium

[0031] (1) High glucose YPD solid medium: YPD, finished product, Haibo Biotechnology Co., Ltd. Chloramphenicol and glucose were added to YPD to make the chloramphenicol content 0.01 g / L and the glucose content 200, 400, and 600 g / L respectively.

[0032] (2) Rose Bengal culture medium: finished product, Haibo Biotechnology Co., Ltd.

[0033] (3) Malt extract medium: finished product, Haibo Biotechnology Co., Ltd.

[0034] (4) SOD production medium: yeast powder 5 g, peptone 10 g, glucose 40 g, ZnSO4·7H2O 0.5 g, CuSO4·5H2O 0.2 g, distilled water 1000 mL.

[0035] (5) Malt extract-MB medium: Based on the malt extract medium, adjust the pH to 4.6 with 0.1 mol / L citrate phosphate buffer, add 20.0 g / L agar and 0.3 g / L methylene blue indicator. Prepare before use and sterilize at 121°C for 20 min.

[0036] 1.1.4 Main reagents and raw materials

[0037] (1) Enzyme activity assay kit, Nanjing Jiancheng Bioengineering Research Institute; (2) Native grape, specifically the purple autumn thorn grape, commercially available.

[0038] 1.2 Methods

[0039] 1.2.1 Strain isolation and purification

[0040] Take 5 mL of the sample and add it to a 250 mL Erlenmeyer flask containing 45 mL of sterile water and shake it well. Take 1 mL of the sample solution for 10-fold gradient dilution. Select the appropriate dilution according to actual needs. Take 0.2 mL of each dilution and spread it on YPD plates containing 200, 400, and 600 g / L glucose, and incubate it at 28°C. After the colonies grow out, select the plate colonies with typical yeast characteristics, good growth and high glucose concentration, streak them on the Bengal Red plate, and incubate them at 28°C. Repeat this process many times until pure colonies are obtained, and store them on the malt juice slant for later use.

[0041] 1.2.2 Strain screening

[0042] (1) Wine and aroma production capacity: fresh grapes were washed, destemmed, and crushed, and the soluble solids were adjusted to 18-20% with sucrose to prepare a grape fermentation matrix. The isolated yeast strain was activated with malt juice liquid medium, and then inoculated into the grape fermentation matrix at 10% (v / v), fermented at 25°C for 3 days, and the juice was taken to determine the alcohol content and conduct sensory evaluation. Among them, the alcohol content was determined according to GB / T 15038 "General Analysis Method for Grape and Fruit Wine". Sensory evaluation method: the fermentation samples were divided into 4 levels according to the flavor characteristics, the first level was pleasant and strong aroma, marked as "+++"; the second level was pleasant and relatively strong aroma, marked as "++"; the third level was light aroma, marked as "+"; the fourth level was almost no aroma, marked as "-"; 12 professionally trained scientific researchers were invited to conduct the evaluation, and the average value was taken for statistical analysis.

[0043] (2) SOD production capacity: The isolated strains were activated with malt extract medium, inoculated into SOD production medium at 5% (v / v), and cultured at 28°C and 150 r / min for 36 h; the SOD production activity of each strain was determined according to the instructions of the SOD assay kit. On this basis, the strains with higher SOD activity were used as test bacteria, sterilized grape juice was used as the fermentation matrix, inoculated at 3% (v / v), and fermented at 28°C for 3 days to determine the SOD production capacity of each strain in grape juice.

[0044] (3) Killing performance study: The sensitive strain, Saccharomyces cerevisiae 1296, was inoculated into malt extract medium at 2% (v / v) and cultured at 28°C for 24 h. The culture medium was diluted to 10% with sterile water. 7 CFU / mL, take 100μL and spread it on malt extract-MB medium; then take 8μL of the test strain to the medium plate and culture it at 25℃ for 48h; observe and measure the diameter of the inhibition zone and the diameter of the colony, and the difference represents the size of the inhibition zone.

[0045] (4) Pectinase production ability: The test bacteria were activated with malt extract medium and inoculated with sterilized grape juice as the fermentation medium at a 3% (v / v) inoculation amount. The fermentation was carried out at 28°C for 3 days, and the pectinase production ability of each bacteria in grape juice was determined.

[0046] 1.2.3 Strain identification

[0047] (1) Morphological observation

[0048] The target strain was inoculated on a malt juice plate, cultured at 28°C, and the colony morphology was observed every day. At the same time, a small amount of culture was picked and observed under a microscope for bacterial cell morphology. The strain was preliminarily classified.

[0049] (2) Gene sequence analysis

[0050] The genomic DNA of the target strain was extracted using a yeast DNA extraction kit, and it was used as a template, and ITS4-TCCTCCGCTTATTGATATGC (as shown in SEQ ID NO.1); ITS5-GGAAGTAAAAGTCGTAACAAGG (as shown in SEQ ID NO.2) was used as primers for PCR amplification, and the PCR amplification product was detected by 1% agarose gel electrophoresis. The purified PCR amplification product was sequenced, and the obtained sequence was homologously compared using BLAST in the GenBank database of NCBI. The sequences of strains with higher homology were selected, and the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA4.0 software.

[0051] 2 Results and analysis

[0052] 2.1 Isolation and screening of yeast strains

[0053] Eleven yeast strains with different morphologies were isolated from 40% and 60% glucose plates. The strain numbers and their brewing characteristics (wine production, aroma production, enzyme production and killing ability) are shown in Table 1. From the perspective of wine production, the alcohol content of each treatment was between 5.5-12.5%, indicating that the wine production ability of the strains varied greatly, among which JLG30116 had the lowest alcohol content of 5.5%, followed by JLG30201, which was 6.5%. The aroma production test results showed that strains JLG30201 and JLG30117 had the most outstanding aroma production ability, emitting a strong banana-like ester aroma; the aroma production ability of other strains was average or almost non-aroma. In the SOD-producing medium, the enzyme activity (>100U) was ranked from high to low in JLG30117, JLG30101, JLG30113, JLG30201 and JLG30116; in the environment with grape juice as the fermentation matrix, JLG30201 had the highest SOD production activity, indicating that the bacteria had a strong ability to secrete SOD outside the cell. The five strains with high SOD production activity were further evaluated for their killing ability. The results showed that JLG30201 had strong killing ability and obvious inhibition zone, JLG30113 had weak killing ability but no inhibition zone, and the other three strains had no killing ability, indicating that JLG30201 had a certain anti-interference ability against other microorganisms to better ensure the normal progress of the fermentation process. Furthermore, the pectinase production ability of JLG30201 and JLG30113 was investigated, and the results showed that the pectinase activity of strain JLG30201 was more than 3.5 times that of JLG30113; in the actual experimental process, it was also found that in the grape matrix fermented with JLG30201 as the strain, grape seeds were more easily separated from the pulp, which should be related to the decomposition of pectin in the pulp by pectinase.

[0054] Comprehensive analysis showed that JLG30201 was an excellent and highly sugar-tolerant strain with good aroma production, strong killing ability, high production of extracellular SOD and low alcohol production. In addition, the bacterium also had a certain decomposition effect on pectin in the raw materials, which helped to increase the juice yield.

[0055] Table 1 Brewing characteristics of isolated strains

[0056]

[0057]

[0058] Note: “ / ” means not determined.

[0059] 2.2 Species identification of target strains

[0060] 2.2.1 Macroscopic and microscopic morphological characteristics of JLG30201

[0061] like Figure 2 , 3 As shown: In malt extract medium, cultured at 28℃ for 48h, the colonies are flat, round, milky white, and have a smooth surface; under an optical microscope, the cells are spherical or oval, mostly arranged singly, and the vegetative cells bud and reproduce, 1.3-2.0μm×3.5-7.5μm.

[0062] 2.2.2 Gene sequence analysis of JLG30201

[0063] The sequencing results of strain JLG30201 were compared by BLAST in NCBI, and the ITS sequence of the strain (as shown in SEQ ID NO.3) was found to have a sequence homology greater than 99.3% with that of Pichia kluyveri. A phylogenetic tree was constructed using MEGA 4.1, as shown in FIG. Figure 4 . JLG30201 and Pichia kluyveri clustered on the same branch, and the homology was as high as 100%. Combined with the morphological characteristics of JLG30201, it was determined that the bacterium was Pichia kluyveri. Pichia kluyveri JLG30201 was deposited in the China Center for Type Culture Collection in Wuhan on October 22, 2024, with the deposit number CCTCC NO: M 20242295.

[0064] Example 2. Biological characteristics of strain JLG30201

[0065] 1 Materials and methods

[0066] 1.1 Materials

[0067] 1.1.1 Bacterial strain and source: Pichia kluyveri JLG30201, self-screened strain, with a deposit number of CCTCC NO: M 20242295.

[0068] 1.1.2 Culture medium

[0069] (1) YPD medium: finished product, Haibo Biotechnology Co., Ltd.

[0070] (2) Malt extract medium: finished product, Haibo Biotechnology Co., Ltd.

[0071] 1.2 Methods

[0072] 1.2.1 Determination of growth curve

[0073] The test strain (bacterial amount was 10 8 CFU / mL, the same below) was inoculated into YPD liquid medium at 5% (v / v), and cultured at 28°C and 150r / min for 48h. The blank medium without inoculation was used as the control for synchronous treatment. The biomass (OD 560 ).

[0074] 1.2.2 Temperature tolerance evaluation

[0075] The test strain was inoculated into YPD liquid medium at 5% (v / v), and cultured at 15, 20, 25, 30, 35, 40, 45, and 50°C with shaking at 150 r / min for 48 h. The blank medium without inoculation was used as a control for synchronous treatment. The biomass (OD 560 ).

[0076] 1.2.3 Study on sugar osmotic pressure tolerance and sugar consumption capacity

[0077] The test strain was inoculated at 5% (v / v) in YPD liquid medium with sucrose concentrations of 20, 30, 40, and 50%, respectively, and cultured at 28°C and 150 r / min for 48 h. The blank medium without inoculation was used as a control for synchronous treatment. The biomass (OD 560 ) and soluble solids content.

[0078] 2 Results and analysis

[0079] 2.1 Growth curve

[0080] The growth curve of strain JLG30201 under suitable conditions is as follows Figure 5In the first 8 hours, the bacterial count increased rapidly, which was the logarithmic growth period. This experiment failed to detect the lag period of the strain, probably because the monitoring time span was slightly longer. From 8 to 40 hours, the bacterial count gradually increased and then tended to stabilize.

[0081] 2.2 Temperature tolerance

[0082] Temperature affects the enzyme activity of yeast and its ability to absorb nutrients. The growth of strain JLG30201 at different temperatures is shown in the figure. Figure 6 . Between 25-40℃, the growth pattern of the bacteria is basically similar, indicating that this is the suitable growth range of the bacteria; at 15℃ and 20℃, the bacterial growth starts a little slower, and after 16h, the bacterial growth is similar to that under 25-40℃ conditions; at the end of fermentation (48h), the bacterial count is the largest at 20℃, and the bacterial count is slightly lower at 15℃, indicating that JLG30201 can still start fermentation at a low temperature of 15℃ and achieve a higher growth amount. When the temperature reaches 45℃, the strain enters the stable period after a short-term proliferation of 16h; at 50℃, the bacterial count remains basically unchanged; the bacteria can tolerate a temperature of 50℃, but has basically no proliferation ability.

[0083] 2.3 Sugar osmotic pressure tolerance

[0084] A certain concentration of sugar can provide an essential carbon source for yeast growth, but a higher sugar concentration will change the osmotic pressure of the yeast cell membrane, thereby inhibiting its growth. The growth trends of strain JLG30201 at different sugar concentrations have their own characteristics, such as Figure 7 20-30% sugar concentration had little effect on strain growth, and the bacterial count OD value was still above 0.95 at 48h; at 40% sugar concentration, bacterial proliferation slowed down, and the final bacterial count OD value was 0.73; at 50% sugar concentration, the bacterial count was only about twice the initial value, reaching 0.23. It can be seen that JLG30201 can tolerate 50% sugar osmotic pressure, but it has a greater impact on its growth.

[0085] 2.4 Sugar consumption capacity

[0086] like Figure 8 As shown in the figure, under CK conditions, the soluble solid content in the matrix decreased linearly and dropped to 0 at 36h, indicating that JLG30201 had consumed all the sugar in the matrix at this time, and the sugar reduction rate reached 100%. As the sugar concentration increased, the sugar reduction rate of the strain slowed down in the early stage of fermentation, and the solid content decreased significantly after 24h; at 48h, the sugar reduction rates of JLG30201 under 20%, 30%, and 40% sugar content were 58.3%, 54.3%, and 32.3%, respectively.

[0087] Example 3: Application of strain JLG30201 (I) Application of JLG30201 in native grape enzyme processing

[0088] 1 Materials and methods

[0089] 1.1 Materials

[0090] 1.1.1 Strains: (1) Pichia kluyveri JLG30201, self-screened strain, with a deposit number of CCTCC NO: M20242295; (2) Lactobacillus plantarum RPC22, with a deposit number of CCTCC NO: M2019297, which has been disclosed in Chinese patent CN112553124B; (3) Commercial Saccharomyces cerevisiae, commercially available.

[0091] 1.1.2 Raw materials: native grapes, specifically the purple autumn thorn grape variety, commercially available.

[0092] 1.1.3 Culture medium: YPD, MRS, malt extract medium, all finished products, Haibo Biotechnology Co., Ltd.

[0093] 1.2 Methods

[0094] 1.2.1 Preparation process of native grape enzyme

[0095] (1) Seed liquid preparation: JLG30201 glycerol seed was inoculated into malt extract medium and cultured in a shaking incubator at 28°C and 150 r / min for 26-28 h; 10% (v / v) was inoculated into YPD liquid medium and cultured in a shaking incubator at 28°C and 150 r / min for 24-22 h; centrifuged at 9000 r / min and 4°C for 10 min, the bacterial sludge was taken, washed twice with sterile saline, and the bacterial cells were suspended with sterile saline and the bacterial count was adjusted to 1.0×10 8 CFU / mL, and the JLG30201 seed solution was made. RPC22 used a laboratory-made direct-injection inoculant as the strain, and the bacterial count was 6.0×10 11 CFU / g or so.

[0096] (2) Raw material processing: sort, wash, remove stems and crush the grapes, add 3-3.5 times the mass of the grapes of purified water, add 30-35% of white sugar (based on the total weight), mix well, and obtain the fermentation matrix.

[0097] (3) Inoculation and fermentation: a) Self-screening bacteria treatment: JLG30201 seed liquid was inoculated with 6% (v / m) in the fermentation matrix, canned at 2 / 3 of the canning volume, and fermented at 28-30°C and 100-120r / min for 3 days; then RPC22 bacterial agent was inoculated with 0.1% (m / m) in the fermentation volume, and fermented at 30-32°C for 10-12 days; the fermented material was filtered, sterilized, etc. to obtain a grape enzyme product. b) Control treatment: Commercial yeast was inoculated with 0.1% (m / m) in the fermentation matrix, and other operations were the same as above.

[0098] 1.2.2 Determination method

[0099] (1) The determination of relevant indicators in enzymes shall be carried out in accordance with QB / T 5323-2018 “Plant Enzymes”.

[0100] (2) Juice yield determination method: Juice yield (%) = filtrate weight / total weight of fermented materials × 100%.

[0101] (3) Analysis of volatile components: The volatile components were extracted by micro solid phase extraction and analyzed by gas chromatography-mass spectrometry (GC-MS). The samples were injected three times and the results were averaged.

[0102] 2 Results and analysis

[0103] The results of the fermentation of native grape enzymes obtained by commercial yeast compound Lactobacillus plantarum RPC22 (CK group) and self-screened Pichia kluyveri JLG30201 compound Lactobacillus plantarum RPC22 (self-screened bacteria group) were as follows: Fig. 9 As shown in Table 2. The basic indicators of grape enzymes obtained from the two treatments all meet the requirements of QB / T 5323-2018 "Plant Enzymes". Compared with CK, the SOD activity, polyphenol content and juice yield of the self-screening bacteria group were significantly improved, which were 3.37, 1.83 and 1.14 times that of the control group, respectively. From the perspective of fermentation flavor, the enzyme product of the self-screening bacteria group has a typical thorny grape fruit aroma and a strong ester aroma; the aroma of the control group is light, and a little odor is produced in the late fermentation period; the volatile component detection also found that the ester substances in the self-screening bacteria group increased significantly. This shows that the fermentation of strain JLG30201 can not only increase the juice yield, but also has the effects of increasing esters and flavoring and improving antioxidant activity.

[0104] Table 2 Related physical and chemical indicators of enzyme samples treated with different

[0105]

[0106] (II) Application of JLG30201 in native wine processing

[0107] 1 Materials and methods

[0108] 1.1 Materials

[0109] 1.1.1 Strains and sources: (1) Pichia kluyveri JLG30201, self-screened strain, deposit number: CCTCC NO: M20242295. (2) Commercial Saccharomyces cerevisiae, commercially available.

[0110] 1.1.2 Raw materials: native grapes, specifically the purple autumn thorn grape variety, commercially available.

[0111] 1.1.3 Culture medium: YPD and malt extract culture medium, both are finished products, Haibo Biotechnology Co., Ltd.

[0112] 1.2 Methods

[0113] 1.2.1 Original wine preparation process

[0114] (1) Seed liquid preparation: JLG30201 and commercial brewer's yeast were inoculated into YPD liquid medium, cultured in a shaking incubator at 28°C and 150 r / min for 24-28 h, centrifuged at 9000 r / min and 4°C for 10 min, and the bacterial sludge was collected and washed twice with sterile saline. The bacterial cells were then suspended with sterile saline and the bacterial count was adjusted to 1.0 × 10 8 CFU / mL, for future use.

[0115] (2) Raw material processing: The grape raw materials are sorted, cleaned, destemmed, and crushed, and white sugar is added to a sugar content of 22%. Sulfur dioxide is added at a rate of 60 mg / kg to obtain the grape fermentation matrix.

[0116] (3) Inoculation and fermentation: a) Self-screening bacteria treatment: Inoculate JLG30201 seed liquid into the fermentation matrix at 1% (v / m), ferment at 24-25°C for 26-28h, then inoculate commercial brewing yeast at 1 / 3 of the inoculation amount of JLG30201, ferment at 24-25°C for 12d, filter to remove the residue, and obtain the original wine. b) Control treatment: Inoculate commercial brewing yeast seed liquid into the fermentation matrix at 1% (v / m), ferment at 24-25°C for 13d, filter to remove the residue, and obtain the original wine.

[0117] 1.2.2 Measurement and evaluation methods

[0118] (1) The determination of relevant indicators in wine shall be carried out in accordance with GB / T 15038 “General analytical methods for wine and fruit wine”.

[0119] (2) Sensory evaluation: The evaluation team consisted of 10 professionally trained personnel who evaluated the wine samples based on their intensity, fruity aroma, floral aroma, aftertaste, green flavor, and sourness.

[0120] 2 Results and analysis

[0121] The original wines obtained by fermentation with commercial yeast (CK group) and self-screened Pichia Kluyveri JLG30201 composite commercial yeast (self-screened bacteria group) are shown in Table 3. Both treatments completed alcohol fermentation, and the relevant indicators met the requirements of dry wine. The residual sugar content of the self-screened bacteria group was lower than that of CK, while the alcohol content was just the opposite, which should be related to the strong sugar consumption ability of strain JLG30201 but low wine production ability. The sensory evaluation results showed that the wine body aroma of the self-screened bacteria group was richer, and in addition to the typical aroma of thorny wine, it also had a unique fruity aroma. This shows that strain JLG30201 metabolizes the sugars in the raw materials and produces aroma substances such as alcohol and esters, thereby improving the quality of the original wine.

[0122] Table 3 Physical and chemical indicators of native wine

[0123]

[0124] In summary, the Pichia kluyveri JLG30201 of the present invention has strong killer instinct and high sugar tolerance, unique aroma production, high extracellular SOD production and low alcohol production; the strain is used for processing fruit wine or edible plant enzymes, which has the effects of increasing ester and flavor, improving juice yield and antioxidant activity of the product.

[0125] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A Pichia kluyveri yeast, characterized in that: The Pichia kluyveri is Pichia kluyveri JLG30201, which was deposited in the China Center for Type Culture Collection in Wuhan on October 22, 2024, with a deposit number of CCTCC NO: M 20242295.

2. A Pichia kluyveri yeast according to claim 1, characterized in that: The application of the Pichia kluyveri JLG30201 in the processing of edible plant enzymes.

3. A Pichia kluyveri yeast according to claim 1, characterized in that: The application of the Pichia kluyveri JLG30201 in fruit wine processing.

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