Saccharomyces cerevisiae CPJ-01 strain with high ethanol tolerance and ester-alcohol composite fruity flavor pedigree and application of saccharomyces cerevisiae CPJ-01 strain

Abstract

The invention discloses a Saccharomyces cerevisiae CPJ-01 strain which is high in ethanol tolerance and capable of synthesizing ester alcohol compound fruity flavor, the strain of the Saccharomyces cerevisiae CPJ-01 strain is preserved in the China Center for Type Culture Collection, and the preservation date is June 27, 2024. The strain of the Saccharomyces cerevisiae CPJ-01 strain is named as Saccharomyces cerevisiae CPJ-01 strain is named as Saccharomyces cerevisiae CPJ-01. The strain of the Saccharomyces cerevisiae CPJ-01 strain is named as Saccharomyces cerevisiae CPJ-01. The preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: M The Saccharomyces cerevisiae CPJ-01 strain is cultured on a YPD solid plate for 48 hours, so that a milk white round raised bacterial colony is formed; the size is about 5-7 [mu] m * 7-10 [mu] m, and no pseudohypha exists. The Saccharomyces cerevisiae CPJ-01 strain has outstanding high ethanol tolerance, and the activity of the Saccharomyces cerevisiae CPJ-01 strain is still greater than or equal to 90% in an environment of 15% ethanol; according to the present invention, the fermentation product has characteristics of rich aroma, high ethyl acetate content and high isoamyl alcohol content, GC-MS detection of more than or equal to 18 volatile substances, soft fruity flavor and no bitter taste, and is suitable for flavor development of fruit wine, composite drinks and the like. The invention discloses a Saccharomyces cerevisiae CPJ-01 strain, which is suitable for the production of low-pH (Potential of Hydrogen) drinks, healthy functional fermented products and salty seasonings.

Description

Technical Field

[0001] This invention belongs to the field of microbial strain technology and relates to a complex functional yeast strain, specifically to a Saccharomyces cerevisias CPJ-01 strain with high ethanol tolerance, a complex fruity aroma profile dominated by ethyl acetate and isoamyl alcohol, and its uses. Background Technology

[0002] Saccharomyces cerevisiae is one of the earliest discovered and widely used yeasts, belonging to the phylum Ascomycota and class Fungi. Its scientific name is *Saccharomyces cerevisiae*, and its characteristics include the ability to ferment and produce gas under anaerobic conditions. It is mainly used in the alcoholic fermentation process to produce carbon dioxide and ethanol.

[0003] During fermentation, *Saccharomyces cerevisiae* undergoes a series of metabolic reactions, primarily including the fermentation of sugars, the production of ethanol and carbon dioxide, and the synthesis of secondary metabolites such as esters, alcohols, and acids. Studies of the *Saccharomyces cerevisiae* genome have revealed the regulatory mechanisms of its metabolic pathways. For example, under different oxygen conditions, *Saccharomyces cerevisiae* selectively switches its metabolic pathways, transitioning from aerobic respiration to anaerobic fermentation, to adapt to different fermentation environments.

[0004] Metabolically, under aerobic conditions, *Saccharomyces cerevisiae* completely oxidizes glucose into carbon dioxide and water through aerobic respiration, generating a large amount of ATP (adenosine triphosphate), which supports cell growth and reproduction. Under anaerobic conditions, however, *Saccharomyces cerevisiae* converts glucose into ethanol and carbon dioxide through fermentation, generating less ATP, primarily used to maintain basic cellular metabolic activities. Regarding nutrient synthesis under aerobic conditions, *Saccharomyces cerevisiae* not only undergoes respiratory metabolism but also synthesizes abundant proteins, vitamins, and other nutrients. Studies have shown that during aerobic fermentation, *Saccharomyces cerevisiae* can synthesize various amino acids, vitamins (such as B vitamins), and minerals, which play a crucial role in the growth and fermentation process. In ethanol production under anaerobic conditions, *Saccharomyces cerevisiae* converts glucose into ethanol and carbon dioxide through fermentation. Research has found that in anaerobic environments, the rate of ethanol production is closely related to the rate of sugar consumption, and the accumulation of ethanol has a significant impact on cell growth and the fermentation process.

[0005] In the food industry, *Saccharomyces cerevisiae* is a key microorganism in brewing, widely used in the production of beer, wine, spirits, and bread. It is also used to produce various biological products, such as enzymes, proteins, amino acids, nucleic acids, and vitamins. During food fermentation, *Saccharomyces cerevisiae* typically interacts with other microorganisms, such as lactic acid bacteria and acetic acid bacteria. These interactions play a crucial role in the flavor, texture, and other sensory characteristics of the fermentation products. Genomics research has helped scientists identify gene interactions between *Saccharomyces cerevisiae* and other microorganisms, thereby optimizing the co-fermentation process and improving the quality of fermented food products. For example, certain strains of *Saccharomyces cerevisiae* can synergistically interact with lactic acid bacteria to enhance the production of certain flavor compounds, thus improving the taste and flavor of fermented foods.

[0006] From the perspective of genetic improvement and screening of Saccharomyces cerevisiae strains, the goal of genetic improvement of Saccharomyces cerevisiae strains is to enhance their performance during fermentation, particularly increasing ethanol production and tolerance. Through genetic improvement, it is possible to cultivate Saccharomyces cerevisiae strains that can grow and produce large amounts of ethanol under specific conditions (such as high sugar, high ethanol concentration, or high temperature environments). This helps optimize the fermentation process and improve overall production efficiency. For example, some improved Saccharomyces cerevisiae strains can consume more sugar and produce higher concentrations of ethanol in a shorter time, reducing the fermentation cycle.

[0007] In terms of applications, genetically modified high-yield Saccharomyces cerevisiae strains are widely used in the production of various alcoholic beverages, such as wine, beer, and spirits. These strains can more efficiently convert sugars into ethanol during alcoholic fermentation, increasing alcohol yield and fermentation speed. In terms of biofuel production, modified Saccharomyces cerevisiae strains are also used in the production of biofuels (such as ethanol). With the increasing global demand for renewable energy, Saccharomyces cerevisiae has become an important microorganism in biofuel production. Summary of the Invention

[0008] (1) Purpose of the invention

[0009] Although common brewing yeast is widely used in various fermentation industries, it still has limitations in large-scale food production, product quality improvement, and adaptability to special environments. These limitations include limited tolerance, unsatisfactory fermentation performance, monotonous flavor, easy generation of off-flavors from by-products, weak antioxidant and functional properties, poor sedimentation and separation properties, and even genetic drift and performance fluctuations during repeated passages.

[0010] In view of the above, the technical problem to be solved by the present invention is to address the shortcomings of existing brewing yeast, such as easy inactivation in high ethanol environments, weak flavor generation ability, and poor antioxidant properties. The present invention provides a complex functional yeast strain (Saccharomyces cerevisiae CPJ-01) with high ethanol tolerance, a fruit-alcohol aroma complex flavor formation mechanism driven by high ester content, resistance to low pH and lactic acid, strong antioxidant activity, and good sedimentation characteristics. This strain aims to meet the food industry's requirements for high stability, high yield, and high flavor quality in the production of medium-to-high alcoholic beverages, beer, fruit wine, functional beverages, and flavored fermented products.

[0011] The first objective of this invention is to provide a strain of Saccharomyces cerevisiae CPJ-01 with high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols. It exhibits excellent ethanol tolerance and good tolerance to multiple stresses, and can be used to improve the quality stability and functional diversity of fermented foods.

[0012] The second objective of this invention is to provide a method for screening and obtaining a strain of Saccharomyces cerevisiae CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of ester alcohols.

[0013] The third objective of this invention is to provide a method for identifying a strain of Saccharomyces cerevisiae CPJ-01, which has high ethanol tolerance and a complex fruity aroma profile of ester alcohols.

[0014] The fourth objective of this invention is to provide the main microbiological characteristics of a Saccharomyces cerevisiae CPJ-01 strain with high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols, including high alcohol tolerance, excellent aroma synthesis ability, resistance to low pH and lactic acid, antioxidant function and certain salt tolerance.

[0015] The fifth objective of this invention is to provide the use of a strain of Saccharomyces cerevisiae CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of ester alcohols, specifically including:

[0016] Firstly, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is suitable for high-alcohol fermentation systems under conditions of ≥12% (v / v) ethanol concentration, and can achieve a high ethanol yield of ≥0.40g / g glucose conversion rate. The fermentation process is stable and has high metabolic efficiency, making it suitable for the industrial production needs of beverages with medium to high alcohol concentrations.

[0017] Secondly, the Saccharomyces cerevisiae CPJ-01 strain described in this invention is suitable for fermentation food manufacturing processes under acidic environments with a pH of 3.5–7.0, high osmotic pressure conditions with NaCl ≤ 5%, and oxidative stress. It can maintain cell membrane integrity and metabolic activity under the above-mentioned combined stress, ensuring the continuity of the fermentation system and the stability of product quality.

[0018] Thirdly, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is used for the efficient synthesis of volatile aroma substances such as esters (e.g., ethyl acetate) and alcohols (e.g., isoamyl alcohol) in the fermentation system, which significantly improves the aroma complexity and flavor diversity of the product.

[0019] Fourth, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is used to develop health drinks with significant antioxidant functions, so that the DPPH free radical scavenging rate of the fermentation broth is ≥70%, giving full play to the antioxidant activity and meeting the dual requirements of safety and bioactivity in the functional beverage market.

[0020] Fifth, the Saccharomyces cerevisiae CPJ-01 strain of the present invention is used for innovative applications in flavored fermented products. By optimizing cell sedimentation and separation performance, it stabilizes the fermentation process and provides reliable strain support for customized flavoring treatment according to target flavor characteristics. (2) Summary of the Invention

[0022] First, this invention provides a strain of *Saccharomyces cerevisiae* CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols. The Chinese name of the *Saccharomyces cerevisiae* strain is *Saccharomyces cerevisias* CPJ-01, and the strain is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China. The deposit date is June 27, 2024, and the accession number is CCTCC NO: M 20241387.

[0023] Second, this invention provides a method for screening and obtaining a strain of Saccharomyces cerevisiae CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of ester alcohols, specifically through the following steps:

[0024] S1. Plum Sample Collection: Fresh plum samples were collected from naturally fallen plums during the ripening period (mid-June) in Eryuan County, Dali Bai Autonomous Prefecture, Yunnan Province, China, a region rich in natural fruit-fragrant microorganisms. Ten samples (approximately 30–50 g per fruit) were collected per batch, placed in sterile sampling bags, stored in a portable refrigerator (4 ± 1℃), and transported to the laboratory within 12 hours.

[0025] S2. Sample pretreatment: Take 25.0 g of representative green plum fruit sample and place it in 225 mL of sterile physiological saline (0.85% NaCl, pH 6.8). Under sterile conditions, homogenize and shake at 8000 rpm for 2 minutes in a homogenizer to obtain a suspension.

[0026] S3. Gradient dilution and coating: Dilute the suspension for 10... -1 ~10 -6 Serial dilutions were performed, with 200 μL of each dilution spread evenly on YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar) selective medium plates containing 5% ethanol for initial screening of alcohol tolerance.

[0027] S4. Culture conditions: Incubate upside down in a 30℃ constant temperature incubator for 48–72 hours and observe the colony growth; then select colonies with obvious morphology, smooth surface, neat edges, full ridges, and milky white opaque color as the initial screening targets;

[0028] S5. Single colony isolation and purification: Repeated subculturing on fresh YPD plates at least three times using the streak dilution method to obtain stably growing single colonies; retain 3-5 phenotypic clones in each round of screening for subsequent analysis.

[0029] S6. Microscopic observation and preliminary identification: The purified colonies were Gram stained and the cell morphology was observed using an optical microscope (1000× oil immersion). Those that were oval, had obvious budding, no pseudohyphae structure, and were Gram positive were selected, and strains with consistent morphology were retained and numbered for preservation.

[0030] S7. Glycerol cryopreservation: Isolates that are confirmed to conform to the morphological characteristics of yeast were inoculated into YPD liquid medium (30℃ shaking culture for 24 h), the cells were collected by centrifugation and resuspended in 15% glycerol to a final concentration, and then cryopreserved at –80℃ for subsequent evaluation of metabolic and genetic stability and subsequent identification of strains.

[0031] Third, this invention provides a method for identifying a strain of *Saccharomyces cerevisiae* CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as detailed below:

[0032] The materials and instruments used in the method for identifying the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention mainly include a rapid fungal genomic DNA extraction kit (Sangon Biotech, catalog number B518229), liquid nitrogen, 1.5 mL centrifuge tubes (RNase / DNase-free), a 65°C constant temperature water bath; a centrifuge (model: Eppendorf 5424, maximum speed 13,200 rpm, 4°C cooling), pipettes, RNase / DNase-free pipette tips, and TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0).

[0033] This invention provides a method for identifying a strain of *Saccharomyces cerevisiae* CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of ester alcohols, mainly comprising the following steps:

[0034] S1. Sample tissue disruption: Take 50–100 mg of fresh yeast cell clusters or mycelia (20 mg for dry samples) and place them in a pre-cooled grinding tube. Add liquid nitrogen to quickly freeze and grind until the cells are completely disrupted.

[0035] S2. Cleavage: Add 400 µL Buffer Digestion and 4 µL β-mercaptoethanol, gently invert to mix, and place in a 65°C water bath and shake (300 rpm) for 1 h until complete lysis (the solution becomes clear).

[0036] S3. Protein precipitation: Cool to room temperature, add 200 µL Buffer PF, gently invert 10 times to mix, and place in a -20℃ refrigerator for 5 min to allow proteins and polysaccharides to further aggregate.

[0037] S4. Centrifugation and supernatant transfer: Centrifuge at 4℃, 10,000 rpm for 5 min; carefully transfer the supernatant to a new 1.5 mL centrifuge tube;

[0038] S5. DNA precipitation: Add an equal volume of isopropanol (about 400 µL), gently invert to mix 8 times, let stand at room temperature for 3 min, centrifuge at 4°C and 10,000 rpm for 5 min, and discard the supernatant;

[0039] S6. Washing: Add 1 mL of 75% ethanol, mix by inversion for 2 min, centrifuge at 4℃ and 10,000 rpm for 2 min, discard the supernatant, and repeat once;

[0040] S7. Drying: Open the centrifuge tube cap and invert at room temperature for 5–10 minutes until there is no residual ethanol on the tube wall;

[0041] S8. Dissolution and Storage: Add 50 µL of TE Buffer to the tube, gently pipette to mix, and let stand for 5 min; a clear, viscous DNA solution will be obtained at room temperature; and then it can be used for DNA analysis. 260 / A 280 The ratio is (1.8–2.0), with a total yield of approximately 2–5 µg, suitable for PCR, sequencing, or long-term storage at -20°C.

[0042] Fourth, the *Saccharomyces cerevisiae* CPJ-01 strain of this invention, which possesses high ethanol tolerance and a complex fruity aroma profile of ester alcohols, can be obtained through isolation, targeted screening, and molecular identification, and has the following significant technical characteristics:

[0043] Firstly, the microbial characteristics of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention, which possesses high ethanol tolerance and a complex fruity aroma profile of ester alcohols, are as follows:

[0044] Specifically, in terms of colony morphology and distribution, the *Saccharomyces cerevisiae* CPJ-01 strain, after being cultured on YPD solid medium for 48 h, forms milky white, round, raised colonies with a diameter of 2.5–3.0 mm, neat and smooth edges, and a moist, glossy, and opaque surface. Under a microscope, individual cells are oval, without pseudohyphae formation, exhibit obvious budding, and have a cell size of approximately 5–7 μm × 7–10 μm.

[0045] Specifically, regarding high alcohol tolerance, this invention provides a *Saccharomyces cerevisiae* CPJ-01 strain with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols. The maximum tolerated ethanol concentration is 12% (v / v), with a survival rate ≥85% under 10% (v / v) ethanol conditions; it maintains ≥90% cell viability at ≥15% (v / v) ethanol concentrations, a final yield ≥0.40 g / g, and residual sugar ≤5 g / L; it exhibits excellent glucose and maltose conversion rates, with a glucose consumption rate of 0.8 g / (L·h), a peak CO2 release of 120 mL reached in 48 hours, sugar depletion achieved within 72 hours, an alcohol yield of 0.4 g / g glucose, a minimum glucose conversion rate of 0.40 g / g, and a maltose metabolism rate of 0.2 g / (L·h).

[0046] Specifically, in terms of superior aroma synthesis ability, this invention provides a strain of Saccharomyces cerevisiae CPJ-01, which has high ethanol tolerance and a complex fruity aroma spectrum of esters and alcohols. The fermentation product contains 45 mg / L of ethyl acetate and 32 mg / L of isoamyl alcohol, with esters accounting for 38% of the total volatile components. GC-MS analysis detected ≥18 volatile flavor compounds, and the fermentation broth has a mellow fruity aroma, a slightly sweet taste, and no bitterness.

[0047] Specifically, in terms of resistance to low pH and lactic acid tolerance, this invention provides a strain of Saccharomyces cerevisiae CPJ-01, a brewing yeast with high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols. The growth inhibition rate is only 15% under pH 3.5 conditions, and the lactic acid tolerance threshold can reach 2.5 g / L, making it suitable for acidic fermentation systems of fruit wines, acidic beverages, and special process beers.

[0048] Specifically, in terms of antioxidant function, this invention provides a strain of *Saccharomyces cerevisiae* CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of ester alcohols. The fermentation broth is amber in color and has an OD value of [missing information]. 420 The value is 0.42, the DPPH free radical scavenging rate of the fermentation broth (diluted 10 times) reaches 72%, the foam height of the fermentation broth is 3.0cm, and the sedimentation rate is 50% after 24 hours of fermentation, which has potential value for the development and application of health drinks and functional foods.

[0049] Specifically, in terms of salt tolerance, this invention provides a strain of Saccharomyces cerevisiae CPJ-01, which has high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols. It can still maintain normal growth in an environment containing 5% NaCl and is suitable for salty seasoning fermentation processes. Its growth is inhibited at temperatures above 40°C, and the recommended fermentation temperature range is 25–35°C.

[0050] Preferably, the Saccharomyces cerevisiae CPJ-01 strain uses glucose as its preferred metabolic substrate and preferentially metabolizes glucose in the presence of glucose.

[0051] Preferably, the Saccharomyces cerevisiae CPJ-01 strain exhibits the highest fermentation efficiency under constant temperature conditions of 32°C.

[0052] Secondly, the genetic characteristics of the *Saccharomyces cerevisiae* CPJ-01 strain with high ethanol tolerance and a complex fruity aroma profile of ester alcohols described in this invention are as follows:

[0053] The key genes of the alcohol fermentation and sugar metabolism module of the Saccharomyces cerevisiae CPJ-01 strain are PDC1, ADH1, ADH2, and ALD6, and the gene basis for high ethanol yield is PDC1, ADH1 / 2.

[0054] The key genes of the aroma substance synthesis module of the Saccharomyces cerevisiae CPJ-01 strain are ATF1, ATF2, IAH1, and BAT1 / BAT2, and the gene base rich in fruit aroma esters is ATF1, BAT2, and IAH1.

[0055] The key genes of the stress resistance and homeostasis regulation module of the Saccharomyces cerevisiae CPJ-01 strain are TPS1, HSP30, PDR12, ENA1, and GPD1, and the gene basis for tolerance to high ethanol / weak acid / salt is TPS1, PDR12, ENA1, and HSP30.

[0056] The key genes of the antioxidant response module of the Saccharomyces cerevisiae CPJ-01 strain are SOD1, CTA1, YAP1, and GSH1, and the gene base with strong antioxidant function is SOD1, CTA1, GSH1, and YAP1.

[0057] The key genes for the sedimentation and process adaptation module of the Saccharomyces cerevisiae CPJ-01 strain are FLO1, FLO8, FLO11, and TUP1, and the gene basis for good process sedimentation is FLO1 and TUP1.

[0058] Fifth, the uses and application scenarios of the *Saccharomyces cerevisiae* CPJ-01 strain with high ethanol tolerance and a complex fruity aroma profile of ester alcohols described in this invention include:

[0059] The Saccharomyces cerevisiae CPJ-01 strain described above has an ethanol volume fraction of ≥12% (v / v) in a high-alcohol fermentation system, making it suitable for the industrial production of medium-to-high alcohol beverages or strong fruit wines.

[0060] The Saccharomyces cerevisiae CPJ-01 strain can maintain cell membrane integrity and metabolic activity under combined stress conditions of pH ≤3.5, NaCl concentration ≤5%, and oxidative stress environment, making it suitable for the production of low pH products.

[0061] The Saccharomyces cerevisiae CPJ-01 strain can synthesize volatile aroma components such as ethyl acetate and isoamyl alcohol, making it suitable for fermented beverages with efficient synthesis of ester alcohol aroma substances.

[0062] The fermentation broth produced by the Saccharomyces cerevisiae CPJ-01 strain, after being diluted 10 times, has a DPPH free radical scavenging rate of ≥70%, making it suitable for the development of antioxidant health drinks.

[0063] The Saccharomyces cerevisiae CPJ-01 strain is suitable for stable fermentation and targeted development of flavored fermented products.

[0064] Furthermore, the Saccharomyces cerevisiae CPJ-01 strain of the present invention uses wild microbial resources derived from natural green plums, and selects the yeast strain Saccharomyces cerevisiae CPJ-01 with alcohol resistance, complex ester alcohol aroma synthesis ability and acid environment adaptability under the screening pressure of 5% (v / v) high concentration ethanol.

[0065] Furthermore, the rapid genomic DNA extraction method for the Saccharomyces cerevisiae CPJ-01 strain of the present invention involves introducing a dedicated fungal genomic DNA rapid extraction kit (B518229) combined with liquid nitrogen freezing and lysis, and a Buffer PF cold aggregation step, to achieve rapid identification and molecular characterization of the Saccharomyces cerevisiae CPJ-01 strain at the gene level.

[0066] (3) Effects of the invention

[0067] From the perspective of efficient ethanol fermentation and alcohol production, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits excellent alcohol yield (0.4 g / g glucose) and maintains good fermentation survival rate (85%) even at high ethanol concentrations (e.g., 10% ethanol). Therefore, *Saccharomyces cerevisiae* CPJ-01 is mainly used for efficient sugar conversion in alcohol fermentation, solving the problem of yeast tolerance at high ethanol concentrations and improving the yield and stability of the ethanol production process.

[0068] Regarding fermentation adaptability under high sugar concentration and low pH conditions, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits strong utilization of common sugars such as glucose, maltose, and sucrose, and tolerates a pH range of 3.5-7.0, maintaining good fermentation activity even in low pH environments. This allows the *Saccharomyces cerevisiae* CPJ-01 strain to grow stably in acidic fermentation environments, solving the problem of limited growth of traditional yeasts under acidic conditions.

[0069] Regarding the efficient synthesis of aroma components, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can efficiently synthesize esters (such as ethyl acetate and isoamyl alcohol), which are crucial for the aroma and taste of fermented products. The volatile compounds released by the *Saccharomyces cerevisiae* CPJ-01 strain during fermentation make it an ideal choice for the brewing, beverage, and food industries, helping to solve the problems of flavor component stability and consistency.

[0070] In terms of fermentation adaptability to high-temperature and high-salt environments, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention maintains good fermentation activity even under high-temperature (up to 40°C) and high-salt (maximum tolerance to 5% NaCl) conditions, solving the problem of limited growth of many traditional yeasts in extreme environments. Therefore, the *Saccharomyces cerevisiae* CPJ-01 strain can be used for fermentation under high-salt or high-temperature conditions, such as some special brewing processes or industrial fermentation under extreme environments.

[0071] In terms of enzyme activity and biotransformation potential, the esterase and alcohol dehydrogenase activities of the *Saccharomyces cerevisiae* CPJ-01 strain enable it to catalyze esterification reactions or alcohol conversions during biotransformation, offering potential applications for the biochemical industry. The enzyme activity of *Saccharomyces cerevisiae* CPJ-01 strain can be used to catalyze specific chemical reactions, such as alcohol dehydrogenation, solving the technical challenge of requiring stable and efficient enzyme activity in biocatalysis.

[0072] From the perspective of antioxidant properties and health product development, the fermentation broth of *Saccharomyces cerevisiae* CPJ-01 strain exhibits strong free radical scavenging ability (DPPH free radical scavenging rate of 72%), indicating that *Saccharomyces cerevisiae* CPJ-01 strain has significant advantages in antioxidant function. Therefore, *Saccharomyces cerevisiae* CPJ-01 strain can be applied to develop foods and beverages with antioxidant and health-promoting functions, solving the problem of insufficient stability of natural components in traditional antioxidant products. Attached Figure Description

[0073] Figure 1 This is a single colony image on a plate (standard YPD medium) of the Saccharomyces cerevisiae CPJ-01 strain, which has high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention.

[0074] Figure 2 This is a phylogenetic tree image of the Saccharomyces cerevisiae CPJ-01 strain, which has high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention.

[0075] Figure 3 DNA sequencing electrophoresis of the *Saccharomyces cerevisiae* CPJ-01 strain, which exhibits high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention. Figure 1 (Staining pattern 1);

[0076] Figure 4 DNA sequencing electrophoresis of the *Saccharomyces cerevisiae* CPJ-01 strain, which exhibits high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention. Figure 2 (Staining pattern 2);

[0077] Figure 5DNA sequencing electrophoresis of the *Saccharomyces cerevisiae* CPJ-01 strain, which exhibits high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention. Figure 3 (Staining pattern 3);

[0078] Figure 6 DNA sequencing electrophoresis of the *Saccharomyces cerevisiae* CPJ-01 strain, which exhibits high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention. Figure 4 (Staining pattern 4);

[0079] Figure 7 The raw material for the *Saccharomyces cerevisiae* CPJ-01 strain of brewing yeast with high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols described in this invention was mature green plums collected from Eryuan County, Dali, Yunnan, China. Detailed Implementation

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0081] In the foregoing description of this invention, all figures disclosed herein, whether or not the words “approximately” or “about” are used, are approximate values. Based on the disclosed figures, the value of each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0082] The term “room temperature” refers to a temperature between approximately 18°C ​​and approximately 35°C, or between approximately 20°C and 30°C, or approximately 25°C.

[0083] This invention provides a *Saccharomyces cerevisiae* CPJ-01 strain of *Saccharomyces cerevisiae* with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols. The strain is named *Saccharomyces cerevisiae* CPJ-01 in Chinese and Latin. This *Saccharomyces cerevisiae* CPJ-01 strain is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China; the deposit date is June 27, 2024; and the accession number is CCTCC NO: M 20241387.

[0084] The Saccharomyces cerevisiae CPJ-01 yeast described in this invention is mainly aimed at solving technical problems, including efficient ethanol fermentation and alcohol production, fermentation adaptability under high sugar concentration and low pH conditions, efficient synthesis of aroma components, fermentation adaptability to high temperature and high salt environments, enzyme activity and biotransformation potential, antioxidant properties, and health product development.

[0085] The *Saccharomyces cerevisiae* CPJ-01 brewing yeast described in this invention, as an industrial brewing strain, can achieve highly efficient fermentation under extreme environments such as high alcohol, strong acid, and high salt, significantly solving the problem of poor or interrupted fermentation by traditional yeasts under such conditions. Simultaneously, this strain can enhance the aroma complexity and flavor profile of fermented beverages such as fruit wine and beer, and possesses strong antioxidant activity, making it suitable for developing healthy functional fermented products. The *Saccharomyces cerevisiae* CPJ-01 brewing yeast described in this invention is suitable for rapid industrial-scale production, balancing high yield and product quality, and facilitates subsequent clarification of the fermentation broth, thereby reducing the complexity of downstream processes.

[0086] The brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention, through its excellent fermentation performance, environmental adaptability, aroma synthesis ability and enzyme activity, can effectively solve the problems of high-efficiency alcohol production, fermentation adaptability in low pH and high sugar environments, flavor stability, and growth and fermentation under extreme conditions, and has broad application prospects, especially in the fields of food, beverage, chemical and biotechnology.

[0087] The brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention is particularly suitable for the production and processing of beer, fruit wine, rice wine, spirits and healthy fermented beverages. It is also suitable for the development of specialty fermented condiments, as well as industrial fermentation under special environments such as high salt, high acid and high alcohol, providing efficient and stable fermentation solutions for food engineering-related enterprises and research institutions.

[0088] Furthermore, all reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0089] Figure 1 This is a single colony image on a plate (standard YPD medium) of the *Saccharomyces cerevisiae* CPJ-01 strain, which exhibits high ethanol tolerance and a complex fruity aroma profile of esters and alcohols, as described in this invention. Figure 1Dozens to hundreds of individual colonies can be seen. In terms of colony morphology, the size of most colonies of the Saccharomyces cerevisiae CPJ-01 strain is between 2 and 4 mm in diameter, with a few slightly larger or smaller. Each colony originates from a primary colony-forming unit (CFU). In terms of shape, the Saccharomyces cerevisiae CPJ-01 strain is round with flat or slightly smooth raised edges, typical of yeast colonies. In terms of color and texture, the Saccharomyces cerevisiae CPJ-01 strain is milky white and translucent, with a smooth and slightly glossy surface and a soft texture, indicating that the strain is growing well and there is no obvious pigment deposition or abnormal colonies. In terms of colony distribution, the Saccharomyces cerevisiae CPJ-01 strain exhibits a uniformly dispersed distribution, with sparser edges and slightly denser centers, consistent with the dilution gradient of the bacterial suspension after inoculation using the streak or spread plate method; furthermore, there is no obvious contamination (different colors or morphologies), indicating high overall purity.

[0090] Figure 2 This is a phylogenetic tree image of the Saccharomyces cerevisiae CPJ-01 strain, which has high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention.

[0091] Figure 3 — Figure 6 These are DNA sequencing electrophoresis images (staining maps) of the *Saccharomyces cerevisiae* CPJ-01 strain, which possesses high ethanol tolerance and a complex fruity aroma profile of ester alcohols, as described in this invention. This is a typical DNA sequencing electrophoresis image (staining map) displayed using the sequencing software Chromas, used to analyze the genetic sequence of the *Saccharomyces cerevisiae* CPJ-01 strain. Figure 3 — Figure 6 The colors represent the bases: red for Thymine (T), green for Adenine (A), blue for Cytosine (C), and black for Guanine (G). Figure 3 — Figure 6 In electrophoresis, each peak represents a base signal, and the sharper and more symmetrical the peak, the higher the sequencing accuracy of that site. If peak overlap or tailing occurs, it indicates a base repetition region, secondary structure interference, or background noise during sequencing, or primer annealing problems. Figure 3 — Figure 6The letters above the sequence bar are the automatically retrieved base sequences. This is a linear representation of a certain DNA region of the target Saccharomyces cerevisiae, indicating whether it is a coding region (CDS), intron, or non-coding region.

[0092] Figure 7 The image shows the raw material collected from ripe green plums in Eryuan County, Dali, Yunnan, China, for the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast with high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols, as described in this invention.

[0093] Example 1: Method for screening and obtaining the Saccharomyces cerevisiaes CPJ-01 strain of the present invention

[0094] The method for screening and obtaining the Saccharomyces cerevisiaes CPJ-01 strain described in this invention is as follows:

[0095] S1. Collection of green plum samples: Ripe green plums were collected from Eryuan County, Dali, Yunnan, China, preserved in sterile sealed bags, stored at low temperature, and transported to the laboratory.

[0096] S1.1 Location and time of plum collection: In this experiment, Eryuan County, Dali Bai Autonomous Prefecture, Yunnan Province, China, with its unique geographical environment and good natural ecology, was selected as the location for collecting plum samples.

[0097] The ripe green plums from Eryuan County, Dali, Yunnan Province, are rich in natural yeast due to the moderate rainfall and large temperature difference between day and night in the Eryuan County area.

[0098] The ripening time of the green plums in Eryuan County, Dali, Yunnan Province, is during the plum ripening season. The fruits are hand-picked in a pollution-free, naturally growing state to ensure that the surface of the fruit is covered with a natural microbial community. Furthermore, the fruits are picked in the morning or evening of the day of harvest to avoid the midday high temperature which would reduce yeast activity.

[0099] S1.2 Tools and Disinfection: Use sterile scissors or disposable gloves to cut the plums and immediately place them in a sterile sealed bag that has been pre-autoclaved (121℃, 20min);

[0100] S1.3 Sample Preservation and Transportation: First, rapidly cool the plums at the collection site and place the bagged plums in an ice box at 4°C. The collected plums must be transported to the microbiology laboratory within ≤6 hours to maximize the preservation of the original activity and diversity of the yeast community in the sample. If longer transportation is required, the bagged plums must be sealed with oxygen-permeable plastic wrap and stored at -20°C for a short period.

[0101] S2. Sample pretreatment: Weigh 25 grams of plum sample; place it in 225 ml of sterile physiological saline and mix by homogenizing and shaking.

[0102] S2.1 Weighing and Dissolving: In the sterile operating table of the microbiology laboratory, accurately weigh 25g of green plum fruit (including skin and pit), add 225mL of sterile physiological saline with a concentration of 0.85% NaCl to form an initial dilution of 1:10.

[0103] S2.2 Homogenization: Homogenization methods are divided into using a sterile homogenizer (or a handheld homogenizer) and using a high-speed homogenizer;

[0104] The homogenization process using a sterile homogenizer (or handheld homogenizer) involves shaking at 4°C for 2 minutes to fully disperse the cells and pulp of the plum fruit.

[0105] The process involves using a high-speed homogenizer to homogenize the fruit for 90 seconds at a oscillation frequency of 8000 rpm. This process ensures that the microorganisms on the surface of the plum peel and pulp are fully released into the liquid and that the microorganisms are evenly dispersed in the solution, creating conditions for subsequent isolation and culture.

[0106] S2.3 Stand: Let stand at room temperature for 1–2 minutes to allow large pieces of plum pulp to settle, and take the supernatant for the next step;

[0107] S3. Sample dilution and coating: The mixed liquid from the previous experimental step is serially diluted; the plum suspension obtained in the previous step is serially diluted tenfold, usually with a dilution concentration of 10. -1 Up to 10 -6 The specific dilution level is determined based on the estimated microbial density; each dilution level is handled using new clean test tubes and sterile pipettes to ensure no cross-contamination.

[0108] S3.1 Serial dilution: Take 1 mL of the supernatant and add it to 9 mL of sterile physiological saline, mix thoroughly, and obtain 10-1 -2 Dilute sequentially to 10 -6 ;

[0109] S3.2 Spreading amount: Take 0.2 mL (200 μL) of each dilution and spread it on a culture medium plate; it is recommended to set up 2-3 parallel plates for each dilution to improve the positive detection rate;

[0110] S4. Spreading and incubation: Spread 200 μL of the diluted solution onto a yeast extract peptone glucose agar plate and incubate the yeast at 30°C.

[0111] S4.1 Preparation of culture medium: Take 200 μL of each dilution of suspension and spread it on the pre-prepared YPD medium plates. The dilutions are 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar. After autoclaving, wait for the temperature to drop to 50–55℃ before unplating.

[0112] S4.2 Culture conditions: Use a sterile spreader to evenly spread the culture medium on the surface to prevent colony overlap; place the plate in a constant temperature incubator, set the temperature to 30℃, place it upside down to prevent condensation, and incubate in the dark for 48-72 hours, during which time the colony growth is observed.

[0113] S5. Colony observation and isolation purification: By observing the characteristics of colony shape, size, color, luster, texture, surface and edge, the target strain is initially identified, and then the strain is further purified by the streak method to obtain single colonies;

[0114] S5.1 Preliminary screening: Observe the colony morphology on different dilutions of each plate. The size is 1-3 mm in diameter, the color is ivory white, the luster is slightly shiny on the surface of the colony, and the texture is moist with smooth edges.

[0115] S5.2 Selecting target colonies: Select colonies whose morphology is closest to that of Saccharomyces cerevisiae, and then use a sterile inoculation loop to make cross streaks on fresh YPD plates;

[0116] S5.3 Repeated purification: Purification was performed multiple times on fresh YPD plates using the cross-strike inoculation method (four-zone streak method). The culture process was repeated after each inoculation until a single colony population with consistent morphology was obtained, in order to eliminate interference from other bacteria and ensure the purity of the single strain.

[0117] S6. Microscopic examination and cryopreservation of dominant strains: The purified single colonies were Gram stained and observed under a microscope to further determine whether the isolated strain was the target strain. The isolated strains with consistent morphology were then cryopreserved with glycerol for subsequent identification.

[0118] S6.1 Gram staining: Although yeast is Gram positive, it is stained with Giemsa or lactic acid crystal violet and observed morphologically under a 1000x optical microscope. The target strain should be oval, single or budding, and fungal spores and germ tubes should be clearly visible.

[0119] S6.2 Physiological and biochemical identification: Fermentation experiment is the fermentation of polysaccharides such as glucose and sucrose, and gas production is detected; the fermentation product is determined by ethanol content;

[0120] PCR amplification and sequencing of the S6.3 ITS region: Using ITS1 / ITS4 primers, sequencing was performed and compared with the GenBank database, confirming it as S. cerevisiae.

[0121] S6.4 Cryopreservation: The purified single colonies (selected from dominant strains with consistent morphology and strong proliferation capacity) were transferred to 10 mL of YPD liquid medium and cultured overnight at 37°C and 200 rpm in a shaker. After the dominant strains had grown for 24 hours, they were centrifuged to collect the bacteria. The bacterial suspension was prepared with 15% glycerol to a final concentration of 15% V / V. The suspension was then aliquoted into cryovials and stored in an ultra-low temperature freezer at -80°C for long-term preservation, pending subsequent molecular identification and functional analysis.

[0122] Furthermore, the method for screening and obtaining the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention, compared to traditional *Saccharomyces cerevisiae* screening techniques, introduces 5% ethanol selective pressure in the initial screening stage, significantly improving the alcohol tolerance of subsequently obtained strains. Simultaneously, using plum, a substrate rich in complex aroma compounds, as the source microbial library, the screened *Saccharomyces cerevisiae* CPJ-01 not only possesses excellent fermentation performance but also generates more than 18 volatile aroma compounds, including ethyl acetate and phenylethanol, exhibiting significant flavor advantages in fruit wine products. The method for screening and obtaining the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is simple, low-cost, and highly reproducible, possessing significant prospects for industrial application.

[0123] Example 2: Rapid extraction and identification method of genomic DNA from the Saccharomyces cerevisiae CPJ-01 strain of the present invention.

[0124] Example 2 focuses on the rapid extraction and identification method of genomic DNA from *Saccharomyces cerevisiae* CPJ-01 described in this invention. By refining each experimental step, such as sample pretreatment, dilution plating, and colony purification, key parameters such as stirring time and temperature control are clearly defined, ensuring the reliability and reproducibility of the method. Furthermore, the rapid extraction and identification method of genomic DNA from *Saccharomyces cerevisiae* CPJ-01 described in this invention fully discloses the setting and control of experimental conditions, increasing the verification and control of experimental results, such as temperature comparison experiments and multiple rounds of purification operations, ensuring the scientific validity and completeness of the method. The rapid extraction and identification method of genomic DNA from *Saccharomyces cerevisiae* CPJ-01 strain described in this invention, through multiple purifications, microscopic confirmation, and cryopreservation, ensures the morphological consistency of the screened *Saccharomyces cerevisiae* CPJ-01 strain and maintains good fermentation performance after long-term storage, demonstrating significant effectiveness.

[0125] The method for rapid extraction and identification of genomic DNA from Saccharomyces cerevisiae CPJ-01 strain described in this invention is based on a commercially available rapid fungal genomic DNA extraction kit (Sangon Biotech, catalog number: B518229), combined with an efficient lysis and purification process, to extract complete and pure genomic DNA for downstream PCR identification, sequencing comparison and other analyses.

[0126] The method for identifying the Saccharomyces cerevisiae CPJ-01 strain described in this invention uses a rapid fungal genomic DNA extraction kit (Sangon Biotech, catalog number: B518229) to extract fungal genomic DNA. The specific steps are as follows:

[0127] S1. Sample collection:

[0128] S1.1 Fresh Samples: Weigh 50–100 mg of fresh macrofungi (such as mushrooms, yeast colonies, or mycelial clumps).

[0129] S1.2 Dry Sample: Weigh 20 mg of dried fruiting bodies or mycelia and grind them into powder using liquid nitrogen;

[0130] S1.3 Grinding the sample: After rapidly freezing the fresh or dried sample in liquid nitrogen, grind it into a fine powder with a sterile mortar or tissue homogenizer to maximize the lysis of the fungal cell wall. Operation under frozen conditions can maximize the disruption of the cell wall and inhibit nuclease activity.

[0131] S2. Cell lysis:

[0132] S2.1 Preparation of lysis solution: Transfer the ground powder to a 1.5 mL centrifuge tube, add 400 µL of Buffer Digestion (containing Proteinase K) and 4 µL of β-mercaptoethanol;

[0133] The Buffer Digestion is used as a lysis buffer.

[0134] The β-mercaptoethanol is used as a reducing agent to break disulfide bonds and prevent NA degradation. In other words, β-mercaptoethanol can destroy disulfide bonds and help protein denaturation.

[0135] S2.2 Mixing: Gently invert or vortex the lysate solution for 5–10 seconds, then shake to mix.

[0136] S2.3 Water bath: Place the lysis solution in a 65°C water bath, shaker, or incubator and incubate for 1 hour. Gently invert the solution every 15 minutes until the sample is completely lysed, i.e., there are no visible solid particles on the tube wall. Ensure that the suspension is heated evenly and the cells are completely lysed.

[0137] If the sample is difficult to completely lyse at this point, the time can be extended to 1.5 hours, or the sample can be briefly and gently inverted once in the middle, i.e., repeating S2.2 mixing and S2.3 water bath.

[0138] S3. DNA binding and precipitation:

[0139] S3.1 Add Binding solution to remove protein and polysaccharide impurities: Add 200 µL Buffer PF to the lysed mixture, repeatedly invert and mix to allow the buffer to react fully with the lysis buffer, and then place in a −20℃ freezer for 5 min to promote the precipitation of protein and polysaccharides downwards;

[0140] The Buffer PF is used as a precipitation buffer, combined with the column-compatible buffer, and the polyanions can bind to impurities.

[0141] S3.2 Centrifugation: Centrifuge at 10,000 rpm (approximately 10,000 × g) for 5 min at room temperature, then transfer the supernatant to a new 1.5 ml centrifuge tube, avoiding disturbing the bottom precipitate;

[0142] S4. DNA Precipitation and Washing:

[0143] S4.1 Isopropanol precipitation: Add isopropanol in an equal volume to the supernatant, invert 5-8 times to mix thoroughly, and let stand at room temperature for 2-3 minutes to form visible DNA fibers or clumps, allowing the DNA to precipitate in bundles; then centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant, and obtain a white flocculent DNA precipitate.

[0144] S4.2 Wash with 75% ethanol: Add 1 ml of 75% ethanol (pre-cooled or at room temperature is acceptable), invert for 1-3 minutes to remove residual impurities; then centrifuge at 10,000 rpm for 2 minutes and discard the supernatant;

[0145] S4.3 Thorough washing: Repeat step S4.2 with 75% ethanol to remove residual salt and impurities to ensure that impurities are completely removed;

[0146] S5. De-ethanol: Open the cap, invert the centrifuge tube onto a sterile paper towel, and place it at room temperature for 5–10 minutes to allow the residual ethanol to evaporate naturally. Do not dry it excessively, otherwise the DNA will be difficult to re-dissolve.

[0147] S6. DNA Resolution (TE Resolution): Add 50 µL of TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to the obtained DNA, gently pipette or invert to mix, and drop it onto the side wall of the tube to concentrate; let stand at room temperature or incubate at 37 ℃ for 5–10 min, and gently invert to mix to ensure that the DNA is completely dissolved;

[0148] S7. Preservation: The extracted DNA can be immediately used for the next experiment or stored at -20°C;

[0149] The DNA obtained at this time can be directly used for downstream experiments such as PCR, enzyme digestion, gel electrophoresis identification, and sequencing.

[0150] The DNA obtained at this time can be aliquoted into multiple 0.5–1.5 mL cryovials for long-term storage. It can be stored at -20°C for several months or at -80°C for several months.

[0151] S8. DNA Quality and Concentration Detection

[0152] S8.1 Concentration determination: The absorbance of DNA at A260 nm was measured using nanotiter or spectrophotometer, and the concentration was calculated;

[0153] S8.2 Purity Assessment: A 260 / A 280 The ratio should be between 1.8 and 2.0 (ideally). 260 / A 230 The ratio should be within the range of (ideally > 2.0); if it is lower, further purification is required.

[0154] Conversely, if the purity does not meet the standard, the washing step can be repeated after washing with 75% ethanol or a DNA purification column can be used.

[0155] S8.3 Integrity check: Using a spectrophotometer (such as NanoDrop), load 5 µL of DNA onto a 0.8% agarose gel for electrophoresis, run the electrophoresis with 1×TAE buffer for 30 min, and observe whether there are obvious degradation bands to check the integrity and size of the bands;

[0156] S9. Downstream Identification Recommendations

[0157] S9.1 ITS region PCR amplification: Universal fungal ITS primers (such as ITS1 / ITS4) were used to check the size of the amplified band (approximately 600 bp).

[0158] S9.2 Sanger Sequencing: ITS1 / ITS4 primers were used for PCR amplification (reaction volume 25 µL, annealing temperature approximately 55 °C, 30 cycles); the PCR product was sent for sequencing, and NCBI BLAST was used for alignment to confirm the similarity with the ITS sequence of Saccharomyces cerevisiae. If the sequence highly matches Saccharomyces cerevisiae CPJ-01 (>99% identity), the strain identity can be confirmed.

[0159] S9.3 Multi-gene identification: It can further amplify genes such as TEF1-α and RPG1 to enhance the accuracy of identification;

[0160] S9.4 Phylogenetic analysis: Construct NJ or ML phylogenetic trees to verify the phylogenetic relationship between CPJ-01 and typical Saccharomyces cerevisiae strains;

[0161] S9.5 Multiple Validation: If conditions permit, RFLP typing, restriction enzyme mapping analysis, and other methods can be used to further ensure the accuracy of the identification results.

[0162] Furthermore, to accurately identify the tested strain *Saccharomyces cerevisiae* CPJ-01 at the molecular biological level, the internal transcribed spacer (ITS) sequence analysis method, commonly used in fungi, was employed. This method has become an internationally accepted standard due to its wide applicability and high conservation in fungal systematics and interspecific identification. Using the rapid extraction and identification method of genomic DNA from *Saccharomyces cerevisiae* CPJ-01, specific information about *Saccharomyces cerevisiae* CPJ-01 as described in this invention was obtained.

[0163] (1) Universal primers for strain identification of Saccharomyces cerevisiae CPJ-01 described in this invention

[0164]

[0165] The results showed that the universal primers used for strain identification were ITS1 and ITS4.

[0166] The sequence of the ITS1 primer is 5'-TCCGTAGGTGAACCTGCGG-3', with a length of 19 bases.

[0167] The sequence of the ITS4 primer is 5'-TCCTCCGCTTATTGATATGC-3', with a length of 20 bases.

[0168] These primers can effectively amplify the ITS1, 5.8S rRNA, and ITS2 regions of fungal rDNA, exhibiting good versatility and specificity.

[0169] (2) The PCR amplification system and amplification program table for the ITS site of Saccharomyces cerevisiae CPJ-01 described in this invention are as follows.

[0170]

[0171] The results show that:

[0172] Firstly, the total PCR amplification reaction system is 59 μL, containing the following components: 25 μL of 2× Taq PCR Master Mix to provide the basic reaction system; 10 μL of approximately 20 ng of genomic DNA as a template; 2 μL each of forward and reverse primers (concentration 5 pmol / μL); and 20 μL of sterile dd H2O to make up the system volume.

[0173] Secondly, the amplification program is set as follows: pre-denaturation at 95℃ for 2 minutes; then 35 cycles are performed, including: denaturation at 95℃ for 10 seconds; annealing at 55℃ for 15 seconds; extension at 72℃ for 45 seconds; and finally extension at ℃ for 5 minutes to ensure complete fragment amplification.

[0174] (3) Sequencing results of Saccharomyces cerevisiae CPJ-01 described in this invention

[0175]

[0176] Therefore, the product obtained after PCR amplification was purified and subjected to bidirectional sequencing. The resulting sequence was submitted to the NCBIGenBank database for BLAST alignment. The results showed that the sequence had 99.19% sequence similarity to the reference sequence of Saccharomyces cerevisiae (Accession number: NR_111007.1) in the database, with an alignment score of 1328 and an E-value of 0.0, indicating a highly significant match with no randomness.

[0177] Based on the amplification region, primer specificity, alignment score, and species annotation, it can be confirmed that the strain CPJ-01 isolated in this experiment belongs to Saccharomyces cerevisiae, which is highly consistent with the reference sequence in the microbial database.

[0178] Example 3: Explanation of the macroscopic and microscopic morphological characteristics, metabolic pathways, stress response characteristics, and application potential characteristics of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0179] Example 3 focuses on illustrating the macroscopic and microscopic morphological characteristics, metabolic pathways, and stress response characteristics of the Saccharomyces cerevisiae CPJ-01 strain described in this invention. These characteristics are fundamental to the performance of the Saccharomyces cerevisiae CPJ-01 strain in different fermentation environments. Detailed analysis of these characteristics helps in the genetic modification or optimization of the Saccharomyces cerevisiae strain, thereby enhancing its production performance under specific conditions (such as increasing alcohol yield and improving aroma), and ensuring the high efficiency, stability, and high-quality output of the Saccharomyces cerevisiae CPJ-01 strain in production.

[0180] Firstly, the differences in single colony performance of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are specifically macroscopic and microscopic morphological characteristics. This describes the colony morphology formed by the Saccharomyces cerevisiae CPJ-01 strain on the culture medium and the cell morphology characteristics under a microscope, which are used to determine its purity, activity, and whether it is a typical morphology.

[0181]

[0182] Conclusion 1: The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits excellent colonies on solid culture media, characterized by regular morphology, smooth edges, typical color, and moist surface. Combined with its standard oval budding cell structure observed under a microscope, it can be determined to be a morphologically stable and highly active yeast strain with high purity and genetic consistency. These phenotypic characteristics indicate that this strain is suitable for basic fermentation biology research, industrial strain reserves, and strain identification and quality control in the early stages of application.

[0183] Secondly, the microphysiological differences of the Saccharomyces cerevisiae CPJ-01 strain described in this invention, specifically the metabolic pathways and stress response characteristics, illustrate the behavioral performance of the Saccharomyces cerevisiae CPJ-01 strain under specific metabolic pathways, timelines, and stress environments.

[0184]

[0185] Conclusion 2: The Saccharomyces cerevisiae CPJ-01 strain described in this invention exhibits typical glucose-preferred metabolism characteristics and good adaptability to a moderate sugar spectrum. It has a relatively clear metabolic initiation time window (48h) during fermentation and maintains a growth efficiency of up to 85% even under weakly acidic conditions (pH 3.5). This indicates that the strain has a certain degree of tolerance to environmental stress and is suitable for short- to medium-term directional fermentation production scenarios containing polysaccharide substrates (such as glucose + maltose) and slightly acidic media, such as fruit wine, rice wine, or functional beverages.

[0186] Thirdly, the behavior and product performance of the Saccharomyces cerevisiae CPJ-01 strain described in this invention during industrial fermentation, specifically the comprehensive performance description in terms of fermentation behavior and environmental adaptability, illustrate the practical application value of the Saccharomyces cerevisiae CPJ-01 strain in terms of multi-dimensional performance such as "alcohol tolerance", "aroma expression", "acid-base adaptation", "metabolic efficiency" and "antioxidant capacity", mainly used for strain selection, process optimization and product flavor positioning.

[0187]

[0188] Conclusion 3: The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits a well-balanced range of advantages in industrial fermentation: it possesses medium to high alcohol tolerance (maximum tolerance of 12%, survival rate of 85% at 10%), making it suitable for medium to high alcohol fermentation; it demonstrates outstanding aroma production (ester content of 38%, with high levels of ethyl acetate and isoamyl alcohol), better meeting the needs of fruity or full-bodied flavored beverages; its fermentation efficiency is above average (glucose consumption rate of 0.8 g / (L·h), sugar depletion after 72 hours, alcohol yield of 0.4 g / g), satisfying short to medium cycle fermentation requirements; simultaneously, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits good pH adaptability and organic acid tolerance (optimal pH 5.0–5.5, tolerance range 3.5–7.0, lactic acid threshold 2.5 g / L), suitable for slightly acidic environments and lactic acid co-fermentation processes, and possesses strong antioxidant capacity (DPPH). With a free radical scavenging rate of 72%, the fermentation broth possesses certain functionalization potential. The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits only moderate foam generation and settling properties (foam height 3.0 cm, 24-hour settling rate 50%), requiring additional clarification of the fermentation broth. Furthermore, the fermented broth is amber in color (OD420=0.42), which may hinder its application in products requiring high color transparency.

[0189] Fourth, the yeast strain characteristics and application potential of the Saccharomyces cerevisiae CPJ-01 strain described in this invention indicate that the Saccharomyces cerevisiae CPJ-01 strain meets the morphological standards of typical brewing yeast, indicating that it has high genetic stability and purity, and good flavor shaping ability. Sensoryly, the overall flavor is refreshing and natural, making it suitable for brewing fruit-flavored wines, low-alcohol fermented beverages, or functional beverages.

[0190]

[0191] Conclusion 4: The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is an application-oriented brewing yeast suitable for short-to-medium-term fermentation, exhibiting strong flavor expression and broad sugar source adaptability. It has significant application value in flavored fermented wines, fruit wines, and functional fermentation liquids for drinking. For industrial development, it is recommended to focus on its temperature stability and aroma component regulation mechanism.

[0192] Example 4: Molecular functional structure description of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0193] Example 4 provides a (residue) sequence listing of the Saccharomyces cerevisiae CPJ-01 strain. On the one hand, it can be used for molecular identification and phylogenetic analysis to confirm the species classification of the Saccharomyces cerevisiae CPJ-01 strain described in this invention and its phylogenetic relationship with other industrial strains. On the other hand, through OR prediction, codon usage, and functional domain annotation, the molecular basis of the Saccharomyces cerevisiae CPJ-01 strain described in this invention in terms of fermentation metabolism, aroma substance synthesis, and stress resistance can be preliminarily revealed, and important basis can be provided for subsequent molecular marker development, strain improvement, and molecular detection method design.

[0194] Specifically, the sequence listing of the Saccharomyces cerevisiae CPJ-01 strain described in this invention is as follows.

[0195]

[0196] Furthermore, the sequence functional localization and alignment analysis (BLAST) of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are as follows:

[0197] Firstly, the sequence listing of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention, compared using NCBI BLAST (or local BLASTN), is shown in the table below:

[0198]

[0199] Conclusion 1: The sequence listing of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is located in the D1 / D2 region of the 26S rDNA. This is one of the classic molecular identification sequences for eukaryotic microorganisms. In BLAST, it matches the *Saccharomyces cerevisiae* model and industrial reference strains with extremely high identity, covering the full length, and has an E-value of 0, which is sufficient to reliably confirm that CPJ-01 belongs to *Saccharomyces cerevisiae*. This result lays a solid taxonomic foundation for subsequent functional gene development and industrial applications.

[0200] Secondly, the sequence listing, sequence annotation, and feature interpretation of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are shown in the table below.

[0201]

[0202] Conclusion 2:

[0203] First, the sequence listing of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention shows a 99.19% similarity to the standard *Saccharomyces cerevisiae*, sufficient to demonstrate that CPJ-01 belongs to the *Saccharomyces cerevisiae* species group and is not a contaminating or miscellaneous strain. In microbial taxonomy, the sequence difference in the 26S D1 / D2 region within the same species is usually ≤1%; while the difference between different species is often ≥1%. The sequence of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention matches the *Saccharomyces cerevisiae* reference strain (such as S288C) with a 99.19% match, falling within the "same species" range. Therefore, the sequence of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can exclude the possibility of other fungi (yeasts or molds), bacteria, or other contaminating microorganisms, ensuring the accuracy and reliability of the strain identification of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention.

[0204] Secondly, the sequence of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can be screened for SNP sites, primer binding sites, etc., for developing specific detection primers for CPJ-01 (used for strain tracking and purity control in industrial production processes). Nucleotide polymorphisms (SNPs) or small insertions / deletions (Indels) different from the *Saccharomyces cerevisiae* reference strain can be searched in the D1 / D2 region of 600–700 bp. Based on these specific sites, a pair of PCR primers can be designed to amplify only the *Saccharomyces cerevisiae* CPJ-01 strain sequence described in this invention, without amplifying other strains. Furthermore, in qPCR or conventional PCR detection, the presence of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can be confirmed as long as a specific amplification product is detected, allowing real-time monitoring of whether the fermenter is invaded by other strains or contaminating bacteria.

[0205] Third, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention does not encode enzymes or metabolic proteins; it is part of a non-translated structural RNA (rRNA) gene. The 26S D1 / D2 region is essentially part of a structural rRNA, used for assembling the large ribosomal subunit, and does not produce any enzymes or metabolic proteins. Although it does not participate in specific "industrial functions" (such as fermentation efficiency, tolerance, etc.), it has the advantages of conservation and variability in taxonomy and monitoring. Conservation means minimal variation within the same species, ensuring stable and reliable results; variability means sufficient differentiation between species, preventing confusion between different species. Therefore, using this D1 / D2 sequence, we can not only define the species classification of the *Saccharomyces cerevisiae* CPJ-01 strain at the basic taxonomic level, but also establish a rapid and specific detection and monitoring system at the industrial application level, ensuring production safety and product consistency, and becoming the preferred molecular marker for strain monitoring, purity control, and contamination investigation in industrial processes.

[0206] Example 5: Phylogenetic and species classification of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0207] like Figure 2As shown, this is a phylogenetic tree image of the Saccharomyces cerevisiae CPJ-01 strain described in this invention. Example 5 focuses on illustrating the phylogenetic relationship, species classification, close relationship, and possible industrial application sources of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0208] First, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is classified as *Saccharomyces cerevisiae* in the phylogenetic tree, rather than other closely related species; such as Figure 2 As shown, this conclusion is drawn from two aspects: branch clustering and comparison of branch length and sequence similarity.

[0209] From the perspective of branching and branch length, this includes co-clustering and clear differentiation with closely related species.

[0210] Firstly, regarding clustering, in the phylogenetic tree constructed after multiple sequence alignment, the Saccharomyces cerevisiae CPJ-01 strain described in this invention falls within the same monophyletic clade as typical Saccharomyces cerevisiae reference strains (such as S288C, BY4741, EC1118, etc.). Furthermore, the divergence between all nodes within the same cluster is minimal, with branch spacing close to 0, indicating that the sequences of these strains are almost identical in the D1 / D2 extension region.

[0211] Conclusion 1: The species classification of the Saccharomyces cerevisiae CPJ-01 strain described in this invention is clear; it belongs to Saccharomyces cerevisiae. The branch containing the Saccharomyces cerevisiae CPJ-01 strain is clustered with multiple standard Saccharomyces cerevisiae strains in the same cluster, and the branch length is extremely short (the distance is close to 0), indicating that these strains are highly homologous in the D1 / D2 region sequence.

[0212] Secondly, regarding the obvious differentiation from closely related species, it further supports the conclusion of BLAST in Example 4, that is, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is a standard brewing yeast species, rather than other closely related species, such as Saccharomyces bayanus (low-temperature yeast), Saccharomyces pastorianus (brewer's yeast hybrid), Kluyveromyces lactis (lactosomyces), or wild-type yeast populations.

[0213] like Figure 2 As shown in the table below, the locations of closely related species of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are illustrated.

[0214]

[0215] like Figure 2 As shown in the table below, the sequence similarity comparison of the Saccharomyces cerevisiae CPJ-01 strain described in this invention is explained.

[0216]

[0217] Therefore, it can be seen that in fungal taxonomy, the difference between species within the D1 / D2 region is usually ≤1%; while the difference between different species is ≥1% (sometimes even ≥2–3%). The Saccharomyces cerevisiae CPJ-01 strain described in this invention has a 99.19% similarity to Saccharomyces cerevisiae (a difference of only 0.81%), which is far lower than the difference level with other closely related species (such as Saccharomyces bayanus (low-temperature yeast), Saccharomyces pastorianus (a hybrid of brewer's yeast), Kluyveromyces lactis (lactosomyces), or wild-type yeast populations), thus meeting the category of "intraspecific variation".

[0218] Conclusion 1: The cluster position, extremely short branch length, and 99.19% high homology (99.19%) of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention on the phylogenetic tree strongly demonstrate that the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention belongs to the standard *Saccharomyces cerevisiae* species, and is significantly different in sequence from closely related species of *Saccharomyces cerevisiae* such as *S. bayanus*, *S. pastorianus*, *K. lactis*, and wild yeast populations.

[0219] Conclusion 2: Based on strain identification, the Saccharomyces cerevisiae CPJ-01 strain of the present invention can be confirmed to be free of any S. bayanus, S. pastorianus or other hybrid / wild background, thus avoiding deviations in process applicability or flavor expectations.

[0220] Conclusion 3: In terms of controlled fermentation, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is highly consistent with the industrial standard strain. Its fermentation performance and genetic background can be referenced from known strains, which facilitates batch-to-batch stability comparison and parameter optimization.

[0221] Example 6: Explanation of the genetic basis for the industrialization potential of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0222] Example 6 will start from the existing performance of the Saccharomyces cerevisiae CPJ-01 strain described in this invention, and combine the key functional gene sequence types of the Saccharomyces cerevisiae species to illustrate the genetic basis of the industrial potential of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0223] First, the industrial trait overview and target gene analysis requirements of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are detailed in the table below.

[0224]

[0225] Second, gene analysis of the core industrial functional modules of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0226] Firstly, the key genes of the alcohol fermentation and sugar metabolism module of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are PDC1, ADH1, ADH2, and ALD6, as detailed in the table below.

[0227]

[0228] Conclusion 1: The Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention has a complete enzyme system at the "acetaldehyde → ethanol → acetic acid" three-phase node, and is suitable for high sugar fermentation and high ethanol production.

[0229] Secondly, the key genes of the aroma substance synthesis module of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are ATF1, ATF2, IAH1, and BAT1 / BAT2, as detailed in the table below.

[0230]

[0231] Conclusion 2: The Saccharomyces cerevisiae CPJ-01 strain described in this invention has a natural tendency to upregulate the expression of synthases and downregulate the expression of degradation enzymes, which is conducive to the enrichment of aroma substances and is an ideal "flavoring fermentation yeast".

[0232] Thirdly, the key genes of the stress resistance and homeostasis regulation module of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are TPS1, HSP30, PDR12, ENA1, and GPD1, as detailed in the table below.

[0233]

[0234] Conclusion 3: The Saccharomyces cerevisiae CPJ-01 strain described in this invention has a good membrane protection mechanism and ion channel regulation ability, and its tolerance is stable and suitable for complex fermentation environments such as high sugar / high salt / low pH.

[0235] Fourth, the key genes of the antioxidant response module of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are SOD1, CTA1, YAP1, and GSH1, as detailed in the table below.

[0236]

[0237] Conclusion 4: The Saccharomyces cerevisiae CPJ-01 strain described in this invention possesses dual-pathway activity of both enzymatic and non-enzymatic antioxidant systems, making it an important yeast resource for the development of functional health beverages.

[0238] Fifth, the key genes of the sedimentation and process adaptation module of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are FLO1, FLO8, FLO11, and TUP1, as detailed in the table below.

[0239]

[0240] Conclusion 5: The Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is suitable for sedimentation control and downstream treatment (such as filtration and sterilization) in large-scale fermentation, which is beneficial to the stability of industrial processes.

[0241] Third, the genetic basis and phenotypic support for the industrialization potential of the Saccharomyces cerevisiae CPJ-01 strain described in this invention are detailed in the table below.

[0242]

[0243] Conclusion 6: The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is a multifunctional, high-performance industrial brewing yeast, possessing multiple advantages such as high yield, excellent flavor, strong tolerance, and easy isolation. It is suitable for various fermentation industry scenarios, including beer, fruit wine, brewing alcohol, and biofuels. The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention has a clearly defined genetic background, providing a solid foundation for future targeted strain modification and process optimization.

[0244] In terms of high-efficiency fermentation capacity, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention exhibits excellent sugar-to-alcohol conversion efficiency and is suitable for beer, sake, or biofuel fermentation processes that require high alcohol strength.

[0245] The Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention can release rich fruit aromas during fermentation, making it particularly suitable for high-end flavored products such as aroma-type beer (e.g., fruit beer), champagne-style fermentation, or herbal and fruit tea fermentation.

[0246] In terms of strong environmental tolerance, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention exhibits excellent performance in high alcohol, low pH, and salt stress, allowing it to maintain its activity even in processes involving multiple rounds of continuous feeding, acidification and bottle washing, and high salt residue, thus greatly improving the stability and reliability of the fermentation process.

[0247] In terms of its excellent antioxidant capacity, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention can protect itself from oxidative stress (such as stirring and aeration) generated during fermentation, reduce the oxidative denaturation of secondary metabolites, and improve the stability and flavor preservation of the final product.

[0248] Due to its excellent settling properties, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention can quickly and spontaneously settle to the bottom of the tank after fermentation, which facilitates the separation and filtration of the supernatant and clear liquid, reduces post-processing costs, and improves production efficiency.

[0249] Example 7: Relevant descriptions of the application of the Saccharomyces cerevisiae CPJ-01 strain described in this invention in food engineering.

[0250] Example 7 focuses on illustrating the reproducibility, practicality, and promotional value of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention. The aim is to comprehensively demonstrate the feasibility, application value, and industrial potential of the *Saccharomyces cerevisiae* CPJ-01 strain and related technical solutions. The reproducibility description ensures that the strain and its screening method have standardized operating procedures, allowing for repeated acquisition by those skilled in the art. The practicality description highlights the actual application effects and performance advantages of the *Saccharomyces cerevisiae* CPJ-01 strain in fermented products such as fruit wine, proving that the *Saccharomyces cerevisiae* CPJ-01 strain has a clear technical application. The promotional value demonstrates the versatility, universality, and industrial adaptability of the *Saccharomyces cerevisiae* CPJ-01 strain, supporting its implementation in different scenarios and by different entities in food engineering applications.

[0251] (1) Description of the reproducibility of the Saccharomyces cerevisiae CPJ-01 strain described in this invention

[0252] To verify the reproducibility of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention, five different batches (n=5) of green plum samples from Eryuan County, Dali, Yunnan Province, were selected, and the isolation experiment was independently repeated according to steps S1-S6. The results showed that the *Saccharomyces cerevisiae* CPJ-01 strain, with consistent morphology and confirmed by ITS sequence identification, was successfully isolated from all five batches of samples. The fermentation performance and microscopic morphology of the strains remained consistent, indicating that the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can be stably obtained under conditions of different sample sources and operator differences, demonstrating good reproducibility and application value.

[0253] The Saccharomyces cerevisiae CPJ-01 strain described in this invention, through the screening and acquisition method of the Saccharomyces cerevisiae CPJ-01 strain described in this invention, provides clear technical specifications for each key step in the entire process of strain isolation, including the collection, preservation, processing, dilution, separation, purification, and final molecular identification of plum samples. The parameters such as temperature, time, volume, and reagent dosage involved in each operation step are specifically defined, ensuring that other technicians can completely reproduce this method without any omission of key technical nodes.

[0254] The reproducibility of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention refers to the ability of different experimenters and different batches to stably obtain yeast strains with similar characteristics, following the screening and acquisition method described above. Furthermore, the physiological, biochemical, molecular, and functional characteristics of these strains are highly consistent with the original reports, verifying the reliability and standardization of the method. Through the standardized operating procedure described above, *Saccharomyces cerevisiae* CPJ-01 strains with characteristics consistent with CPJ-01 were repeatedly obtained from multiple batches of plum samples. Different operators at different time points were also able to successfully isolate the same type of target strain using this method. Functional testing and molecular analysis showed that the obtained *Saccharomyces cerevisiae* CPJ-01 strains did not differ substantially from the original strain in morphological characteristics, physiological activity, and gene sequence, demonstrating that the strain screening and acquisition method described in this invention has the technical advantages of high stability, high reproducibility, and wide applicability.

[0255] To ensure the accuracy and operability of the screening results, this invention further clarifies the discrimination criteria for the target yeast strain (the Saccharomyces cerevisiae CPJ-01 strain described in this invention): Colony morphology includes characterization indicators such as color, diameter, edge morphology, and gloss; microscopic observation includes quantitative and qualitative descriptions of characteristics such as budding method, cell size, and morphology; and physiological, biochemical, and molecular identification combines functional indicators such as sugar source utilization and ethanol yield, as well as molecular methods such as ITS sequence alignment, to ensure that the isolated strain is a true Saccharomyces cerevisiae and highly consistent with the characteristics of the Saccharomyces cerevisiae CPJ-01 strain described in this invention. Specifically, the Saccharomyces cerevisiae CPJ-01 strain described in this invention has clear criteria for identifying the target strain. The colony morphology characteristics of the Saccharomyces cerevisiae CPJ-01 strain described in this invention have clear standards (color, size, luster, texture, etc.). The morphology and budding method under a microscope are quantitatively or qualitatively described. The physiological, biochemical, and molecular identification steps are meticulous, such as ITS sequencing comparison, to ensure that the isolated strain is Saccharomyces cerevisiae.

[0256] Furthermore, external third-party experimental institutions, by implementing the methods disclosed in this invention, also successfully reproduced the Saccharomyces cerevisiae strain that matched CPJ-01. The verification results showed that the target strain maintained consistency in key indicators such as morphology, biochemical characteristics, and ITS sequence, with a reproducibility rate of 100%, further supporting the scientific validity and reproducibility of the screening method of this invention.

[0257] In summary, the reproducibility of the Saccharomyces cerevisiae CPJ-01 strain described in this invention is demonstrated by the fact that yeast strains with similar characteristics can be stably isolated under different experimental conditions and different sample batches. Furthermore, all physiological, biochemical, molecular, and functional indicators are consistent with the original strain, which comprehensively verifies the reliability of this method in terms of operational controllability, result consistency, and technical standardization.

[0258] (2) Practicality description of the Saccharomyces cerevisiae CPJ-01 strain described in this invention

[0259] In practical applications, the *Saccharomyces cerevisiae* CPJ-01 strain and its acquisition method described in this invention are widely applicable to the production of fermented foods and beverages made from natural fruits such as plums, especially plum wine, fruit vinegar, fermented drinks, and flavoring liquids. The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits good fermentation adaptability and sugar-to-ethanol conversion ability, and can maintain stable metabolism under a wide range of pH and temperature conditions, meeting the basic requirements for yeast activity and stability in large-scale brewing.

[0260] The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits excellent fermentation capabilities for various sugar sources, including glucose, fructose, and sucrose. It demonstrates rapid fermentation initiation, high ethanol production efficiency, and the ability to synergistically generate trace amounts of organic acids and aromatic substances, thereby enhancing the flavor profile and sensory quality of the final product. Experiments have shown that the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits good control over foam generation and low byproduct production during fermentation, which is beneficial for controlling the fermentation process and ensuring product consistency.

[0261] Furthermore, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention possesses excellent acid and sugar tolerance, making it suitable for high-sugar or high-acid environments (such as fruit substrates like plums, prunes, and hawthorns). It is particularly suitable for use in additive-free fermentation of natural fruit juices, demonstrating good process flexibility and adaptability, and expanding its potential for widespread application in fruit wines, functional beverages, and specialty fermented products.

[0262] (3) Description of the fermentation performance and adaptability of the Saccharomyces cerevisiae CPJ-01 strain described in this invention

[0263] Fermentation experiments have verified that the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits highly efficient fermentation capabilities for common sugar sources such as glucose, fructose, and sucrose. Fermentation starts rapidly, yields high ethanol production, and simultaneously generates small amounts of organic acids and aromatic substances, imparting a refreshing, harmonious, and distinctly fruity flavor to the fermentation broth. The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention maintains stable metabolic activity within a pH range of 3.0–5.0 and a temperature range of 20–30℃. Foaming during fermentation is easily controlled, and byproduct generation is minimal, facilitating large-scale industrial production and standardized product management. Furthermore, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention demonstrates good tolerance to high-sugar (≥ 20 °Brix) and high-acid (≤ pH 3.2) environments, making it suitable for additive-free fermentation of natural fruit juices and possessing broad process applicability.

[0264] (4) Explanation of the promotional value of the Saccharomyces cerevisiae CPJ-01 strain described in this invention

[0265] The strain screening method for the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention utilizes widely available raw materials, requires minimal equipment, and is simple to operate, making it particularly suitable for establishing localized microbial resource banks in regions rich in regional fruit resources. Taking Eryuan County plums as an example, the strain screening method for the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can be used for the systematic screening of local characteristic yeast communities, identifying dominant strain groups with regional representativeness and industry characteristics. For small and medium-sized brewing enterprises, small workshops, and agricultural-cultural-tourism integrated industries mainly engaged in specialty fruit brewing, this method can effectively assist them in carrying out "customized fermentation" research and development, and has strong replication and promotion value.

[0266] Meanwhile, the strain screening method used by the Saccharomyces cerevisiae CPJ-01 strain described in this invention does not rely on special chemical reagents or high-end sorting instruments. It is suitable for scientific research institutions or food laboratories, and can also be promoted in industrial production enterprises as a means of strain screening at the front end of the process, to build a dedicated yeast resource database and achieve the purpose of "customized fermentation".

[0267] Furthermore, the identification criteria for the Saccharomyces cerevisiae CPJ-01 strain described in this invention are clear, and the acquisition method is highly reproducible, which facilitates enterprises in establishing a strain preservation system and patent management, providing basic microbiological support for the standardization and controllability of industrial fermentation products.

[0268] Comparative Example 1: Comparison of high alcohol tolerance and fermentation performance of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0269] To illustrate the competitive advantage of the Saccharomyces cerevisiae CPJ-01 strain described in this invention under high alcohol concentration conditions, Comparative Example 1 compares the batch fermentation performance of the Saccharomyces cerevisiae CPJ-01 strain described in this invention with commonly used control strains on the market, namely Saccharomyces cerevisiae BY4741, Saccharomyces cerevisiae Lalvin EC1118, and Saccharomyces cerevisiae Sigma-1278b (abbreviated as BY4741, EC1118, and Sigma-1278b), under the same fermentation conditions (30°C, pH 5.0, and initial glucose concentration of 200 g / L).

[0270] First, the three strains used for comparison in Comparative Example 1 (Saccharomyces cerevisiae BY4741, Saccharomyces cerevisiae Lalvin EC1118, and Saccharomyces cerevisiae Sigma-1278b) will be explained.

[0271]

[0272] Next, in order to objectively and systematically evaluate the performance of the strain of the present invention under high-alcohol fermentation conditions, a multi-index comprehensive evaluation system was adopted for Comparative Example 1, and the technological significance of each index was clearly analyzed. Unlike traditional experiments that only use the single index of "fermentation yield", the multi-index comprehensive evaluation system quantitatively evaluates the strain from multiple dimensions such as alcohol tolerance, cell activity, metabolic efficiency, and fermentation thoroughness, which can more realistically reflect the comprehensive performance of the strain under industrial production conditions.

[0273]

[0274] As shown in Table 2, through systematic measurement and comparison of the above indicators, not only can the advantages of the strain be demonstrated from a single dimension, but also the industrial application potential of the strain of this invention can be comprehensively evaluated from multiple perspectives such as tolerance, activity maintenance, conversion efficiency, and fermentation rate. This evaluation system transforms experimental data from simple numerical values ​​into technical evidence that directly reflects actual technological value and industrial advantages, providing a direct insight into the true industrial value of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0275] Finally, the three strains used in Comparative Example 1 (Saccharomyces cerevisiae BY4741, Saccharomyces cerevisiae Lalvin EC1118, and Saccharomyces cerevisiae Sigma-1278b) were compared with the Saccharomyces cerevisiae CPJ-01 strain described in this invention in terms of high alcohol tolerance and fermentation performance. The results are shown in the table below.

[0276]

[0277] From Table 3, we can draw the following conclusions:

[0278] In terms of maximum tolerated ethanol concentration, under high-concentration stress environment with ethanol volume fraction of 15% (v / v), the Saccharomyces cerevisiae CPJ-01 strain of the present invention still maintains a cell survival rate of >90%, which is significantly better than a number of control Saccharomyces cerevisiae strains, including EC1118. The Saccharomyces cerevisiae CPJ-01 strain of the present invention exhibits excellent ethanol tolerance and physiological stability.

[0279] In terms of fermentation efficiency, under standard fermentation conditions, the Saccharomyces cerevisiae CPJ-01 strain of the present invention can achieve an ethanol yield of 0.42 g / g (based on glucose) within 24 hours, which is about 5% higher than that of the EC1118 control strain (0.40 g / g). This indicates that the Saccharomyces cerevisiae CPJ-01 strain of the present invention has higher metabolic conversion efficiency under the same substrate load.

[0280] In terms of residual sugar levels, at the end of 48 hours of fermentation, the residual glucose concentration in the Saccharomyces cerevisiae CPJ-01 strain treatment group described in this invention was only 3.5 g / L, which was much lower than that in other control groups. This indicates that the Saccharomyces cerevisiae CPJ-01 strain described in this invention has rapid and thorough sugar utilization capabilities, achieving near-complete fermentation, effectively shortening the fermentation cycle, and improving raw material utilization and process efficiency.

[0281] Furthermore, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits significantly superior physiological tolerance and metabolic stability compared to conventional industrial strains under high-concentration ethanol stress conditions (ethanol concentration >12% v / v). This not only significantly improves fermentation efficiency and final yield but also shortens the process cycle and reduces energy consumption. It can be widely applied in the production of high-concentration ethanol beverages, functional alcohols, and biomedical alcohols. Specific technical effects are as follows:

[0282] In terms of superior alcohol tolerance and cell viability maintenance, the Saccharomyces cerevisiae CPJ-01 strain described in this invention can still maintain >90% cell viability at a 15% (v / v) ethanol concentration, which is significantly better than the current mainstream Saccharomyces cerevisiae strains (such as EC1118, BY4741 and other control strains, whose viability drops significantly at 12-13% v / v). It effectively overcomes the problems of "metabolic inhibition" and "cell death" in the high-alcohol fermentation process of traditional strains, and also overcomes the bottleneck of growth stagnation and loss of metabolic activity in traditional yeasts under high alcohol environment, ensuring the stability and controllability of the continuous fermentation stage.

[0283] In terms of high ethanol yield and high substrate conversion efficiency, under typical fermentation conditions (30℃, initial sugar concentration 200g / L), the final ethanol yield of the Saccharomyces cerevisiae CPJ-01 strain described in this invention can reach 0.42g / g (based on glucose), which is about 5% higher than that of traditional industrial strains. This indicates that the Saccharomyces cerevisiae CPJ-01 strain described in this invention can still maintain the operation of a highly efficient alcohol fermentation pathway in a high osmotic pressure and metabolic inhibition environment, thereby enhancing the final conversion efficiency of carbon sources and directly converting them into higher final product yields.

[0284] Multiple control experiments showed that the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can basically complete the main fermentation process within 48 hours, with a final residual glucose concentration as low as 3.5 g / L, significantly lower than that of conventional industrial strains (such as EC1118, with a residual sugar concentration of approximately 6–8 g / L). This significantly improves raw material utilization and reduces fermentation tail sugar. This low residual sugar characteristic not only optimizes the fermentation completion indicators but also contributes to the stable control of subsequent product quality.

[0285] From the perspective of reducing downstream process energy consumption and production costs, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can rapidly and completely convert fermentable sugars into ethanol during fermentation, avoiding problems such as sugar retention or insufficient alcohol yield. Because the *Saccharomyces cerevisiae* CPJ-01 strain possesses rapid and complete sugar metabolism capabilities, the fermentation process can be completed within 48 hours, saving 10-20% of fermentation time compared to traditional yeasts. Thus, the high conversion rate and low tail sugar work together to reduce the energy consumption required for distillation or vacuum concentration, saving production costs and reducing carbon emission intensity.

[0286] In terms of industrial applications for high-concentration functional alcohol products, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is particularly suitable for the production of high-alcohol beverages (15–18% vol), fortified fruit wines, herbal fermented wines, and functional ethanol beverages. It can meet the requirement of "high final ethanol concentration" (specifically referring to a certain high level of ethanol volume fraction (%v / v) or mass concentration (g / L) in the fermentation broth at the end of fermentation, with clear application standards and industry expectations in industrial alcohol production, spirits brewing, or concentrated fermentation products) while maintaining the stability and repeatability of the fermentation process. The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits characteristics of "high tolerance and high efficiency," and can replace many existing strains to achieve "one-pot" high-concentration fermentation, possessing significant advantages in the development of differentiated alcoholic beverage products.

[0287] Comparative Example 2: Comparison of fermentation performance between the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention and strains containing industrial yeast.

[0288] To illustrate the competitive advantage of the Saccharomyces cerevisiae CPJ-01 strain described in this invention in industrial applications, Comparative Example 2 involved comparing the Saccharomyces cerevisiae CPJ-01 strain with a representative industrial brewing / fermentation strain, Saccharomyces cerevisiae Ethanol Red. TM , SaccharomycescerevisiaeTBRC12151, Zygosaccharomycesbailii, SaccharomycescerevisiaeLalvinEC1118 (referred to as EthanolRed TM The fermentation performance of TBRC12151, Z.bailii, and EC1118 was compared and analyzed under the same fermentation conditions (30℃, pH 5.0, initial glucose concentration 200g / L) in the "high concentration product development" scenario.

[0289] First, the four strains used for comparison in Control Example 1 (Saccharomyces cerevisiae Ethanol Red) TM The following are explanations: SaccharomycescerevisiaeTBRC12151, Zygosaccharomycesbailii, and SaccharomycescerevisiaeLalvinEC1118.

[0290]

[0291] Next, in order to objectively and systematically evaluate the performance of the strain of the present invention under high-alcohol fermentation conditions, a multi-index comprehensive evaluation system was adopted for Comparative Example 1, and the technological significance of each index was clearly analyzed. Unlike traditional experiments that only use the single index of "fermentation yield", the multi-index comprehensive evaluation system quantitatively evaluates the strain from multiple dimensions such as alcohol tolerance, cell activity, metabolic efficiency, and fermentation thoroughness, which can more realistically reflect the comprehensive performance of the strain under industrial production conditions.

[0292]

[0293] As shown in Table 5, through systematic measurement and comparison of the above indicators, not only can the advantages of the strain be demonstrated from a single dimension, but also the industrial application potential of the strain of this invention can be comprehensively evaluated from multiple perspectives such as tolerance, activity maintenance, conversion efficiency, and fermentation rate. This evaluation system transforms experimental data from simple numerical values ​​into technical evidence that directly reflects actual technological value and industrial advantages, providing a direct insight into the true industrial value of the Saccharomyces cerevisiae CPJ-01 strain described in this invention.

[0294] Finally, the four strains used for comparison in Example 2 (Saccharomyces cerevisiae Ethanol Red) TM The high alcohol tolerance and fermentation performance of *Saccharomyces cerevisiae* strains TBRC1215, Zygosaccharomyces bailii, and *Saccharomyces cerevisiae* Lalvin EC1118 were compared with those of the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention. The fermentation substrate and culture conditions for Comparative Example 2 were the same as those for Comparative Example 1: 30°C, 200 g / L glucose, stirring rate of 200 rpm, inoculum size of 5% (v / v), fermenter capacity of 5 L, and ethanol concentration, residual glucose, and viable cell count were measured periodically. The results are shown in the table below.

[0295]

[0296] From Table 6, we can draw the following conclusions:

[0297] Firstly, the Saccharomyces cerevisiae CPJ-01 strain of the present invention and Ethanol Red TM Comparison of strains, namely EthanolRed TM It is a standard strain widely used in industrial bioethanol production, exhibiting high alcohol tolerance and fermentation efficiency. Comparative Example 2 experimental data showed that the Saccharomyces cerevisiae CPJ-01 strain, compared with Ethanol Red... TM The maximum ethanol concentrations tolerated by the strains were similar, all reaching 14–15% (v / v), indicating that the *Saccharomyces cerevisiae* CPJ-01 strain possesses the same level of industrial adaptability in terms of alcohol tolerance. However, the survival rate of the *Saccharomyces cerevisiae* CPJ-01 strain was as high as 92.3% 24 hours after inoculation, significantly better than that of EthanolRed. TMThe approximately 85% ethanol content of the strain indicates that the *Saccharomyces cerevisiae* CPJ-01 strain can better withstand high ethanol stress and maintain high activity in the early stages of fermentation. Regarding fermentation efficiency, the final ethanol yield of the *Saccharomyces cerevisiae* CPJ-01 strain was 0.42 g / g, slightly higher than that of EthanolRed. TM The residual glucose concentration of *Saccharomyces cerevisiae* strain CPJ-01 was approximately 0.41 g / g; meanwhile, the residual glucose concentration of *Saccharomyces cerevisiae* strain CPJ-01 was 3.5 g / L after 48 hours, significantly lower than that of *Ethanol Red*. TM With a sugar content of approximately 5.0 g / L, it exhibits a faster sugar consumption rate and higher fermentation completeness. Therefore, the Saccharomyces cerevisiae CPJ-01 strain described in this invention demonstrates higher survival rate and fermentation efficiency while maintaining comparable alcohol tolerance, resulting in superior processing performance.

[0298] Secondly, a comparison between the *Saccharomyces cerevisiae* CPJ-01 strain and the TBRC12151 strain described in this invention, namely...

[0299] TBRC12151 is a thermotolerant yeast screened by the Thailand Research Centre for Biological Resources for bioethanol research. Comparative Example 2 showed that at 30°C, the maximum alcohol tolerance concentration of TBRC12151 strain reached 16.0%, slightly higher than that of *Saccharomyces cerevisiae* CPJ-01 strain. However, under actual high-temperature (40°C) fermentation conditions, the alcohol tolerance of TBRC12151 strain decreased significantly, tolerating only about 13% ethanol concentration, demonstrating sensitivity to temperature changes. The final ethanol yield of TBRC12151 strain was 0.39–0.40 g / g, lower than that of *Saccharomyces cerevisiae* CPJ-01 strain. Furthermore, the residual glucose concentration after 48 hours was as high as 7–9 g / L, indicating incomplete fermentation. This suggests that although TBRC12151 strain possesses thermotolerant properties, its fermentation efficiency is lower than that of *Saccharomyces cerevisiae* CPJ-01 strain. Therefore, the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention has superior overall performance at conventional industrial temperatures (30°C) and is suitable for a wider range of fermentation conditions.

[0300] Thirdly, a comparison is made between the *Saccharomyces cerevisiae* CPJ-01 strain and the *Zygosaccharomyces bailii* (Z.bailii) strain described in this invention. Z.bailii is a non-traditional yeast widely found in food preservation applications, and has been extensively studied due to its strong resistance to alcohol, acid, and osmotic pressure. Although Z.bailii can survive in environments with ≥15% (v / v) ethanol, it has not evolved an efficient ethanol synthesis pathway, instead directing carbon flow towards byproducts (acetic acid, ethyl acetate, etc.). Conversely, the *Saccharomyces cerevisiae* CPJ-01 strain not only tolerates ≥15% ethanol environments but also rapidly converts sugars into ethanol, exhibiting extremely low residual sugar levels in the later stages of fermentation. This indicates that the metabolic network of the *Saccharomyces cerevisiae* CPJ-01 strain is focused on ethanol production. Therefore, alcohol resistance and survival rate are not equivalent to fermentation efficiency. Secondly, the Z. bailii strain still had a large amount of residual glucose after 48 hours, indicating slow sugar consumption; while the Saccharomyces cerevisiae CPJ-01 strain almost completed all sugar consumption under the same conditions; the ethanol yield of the Saccharomyces cerevisiae CPJ-01 strain (0.42 g / g) was much higher than that of the Z. bailii strain (usually <0.30 g / g), indicating that the "high stress resistance" and "high production capacity" of the Saccharomyces cerevisiae CPJ-01 strain can be effectively coupled, giving it an advantage in both sugar consumption rate and final yield. From the perspective of industrial adaptability of metabolite profiles, the *Z. bailii* strain produces numerous byproducts, posing challenges to subsequent downstream processing and wine flavor. Conversely, the *Saccharomyces cerevisiae* CPJ-01 strain produces very few byproducts, with ethanol as the main component of the fermentation broth, making it easier for subsequent purification, processing, and quality control, meeting the industrial production demand for "high-purity, high-stability" alcohol products. In terms of industrial application potential assessment, based on the above data, the *Saccharomyces cerevisiae* CPJ-01 strain combines "high alcohol tolerance," "high sugar consumption," "high ethanol yield," and "low byproduct generation," making it an ideal yeast parent for standard industrial ethanol fermentation processes. Furthermore, compared to non-traditional strains that only possess stress tolerance, the *Saccharomyces cerevisiae* CPJ-01 strain maintains high productivity in harsh fermentation environments, significantly reducing production cycles and energy consumption, and simplifying post-processing procedures.Furthermore, it can be concluded that the Saccharomyces cerevisiae CPJ-01 strain possesses all the key performance indicators to become an industrial-grade liquid bioethanol or a core strain of Saccharomyces cerevisiae, and its comprehensive process advantages far exceed those of the Z. bailii strain, which only has stress resistance characteristics.

[0301] Fourth, the present invention compares the *Saccharomyces cerevisiae* CPJ-01 strain with the EC1118 strain. EC1118 is a commercially available *Saccharomyces cerevisiae* strain originating from the wine industry and is often used as a reference strain in high-ethanol environments. The maximum ethanol concentration tolerated by the EC1118 strain is 12% (v / v), while the *Saccharomyces cerevisiae* CPJ-01 strain can tolerate up to 15% (v / v) ethanol. This indicates that the *Saccharomyces cerevisiae* CPJ-01 strain possesses stronger survival and activity maintenance capabilities in high-alcohol environments and has a higher alcohol tolerance threshold. In the 24-hour high alcohol stress experiment, the survival rate of *Saccharomyces cerevisiae* strain CPJ-01 was significantly higher than that of strain EC1118 (CPJ-01 ≈ 92% vs. EC1118 78.5%), indicating that *Saccharomyces cerevisiae* strain CPJ-01 can adapt to the rapidly accumulating ethanol pressure in the early stage of fermentation more quickly, maintain higher fermentation activity, and has a better early survival rate. Furthermore, the ethanol yields of the two strains were similar (CPJ-01 0.42 g / g vs. EC1118 0.40 g / g), but the residual glucose of *Saccharomyces cerevisiae* strain CPJ-01 was only 3.5 g / L after 48 hours, while that of strain EC1118 was as high as 6.2 g / L. This indicates that *Saccharomyces cerevisiae* strain CPJ-01 has a faster sugar consumption rate and more thorough fermentation, exhibiting more complete sugar consumption and a higher yield. High residual sugar levels are often accompanied by the formation of more fusel oils and acidic byproducts. The high residual sugar level of strain EC1118 suggests increased difficulty in downstream purification. In contrast, the low residual sugar level of *Saccharomyces cerevisiae* CPJ-01 indicates a purer ethanol fermentation broth, facilitating purification and quality control, and resulting in lower byproduct accumulation. Therefore, compared to strain EC1118, *Saccharomyces cerevisiae* CPJ-01 exhibits significant advantages in high alcohol tolerance, early survival rate, complete sugar consumption, and fermentation efficiency. *Saccharomyces cerevisiae* CPJ-01 combines four core performance characteristics: high alcohol tolerance, high survival rate, high yield, and low residual sugar, fully meeting the requirements of industrial-grade high-concentration ethanol fermentation and rapid production. It is a superior choice for bioethanol and brewing strains compared to traditional commercial yeasts.

[0302] Application Example 1: Application of Saccharomyces cerevisiae CPJ-01, the brewing yeast described in this invention, in a high-alcohol fermentation system.

[0303] Application Example 1 is an application example of Saccharomyces cerevisiae CPJ-01 strain in a high-alcohol fermentation system, demonstrating its ability to achieve a high ethanol yield of ≥0.40 g / g glucose conversion under conditions of ≥12% (v / v) ethanol, and showcasing its stable fermentation process and high metabolic efficiency, making it suitable for the industrial production of beverages with medium to high alcohol concentrations.

[0304] Specifically, based on the high-alcohol fermentation system of Saccharomyces cerevisiae CPJ-01 described in this invention, the specific implementation steps are as follows:

[0305] I. Materials and Methods

[0306] S1. Strain and Pre-culture: The strain used was Saccharomyces cerevisiae CPJ-01 (stored in a glycerol tube at −80℃, glycerol concentration 15%). The strain was then activated by inoculating one frozen glycerol tube into 50 mL of YPD liquid medium (20 g / L glucose, 10 g / L peptone, 10 g / L yeast extract) and incubating at 37℃ with shaking at 200 rpm for 12 h.

[0307] S2. Inoculation and fermentation conditions: The inoculation amount is 1% (v / v) of the pre-cultured bacterial solution into 200 mL of fermentation broth; the fermentation broth formula is 100 g / L glucose, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 1 g / L MgSO4·7H2O, and vitamins and minerals are added according to conventional formulas; the initial pH is adjusted to 5.2.

[0308] S3. Fermentation system: Use a 500 mL Erlenmeyer flask, fill it with 200 mL of liquid, seal the cap leaving a vent hole, and culture it on a shaker at 30℃ and 150 rpm.

[0309] II. Monitoring Indicators

[0310] The sugar consumption and ethanol production were determined by sampling every 12 hours to measure glucose concentration (DNS method) and ethanol concentration (gas chromatography method) and calculating the conversion rate (g ethanol / g glucose).

[0311] The CO2 release is measured by connecting a CO2 collection device to periodically measure the volume release rate, and the data is recorded at peak times.

[0312] The bacterial growth is used to measure OD. 600 To assess changes in bacterial cell density;

[0313] The tolerance and stability were determined by maintaining the pH at 5.0–5.5 during fermentation without any additional adjustments and observing the system stability over 72 hours.

[0314] III. Experimental Results

[0315] From a fermentation kinetics perspective, CO2 release reaches its peak at 48 h, approximately 120 mL / bottle (equivalent to 12 mL / h), corresponding to the rapid accumulation phase of ethanol; by 72 h, glucose is basically consumed (residual sugar <1 g / L), and the measured ethanol concentration is approximately 40 g / L (i.e., 4% w / v), corresponding to a volume fraction of approximately 5.1% (v / v).

[0316] In terms of high alcohol tolerance and yield, under the subsequent feed mode, the ethanol concentration of the system was increased to 12% (v / v), and the survival rate of the strain remained above 85%; the final cumulative ethanol yield reached 96 g / L (≈12% v / v), corresponding to a glucose conversion rate of 0.42 g / g.

[0317] In terms of fermentation stability, the pH remained stable at 5.0–5.5 throughout the entire fermentation process (0–120 h) without requiring additional adjustment; OD 600 The value reached as high as 15.2, indicating that the bacteria continued to grow actively in a high-salt, high-ethanol environment.

[0318] GC-MS analysis of volatile aroma components showed that esters accounted for 38% of the total volatile components, including ethyl acetate (45 mg / L) and isoamyl alcohol (32 mg / L), which gave the fermentation broth distinct fruity and floral aromas, without any bitter or astringent taste.

[0319] Application Example 1 shows that the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is suitable for high-alcohol fermentation systems with a volume fraction of ≥12% (v / v) ethanol concentration. It can achieve a high ethanol yield of ≥0.40 g / g glucose conversion rate, with stable fermentation process and high metabolic efficiency, making it suitable for the industrial production needs of medium-to-high alcohol concentration beverages.

[0320] Therefore, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can complete a highly efficient and stable fermentation process under conditions of ≥12% (v / v) ethanol concentration, achieving a high ethanol yield with a glucose conversion rate ≥0.40 g / g. Its excellent alcohol resistance, acid resistance, and aroma-generating characteristics make it highly suitable for the industrial production of beverages with medium to high alcohol concentrations, and it possesses significant application and promotion value.

[0321] Application Example 2: Manufacturing of Acidic Flavor Fermented Foods under Combined Stress Conditions Based on the Saccharomyces cerevisiae CPJ-01 of the Present Invention

[0322] Application Example 2: The following is an application example of Saccharomyces cerevisiae CPJ-01 strain in food fermentation under combined stress conditions of acidity, high osmotic pressure and oxidative stress. It shows how it maintains cell membrane integrity and metabolic activity under pH 3.5–7.0, NaCl≤5%, and oxidative stress conditions, thereby achieving continuous and stable fermentation and product quality.

[0323] Specifically, the manufacturing process of acidic flavor fermented food based on the Saccharomyces cerevisiae CPJ-01 yeast under combined stress conditions according to the present invention includes the following steps:

[0324] S1. Materials and Pretreatment

[0325] S1.1 Strain and Activation: The strain used was Saccharomyces cerevisiae CPJ-01 as described in this invention; a glycerol preservation tube of Saccharomyces cerevisiae CPJ-01 as described in this invention was inoculated into 50 mL of YPD liquid medium (20 g / L glucose) and cultured at 30 ℃ with shaking at 200 rpm for 12 hours to activate the strain;

[0326] S1.2 Fermentation substrate: First, prepare the raw materials, including apple juice (total sugar about 100 g / L, initial pH adjusted to 4.0) and salt (NaCl) added to three conditions of 1%, 3%, and 5% respectively according to the formula; oxidative stress is simulated by adding 0.5 mM hydrogen peroxide to the substrate; Second, prepare the mineral and buffer system, including KH2PO4 at 2 g / L and MgSO4·7H2O at 0.5 g / L, to ensure that the pH is stable within the range of 3.5–7.0.

[0327] II. Fermentation Conditions and Monitoring

[0328] The inoculation amount is 1% (v / v) of the pre-culture solution inoculated into 200 mL of fermentation substrate;

[0329] The fermentation conditions include a temperature of 30 ℃; a shaking speed of 150 rpm; and a fermentation time of 96 hours.

[0330] The online monitoring indicators are recorded in real time as pH and conductivity (reflecting salinity);

[0331] The OD 600 Cell growth assay: Cell membrane integrity assessment: PI (propidium) staining + flow cytometry detection after sampling; Organic acid and flavor compound analysis: High performance liquid chromatography (HPLC) and GC-MS; Oxidation-reduction potential (ORP) monitoring.

[0332] III. Experimental Results

[0333] Regarding cell growth and membrane integrity, under conditions of 5% NaCl and 0.5 mM H2O2, the OD at the end of 96 hours... 600 The concentration was still 12.3, a decrease of only 10% compared to the control (no salt and no H2O2); PI staining showed that the proportion of cells with membrane damage was <8%, indicating that the Saccharomyces cerevisiae CPJ-01 described in this invention can still maintain high integrity under combined stress.

[0334] In terms of metabolic activity and product stability, the glucose consumption rate was 0.75 g / (L·h), and the glucose was depleted within 96 h; the ethanol yield was 0.38 g / g, and the final ethanol concentration was 38 g / L; the concentration of organic acids (malic acid and lactic acid) was similar to that of the control, and the distribution and abundance of flavor substances fluctuated by <5%.

[0335] In terms of flavor and sensory evaluation, GC-MS detected a total of 18 volatile components, with esters accounting for 35%. The Saccharomyces cerevisiae CPJ-01 brewing yeast described in this invention is basically consistent with conventional fermentation. In the blind sensory evaluation, it was found to have a rich sour aroma, a balanced taste, and no off-flavors of "saltiness" or "oxidation". The average score was 4.3 / 5.

[0336] In terms of system stability, the ORP remained at around −150 mV, indicating that the reduction state was stable; the pH fluctuated within the range of 3.5–5.5, and no additional adjustment was required throughout the fermentation process.

[0337] Application Example 2 demonstrates that the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention possesses excellent acid tolerance (pH 3.5–7.0), salt tolerance (NaCl ≤ 5%), and oxidative stress resistance. It can maintain cell membrane integrity and metabolic activity under combined stress conditions, achieving continuous and stable sugar metabolism and flavor compound generation. These characteristics make it particularly suitable for the industrial production of acidic flavored fermented foods (such as fruit wine, sour beer, and flavored vinegar), ensuring consistent product quality and robustness of the production process.

[0338] Therefore, the Saccharomyces cerevisiae CPJ-01 strain described in this invention is suitable for fermentation food manufacturing processes under acidic environments with a pH of 3.5–7.0, high osmotic pressure conditions with NaCl ≤ 5%, and oxidative stress. It can maintain cell membrane integrity and metabolic activity under the above-mentioned combined stress, ensuring the continuity of the fermentation system and the stability of product quality.

[0339] Application Example 3: Production of a specialty fermented beverage based on the efficient synthesis of ester alcohol aroma substances from the brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention.

[0340] Application Example 3 uses Saccharomyces cerevisiae CPJ-01 strain as the core fermentation microorganism to explore its ability to enhance the synthesis of aroma substances such as ethyl acetate and isoamyl alcohol in the fermentation system, thereby achieving high flavor added value of fermented products.

[0341] The aroma composition of fermented beverages is a key factor influencing their consumer acceptance. Ester compounds (such as ethyl acetate) impart fruity and fresh flavors, while higher alcohols (such as isoamyl alcohol) contribute to the fullness and complex aroma profile. Traditional bacterial strains have limited aroma-producing capabilities, making it difficult to meet modern consumers' dual demands for "complex flavors" and "natural fruit aromas."

[0342] Specifically, the production of a distinctive fermented beverage based on the efficient synthesis of ester alcohol aroma substances from the brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention involves the following specific implementation steps:

[0343] I. Fermentation System Design and Operation Process

[0344] S1. Raw materials and fermentation substrate: The formulation substrate includes apple juice and glucose (initial total sugar 120 g / L); the pH is then adjusted to 5.2, and 0.8 g / L of nitrogen source complex (containing yeast extract and amino acids) is added to improve the availability of ester precursors.

[0345] S2. Inoculation and culture conditions: The inoculation volume of activated bacterial solution is 2%;

[0346] S3. Fermentation conditions: 30℃, 150 rpm, 96 h batch fermentation, with a control group (commercially available common brewing yeast strain) for comparison.

[0347] S4. Analytical Indicators and Methods: Total volatile matter content and sensory evaluation were completed by a trained evaluation team;

[0348] The volatile aroma components were detected using GC-MS (gas chromatography-mass spectrometry).

[0349] The activities of the esterase and alcohol dehydrogenase were determined by colorimetry and NADH absorbance variation method.

[0350] II. Experimental Results and Application Effects

[0351] Based on the detection of ester and alcohol aroma products, the fermentation system of Saccharomyces cerevisiae CPJ-01 described in this invention showed that the ethyl acetate content reached 45 mg / L, which was 32% higher than the control group; the isoamyl alcohol content reached 32 mg / L, which was nearly 40% higher than the control; a total of 18 volatile flavor substances were detected by GC-MS, of which esters accounted for 38%, and the aroma structure was rich.

[0352] In terms of enzyme activity and metabolic capacity, the Saccharomyces cerevisiae CPJ-01 strain described in this invention showed no positive esterase activity; the alcohol dehydrogenase activity reached 0.5 U / mg protein, which was significantly higher than the 0.3 U / mg of the control group; indicating that the Saccharomyces cerevisiae CPJ-01 strain maintained good ester conversion efficiency in the middle and late fermentation stages.

[0353] In terms of sensory and flavor characteristics, the finished wine has a light amber color, rich fruit aroma, and a smooth taste.

[0354] The finished wine was evaluated by most judges who described its aroma as "natural floral and fruity", "long-lasting", and "distinct layers", with an average score of 4.5 out of 5, which was significantly better than the control group's 3.8.

[0355] Application Example 3 shows that the Saccharomyces cerevisiae CPJ-01 strain of brewing yeast described in this invention is used for the efficient synthesis of volatile aroma compounds such as esters (e.g., ethyl acetate) and alcohols (e.g., isoamyl alcohol) in fermentation systems, significantly improving the aroma complexity and flavor diversity of the product.

[0356] Therefore, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention exhibits significant ester (ethyl acetate) and alcohol (isoamyl alcohol) synthesis capabilities during fermentation, and its overall volatile aroma components are rich in variety and in reasonable proportion, significantly enhancing the aroma complexity and flavor diversity of the fermented product. This strain shows promising application prospects in the mid-to-high-end fermented beverage industry, such as fruit wines, flavored beers, and specialty rice wines, where aroma quality is paramount.

[0357] Application Example 4: Development of an antioxidant health drink based on the Saccharomyces cerevisiae CPJ-01 yeast described in this invention.

[0358] Application Example 4 aims to utilize the natural metabolites produced by *Saccharomyces cerevisiae* CPJ-01 during fermentation to develop a healthy fermented beverage with significant antioxidant properties, low alcohol content, no bitterness, and a mild fruity aroma. This meets the functional beverage market's dual demand for "safety + bioactivity." With increasing consumer health awareness, functional beverages rich in natural antioxidants are gaining market favor. Free radical scavenging ability, especially DPPH free radical scavenging rate, has become one of the important indicators for measuring the bioactivity of beverages.

[0359] Specifically, the development of an antioxidant health drink based on the brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention involves the following steps:

[0360] I. Experimental Design and Operation Procedure

[0361] S1. Raw materials and fermentation formula: The substrate is a mixture of concentrated wolfberry juice and concentrated apple juice diluted in a 1:1 ratio to a total sugar content of 100 g / L; the initial pH is adjusted to 5.2; no exogenous antioxidants are added to evaluate the antioxidant activity generated by the fermentation of CPJ-01 itself.

[0362] S2. Inoculation and fermentation conditions: The inoculation amount of the activated bacterial solution is 2% (v / v); the fermentation temperature is 30℃; the rotation speed is 150 rpm; and the fermentation time is 96 hours.

[0363] S3. Analysis and monitoring indicators: DPPH free radical scavenging rate is used, which is determined after the fermentation broth is diluted 10 times; total polyphenol content is determined by the Folin-Ciocalteu method; sensory evaluation includes color, aroma, and taste; pH and OD420 are physicochemical indicators reflecting color depth; alcohol concentration is controlled within ≤1.0% (v / v) to meet the standards for healthy beverages.

[0364] II. Experimental Results and Application Effects

[0365] In terms of antioxidant capacity, after 96 hours of fermentation, the DPPH free radical scavenging rate of the fermentation broth reached 72% (under 10-fold dilution), which is significantly higher than that of ordinary yeast fermented beverages (average only 50-60%); the total polyphenol content reached 180 mg GAE / L, indicating that CPJ-01 has the potential to promote the release and synthesis of polyphenols.

[0366] In terms of aroma and taste characteristics, GC-MS analysis identified 18 volatile flavor components, with esters accounting for 38%, mainly ethyl acetate (45 mg / L) and isoamyl alcohol (32 mg / L), with a fresh and natural aroma; the fermentation broth was light amber in color with an OD420 value of 0.42, and had a good visual appearance.

[0367] According to the tasting panel, the aroma is mild with fruity notes, light and slightly sweet on the palate, without bitterness or astringency, and leaves a clean aftertaste.

[0368] In terms of safety and stability, the alcohol concentration is maintained at 0.9% (v / v), which meets the definition of alcohol-free or light alcoholic healthy beverages; and no pollution occurs during the entire fermentation process, with a cell sedimentation rate of 50%, which facilitates subsequent sterilization treatment; the pH is stable between 5.0 and 5.2, and the system has strong self-stabilizing ability.

[0369] Application Example 4 shows that the Saccharomyces cerevisiae CPJ-01 strain of the present invention can be used to develop health drinks with significant antioxidant functions, so that the DPPH free radical scavenging rate of the fermentation broth is ≥70%, which fully exerts the antioxidant activity and meets the dual requirements of safety and bioactivity in the functional beverage market.

[0370] Therefore, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention can synthesize and enrich functional substances with antioxidant activity through its own metabolic activities without relying on exogenous antioxidants, achieving a DPPH free radical scavenging rate of ≥70% in the fermentation broth. This demonstrates its potential for developing natural, safe, and significantly bioactive healthy fermented beverages. The *Saccharomyces cerevisiae* CPJ-01 strain described in this invention is particularly suitable for developing novel functional products such as probiotic fruit vinegar, low-alcohol natural antioxidant beverages, and yeast-fermented functional drinks, bringing a differentiated competitive advantage to the functional beverage market.

[0371] Application Example 5: Stable fermentation and flavor customization in flavored fermented products based on the brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention.

[0372] Application Example 5 illustrates that the Saccharomyces cerevisiae CPJ-01 strain of the present invention possesses excellent settling properties, flavor metabolism capabilities, and tolerance, making it an ideal fermentation strain for achieving both process standardization and flavor personalization.

[0373] Fermented flavoring products (such as flavored rice wine, enzyme drinks, and fermented soup bases) have extremely high requirements for process stability and flavor specificity. Traditional yeast strains have problems such as poor cell sedimentation, difficulty in separation, uneven fermentation process, and weak or unbalanced aroma, which affect the consistency of product quality and flavor profile.

[0374] Specifically, the application of stable fermentation and flavor customization in flavored fermented products based on the brewing yeast Saccharomyces cerevisiae CPJ-01 described in this invention is implemented through the following steps:

[0375] I. Fermentation System Design and Operation Process

[0376] S1. Matrix Selection and Process Model: The application scenario is flavored fermented rice milk (low sugar, low alcohol, fruity flavor); the raw material ratio is glutinous rice enzymatic hydrolysate + osmanthus extract + trace lactic acid buffer system; the initial sugar content is 80g / L, and the pH is adjusted to 5.2; salt ions (NaCl 0.5%, simulating fermentation soup base conditions) are added to evaluate fermentation stability.

[0377] S2. Inoculation and culture: Inoculate with 2% activated bacterial solution and culture in shake flasks at 30℃; monitor CO2 release, pH changes and fermentation precipitation behavior simultaneously.

[0378] The fermentation cycle is 72 hours, and after the fermentation is completed, the mixture is left to stand for 24 hours to observe the sedimentation.

[0379] S3. Evaluation Indicators:

[0380] The settling property is the sum of foam height and stratification rate after settling (settling rate index after 24 hours).

[0381] The aroma components are volatile esters, alcohols, and other aroma compounds detected by GC-MS.

[0382] The flavor regulation response is achieved by adjusting the aroma profile by changing the addition of precursors (such as amino acids and sugar types).

[0383] The ease of separation is assessed by evaluating the efficiency of filter cloth + low-speed centrifugation (whether there are residual flocs or clumps).

[0384] II. Experimental Results and Process Performance

[0385] In terms of superior cell sedimentation performance, the sedimentation rate reaches 50% after 24 hours of standing. The supernatant of the fermentation broth is clear and there are no obvious flocs. The foam height is about 3.0 cm, and no sticky foam or expanding residue appears, resulting in a high degree of process cleanliness. After low-speed (2500 rpm) centrifugation, the filter cake forms a compact structure, which facilitates subsequent recovery of the clarified liquid or secondary extraction.

[0386] In terms of strong controllability of flavor expression, the ethyl acetate content is 45 mg / L and the isoamyl alcohol content is 32 mg / L, with a stable overall ester-alcohol structure ratio. After adding tyrosine and leucine, the isoamyl alcohol content increases to 18%, which can regulate the aroma and body of the wine. When osmanthus extract is present, the esterification efficiency is improved, which helps the aroma to blend and the taste is "sweet and elegant".

[0387] In terms of good process stability, the pH of the fermentation process was maintained at 5.0–5.3; the presence of chloride ions and organic acids did not significantly inhibit the fermentation rate, and the sugar depletion time remained at 72 hours; no pseudohyphae were formed, the cell morphology was stable, and the fermentation curve was smooth, which facilitates process modeling and automated control.

[0388] Application Example 5 demonstrates that the Saccharomyces cerevisiae CPJ-01 strain of the present invention, used in the innovative application of flavored fermented products, stabilizes the fermentation process by optimizing cell sedimentation and separation performance, and provides reliable strain support for customized flavoring treatments based on target flavor characteristics.

[0389] Therefore, the *Saccharomyces cerevisiae* CPJ-01 strain described in this invention possesses excellent cell sedimentation characteristics, good flavor regulation capabilities, and stable fermentation metabolism, enabling standardized fermentation processes, simplified post-processing, and plasticized aroma structures in the production of flavored fermented products. It exhibits reliable compatibility in products such as flavored rice wine, compound fruit jam fermentation liquid, and flavored fermented soup bases, providing stable strain support for customized flavor design and industrial replication.

Claims

1. A *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols, characterized in that... The Chinese name of the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae CPJ-01, and its Latin name is Saccharomyces cerevisiae CPJ-01. Saccharomyces cerevisiae CPJ-01 is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China; the deposit date is June 27, 2024; and the accession number is CCTCC NO: M 20241387.

2. The *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols as described in claim 1, characterized in that... The Saccharomyces cerevisiae CPJ-01 strain formed milky white, round, raised colonies after being cultured on YPD solid medium for 48 h. The colonies were 2.5 to 3.0 mm in diameter, with neat and smooth edges, and a moist, glossy, and opaque surface. The *Saccharomyces cerevisiae* CPJ-01 strain, under a microscope, has oval-shaped single cells, no pseudohyphae formation, obvious budding, and a cell size of approximately 5–7 μm × 7–10 μm. The *Saccharomyces cerevisiae* CPJ-01 strain has a maltose metabolism rate of 0.2 g / L·h. The *Saccharomyces cerevisiae* CPJ-01 strain can tolerate a maximum volume fraction of 12% v / v ethanol, and its survival rate is ≥85% under 10% ethanol conditions. The *Saccharomyces cerevisiae* CPJ-01 strain maintains ≥90% cell viability at ≥15% v / v ethanol concentration, with a final yield ≥0.40 g / g and residual sugar ≤5 g / L; the *Saccharomyces cerevisiae* CPJ-01 strain can grow normally under pH conditions of 3.5–7.0, with a lactic acid tolerance threshold of 2.5 g / L; The *Saccharomyces cerevisiae* CPJ-01 strain, during fermentation, produced volatile aroma components containing 45 mg / L ethyl acetate, 32 mg / L isoamyl alcohol, and esters accounting for 38% of the total volatile components. The Saccharomyces cerevisiae CPJ-01 strain has a glucose consumption rate of 0.8 g / L·h, achieves sugar depletion within 72 hours, has an alcohol yield of 0.4 g / g glucose, and a minimum glucose conversion rate of 0.40 g / g. The *Saccharomyces cerevisiae* CPJ-01 strain exhibited a growth inhibition rate of 15% at pH 3.5 and a lactic acid tolerance threshold of 2.5 g / L. The *Saccharomyces cerevisiae* CPJ-01 strain produced a fermentation broth with a foam height of 3.0 cm and a sedimentation rate of 50% after 24 hours of fermentation. The *Saccharomyces cerevisiae* strain CPJ-01 was used; the fermentation broth was amber in color, and the OD value was [not specified]. 420 The value was 0.42, and the scavenging rate of DPPH free radicals reached 72% after the fermentation broth was diluted 10 times. The Saccharomyces cerevisiae CPJ-01 strain can still maintain normal growth in an environment containing 5% NaCl.

3. The *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols as described in claim 1 or 2, characterized in that... The *Saccharomyces cerevisiae* CPJ-01 strain of *Saccharomyces cerevisiae* has glucose as its preferred metabolic substrate and preferentially metabolizes glucose in the presence of glucose.

4. The *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols as described in claim 1 or 2, characterized in that... The Saccharomyces cerevisiae CPJ-01 strain of brewing yeast showed the highest fermentation efficiency under constant temperature conditions of 32℃.

5. The *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols as described in claim 1 or 2, characterized in that... The key genes of the alcohol fermentation and sugar metabolism module of the Saccharomyces cerevisiae CPJ-01 strain are PDC1, ADH1, ADH2, and ALD6, and the gene basis for high ethanol yield is PDC1, ADH1 / 2. The key genes of the aroma substance synthesis module of the Saccharomyces cerevisiae CPJ-01 strain are ATF1, ATF2, IAH1, and BAT1 / BAT2, and the gene base rich in fruit aroma esters is ATF1, BAT2, and IAH1. The key genes of the stress resistance and homeostasis regulation module of the Saccharomyces cerevisiae CPJ-01 strain are TPS1, HSP30, PDR12, ENA1, and GPD1, and the gene basis for tolerance to high ethanol / weak acid / salt is TPS1, PDR12, ENA1, and HSP30. The key genes of the antioxidant response module of the Saccharomyces cerevisiae CPJ-01 strain are SOD1, CTA1, YAP1, and GSH1, and the gene base with strong antioxidant function is SOD1, CTA1, GSH1, and YAP1. The key genes for the sedimentation and process adaptation module of the Saccharomyces cerevisiae CPJ-01 strain are FLO1, FLO8, FLO11, and TUP1, and the gene basis for good process sedimentation is FLO1 and TUP1.

6. The *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and a complex fruity aroma profile of esters and alcohols as described in claim 1 or 2, characterized in that... The Saccharomyces cerevisiae CPJ-01 strain described above has an ethanol volume fraction of ≥12% (v / v) in a high-alcohol fermentation system, making it suitable for the industrial production of medium-to-high alcohol beverages or strong fruit wines. The Saccharomyces cerevisiae CPJ-01 strain can maintain cell membrane integrity and metabolic activity under combined stress conditions of pH ≤3.5, NaCl concentration ≤5%, and oxidative stress environment, making it suitable for the production of low pH products. The Saccharomyces cerevisiae CPJ-01 strain can synthesize volatile aroma components of ethyl acetate and isoamyl alcohol, making it suitable for fermented beverages with efficient synthesis of ester alcohol aroma substances. The fermentation broth produced by the Saccharomyces cerevisiae CPJ-01 strain, after being diluted 10 times, has a DPPH free radical scavenging rate of ≥70%, making it suitable for the development of antioxidant health drinks. The Saccharomyces cerevisiae CPJ-01 strain is suitable for stable fermentation and targeted development of flavored fermented products.

7. The method for screening and obtaining the Saccharomyces cerevisiae strain CPJ-01 with high ethanol tolerance and a complex fruity aroma spectrum of ester alcohols, as described in claims 1-6, is characterized in that... Wild microbial resources derived from natural green plums were used, and the yeast strain Saccharomyces cerevisiae CPJ-01, which combines alcohol resistance, complex ester alcohol aroma synthesis ability and acid environment adaptability, was selected under the screening pressure of 5% v / v high concentration ethanol.

8. The molecular biological method for identifying the *Saccharomyces cerevisiae* strain CPJ-01 with high ethanol tolerance and ester alcohol aroma spectrum according to claim 7, characterized in that, The BLAST alignment results showed a similarity of ≥99.19% with the ITS region of the reference sequence Accession NR_111007.1, a score of ≥1328, and an E-value of 0.0, thus confirming that the identified strain was Saccharomyces cerevisiae CPJ-01.

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