Saccharomyces cerevisiae CPJ-02 with high acid resistance, high ethanol content and strong ester aroma generation and application thereof

Abstract

The invention discloses a strain of saccharomyces cerevisiae CPJ-02 with high acid resistance, high ethanol content and strong ester aroma generation and application of the saccharomyces cerevisiae CPJ-02, the strain of the saccharomyces cerevisiae CPJ-02 has been preserved in China Center for Type Culture Collection on June 27, 2024; the preservation number of the strain is CCTCC NO: M 20241388. The saccharomyces cerevisiae CPJ-02 strain is milky white to faint yellow after being cultured on a YPD solid plate for 48 hours, the surface is dry and matte, the texture is slightly rough, and the colonial morphology is round. The saccharomyces cerevisiae CPJ-02 strain has prominent high ethanol tolerance, and still has activity greater than or equal to 92% in a 12% ethanol environment; and the fermentation product is rich in aroma and high in ethyl caprylate and phenethyl alcohol content, more than or equal to 25 volatile substances are detected by GC-MS (gas chromatography-mass spectrometry), and the fermented product is rich in fruit fragrance and has ester flower fragrance. The saccharomyces cerevisiae CPJ-02 strain is suitable for production of high-concentration wines and directional production of low-concentration healthy fermented beverages, fermented foods and aroma compounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial strains, and relates to a composite functional yeast strain, in particular to a high-acid-tolerant high-ethanol strong-ester-aroma-generating Saccharomyces cerevisiae CPJ-02 with excellent sugar metabolism, alcohol and aroma production capacity, high ester aroma substance generation capacity, and broad-spectrum environmental adaptation capacity, and uses thereof. BACKGROUND

[0002] Saccharomyces cerevisiae is the most widely used industrial fermentation microorganism in the world, and is widely used in the traditional brewing fields of fruit wine, yellow wine and beer, as well as the development of functional fermented drinks and new type of low-alcohol refreshing wine foods in recent years. In the fermentation process, Saccharomyces cerevisiae not only bears the main function of sugar conversion and ethanol generation, but also synthesizes a large amount of aromatic compounds such as ethyl acetate, phenethyl alcohol and ethyl octanoate through its complex metabolic activity. These aroma substances have a decisive influence on the flavor characteristics and consumer acceptance of the final product.

[0003] With the consumers' higher requirements for the flavor levels and sensory properties of wine and fermented drinks, Saccharomyces cerevisiae strains with high aroma generation capacity (especially ester substances) have become the research focus of microbial breeding and targeted improvement. However, the metabolic activity of traditional Saccharomyces cerevisiae strains is often significantly inhibited under the stress of high sugar, high acid or high ethanol, resulting in limited generation of aroma products, which has become one of the main bottlenecks of flavor expression in industrial production.

[0004] In view of the above problems, domestic and foreign scholars have carried out a lot of research on the screening and improvement of high-ethanol-tolerant, acid-tolerant and salt-tolerant Saccharomyces cerevisiae. For example, Wang Fei (2017) obtained a Saccharomyces cerevisiae strain with pH tolerance of 3.0 through step-by-step domestication, which significantly improved its growth and fermentation capacity in low-pH environment; Zhang Xiaofei (2019) analyzed the aroma metabolic products of high-ethanol-tolerant beer yeast, revealing its metabolic characteristics under high-alcohol concentration; Xie Ronghua (2015) discussed the correlation between the antioxidant activity (DPPH free radical scavenging rate) of yeast fermentation products and their metabolites. In addition, some studies have improved the synthesis efficiency of aroma products by regulating the expression of esterases (such as ATF1, ATF2 and EHT1) (Liu Hong, 2016), but under high-acid and high-ethanol extreme conditions, the effect of these methods is still limited.

[0005] However, there are still some technical bottlenecks and deficiencies in the existing Saccharomyces cerevisiae strains. First, it is extremely rare to have a strain that simultaneously achieves high acid tolerance, high ethanol tolerance, and high ester aroma generation ability with stable genetic traits. Some of the strains reported in the current literature often exhibit a decrease in aroma production or a decrease in growth rate after improving tolerance, while strains that focus on high ester aroma production often sacrifice acid or alcohol tolerance. From the perspective of metabolic regulation, there is a contradiction between acid tolerance, alcohol tolerance, and high ester aroma production in the metabolic network. For example, in a high ethanol environment, yeast cells are easily affected by metabolic stress, which affects lipid metabolism, inhibits the expression of ester synthesis-related enzymes, and thus reduces the production of ester aroma substances. Therefore, in high alcohol and low pH fermentation systems (such as baijiu and highly fruit wine), traditional yeast strains often exhibit growth inactivation or single aroma, limiting the innovation of multi-flavor in high-end wine.

[0006] In addition, there are challenges in the control of by-products and the improvement of flavor purity. Saccharomyces cerevisiae often produces a large amount of higher alcohols (such as isoamyl alcohol and n-propanol) and fusel oil by-products while synthesizing ester aroma substances. The above by-products help to improve the complex flavor of the wine body at a certain concentration, but once it exceeds the standard, it will cause the wine body to have increased irritation, rough flavor, and even food safety risks. The synthesis of ester substances and the production of higher alcohols share part of the amino acid decarboxylation and fatty acid acetylation metabolic pathway, and regulating one pathway easily affects the overall metabolic balance. High content of fusel oil has become one of the key obstacles for the export of high-quality alcohol and food-grade wine products. Although there have been studies to reduce by-product production through gene knockout or metabolic regulation, the genetic stability and industrial application of related strains still need to be further improved.

[0007] Therefore, there are still many technical difficulties in Saccharomyces cerevisiae in achieving the simultaneous optimization of high acid tolerance, high ethanol, and high ester aroma, precise regulation of by-products, maintenance of genetic stability, and consideration of production economy, which need to be overcome. SUMMARY

[0008] (1) Invention purpose

[0009] The existing Saccharomyces cerevisiae technology mainly exists in the technical bottleneck that traditional yeast is prone to growth inactivation, reduction of ethanol and aroma yield under extreme fermentation conditions such as high sugar, high acid, and high ethanol. With the rise of low pH and high acid fermentation products, traditional Saccharomyces cerevisiae is difficult to maintain activity at low pH, the risk of contamination by miscellaneous bacteria increases, and the quality and safety of the final product are affected. At the same time, some high-performance Saccharomyces cerevisiae strains also have problems such as complex process, high cost, and unstable yield in production, which affect the scale-up and high-value conversion application of Saccharomyces cerevisiae.

[0010] In view of the above, the technical problem to be solved by the present application is to provide a high-acid-tolerant high-ethanol strong-ester-aroma-producing Saccharomyces cerevisiae CPJ-02 strain with excellent sugar metabolism, ethanol production and aroma production capacity, high-ester-aroma production capacity, and broad-spectrum environmental adaptation capacity, to solve the problems of the prior art, such as the difficulty in achieving multiple trait coordination, the limitation of metabolic activity under extreme environment, and the significant impact of stress on aroma expression capacity, so as to meet the needs of fermentation of Saccharomyces cerevisiae strains in food engineering to achieve high-acid-tolerant high-ethanol high-ester-aroma three trait coordination optimization.

[0011] The first object of the present application is to provide a high-acid-tolerant high-ethanol strong-ester-aroma-producing Saccharomyces cerevisiae CPJ-02 strain, which has excellent ethanol tolerance and exhibits good multiple stress tolerance, and is used to improve the quality stability and functional diversity of fermented foods.

[0012] The second object of the present application is to provide a strain screening and obtaining method of the high-acid-tolerant high-ethanol strong-ester-aroma-producing Saccharomyces cerevisiae CPJ-02 strain.

[0013] The third object of the present application is to provide a strain identification method of the high-acid-tolerant high-ethanol strong-ester-aroma-producing Saccharomyces cerevisiae CPJ-02 strain.

[0014] The fourth object of the present application is to provide the main microbiological characteristics of the high-acid-tolerant high-ethanol strong-ester-aroma-producing Saccharomyces cerevisiae CPJ-02 strain, including high-ethanol-tolerant performance, excellent aroma synthesis capacity, low-pH and lactic acid resistance, antioxidant function, and certain salt tolerance.

[0015] The fifth object of the present application is to provide the use of the high-acid-tolerant high-ethanol strong-ester-aroma-producing Saccharomyces cerevisiae CPJ-02 strain, and the specific uses include:

[0016] Firstly, the Saccharomyces cerevisiae CPJ-02 strain can stably ferment in a high-alcohol fermentation system with a volume fraction of ≥12% (v / v), and achieve a high-ethanol yield of glucose conversion rate ≥0.40 g / g, with stable fermentation process and high metabolic efficiency, which can provide a more stable and efficient brewing solution for high-concentration alcohol production, and is suitable for industrial production.

[0017] Secondly, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application has good resistance in an acidic environment (pH 3.5-7.0), can tolerate high osmotic pressure conditions of NaCl ≤ 5% and fermentation environments under oxidative stress, has good antioxidant capacity and stability, can provide stable fermentation performance and efficient antioxidant capacity for low-alcohol drinks and healthy beverages, and enhances the added value of products.

[0018] Thirdly, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application exhibits excellent adaptability in an acidic (pH 3.5-7.0) and high-salt (NaCl ≤ 5%) fermentation environment, can improve the production efficiency and product quality of fermented foods, and is particularly stable under high-salt and acidic conditions, making it an ideal choice for the fermented food industry.

[0019] Fourthly, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application can efficiently synthesize a variety of volatile aroma substances, especially esters (such as ethyl octanoate) and alcohols (such as phenethyl alcohol). This characteristic makes the Saccharomyces cerevisiae CPJ-02 strain very suitable for the directed production of aroma compounds, helps to improve the flavor of products such as wine, and meets the market demand for high-quality, complex-flavor beverages.

[0020] Fifthly, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application is used to develop healthy beverages with significant antioxidant function, so that the DPPH free radical scavenging rate of the fermentation broth is ≥ 70%, fully exerting the antioxidant activity, and meeting the dual requirements of safety and biological activity in the functional beverage market. (2) Summary of the invention

[0022] Firstly, the present application provides a Saccharomyces cerevisiae CPJ-02 strain with high acid tolerance and high ethanol tolerance, and strong ester aroma generation. The Chinese name of the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae CPJ-02, and the Latin name is Saccharomyces cerevisias CPJ-02. The Saccharomyces cerevisias CPJ-02 strain is preserved in the China Center for Type Culture Collection, located in Wuhan University, Wuhan, Hubei Province, and was preserved on June 27, 2024, with the preservation number CCTCC NO: M 20241388.

[0023] Secondly, the application provides a strain screening and obtaining method of a high-acid-tolerant high-ethanol and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain, which specifically comprises the following steps:

[0024] S1. Green plum sample collection: Select a region rich in fruit aroma microorganisms, namely Eryuan County, Dali Bai Autonomous Prefecture, Yunnan Province, China, and collect natural fallen green plum fruit samples in the mature period (mid-June). Collect 10 samples (single fruit weight about 30-50 g) per batch, place them in sterile sampling bags, store them in a portable cooler (4 ± 1°C), and transport them to the laboratory within 12 hours;

[0025] S2. Sample pretreatment: Take 25.0 g of representative green plum fruit samples and place them in 225 mL of sterile normal saline (0.85% NaCl, pH 6.8). Homogenize the mixture in a homogenizer under sterile conditions at a speed of 8000 rpm for 2 minutes to obtain a suspension;

[0026] S3. Gradient dilution and plating: Dilute the suspension by 10 -1 ~10 -6 Gradient dilution, and take 200 μL of each gradient dilution and evenly plate it on a YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar) selective medium plate containing 5% ethanol for initial screening of alcohol tolerance.

[0027] S4. Culture conditions: Incubate in a 30°C constant temperature incubator for 48-72 hours, and observe the growth of the colonies. Then select colonies with obvious morphology, smooth surface, neat edges, full and raised, and milky white opacity as initial screening objects;

[0028] S5. Single colony isolation and purification: Repeat the passage at least three times on fresh YPD plates by streak dilution method to obtain single colonies with stable growth. Keep 3-5 strains of clones with consistent phenotype for subsequent analysis.

[0029] S6. Microscope observation and preliminary identification: Perform Gram staining on the purified colonies, observe the cell morphology using an optical microscope (1000× oil lens), select oval, obvious budding, no pseudohyphal structure, and Gram-positive ones, and keep the strains with consistent morphology and number for storage;

[0030] S7. Glycerol freezing preservation: the isolated strain confirmed to meet the morphological characteristics of yeast was inoculated in YPD liquid medium (30℃ shaking culture for 24 h), the bacterial cells were collected by centrifugation and resuspended in glycerol with a final concentration of 15%, and then stored at -80℃ for subsequent metabolic and genetic stability evaluation and subsequent strain identification.

[0031] Thirdly, the present application provides a strain identification method of a Saccharomyces cerevisiae CPJ-02 strain with high acid tolerance and high ethanol tolerance and strong ester aroma generation, which specifically comprises the following steps:

[0032] The materials and instruments used in the strain identification method of the Saccharomyces cerevisiae CPJ-02 strain provided by the present application mainly include a fungal genomic DNA rapid extraction kit (Shengong, item number B518229), liquid nitrogen, a 1.5 mL centrifuge tube (RNase / DNase-free), a 65℃ constant temperature water bath, a centrifuge (model number: Eppendorf 5424, maximum speed 13,200 rpm, 4℃ refrigeration), a pipettor, an RNase / DNase-free pipette tip, and TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0033] The present application provides a strain identification method of a Saccharomyces cerevisiae CPJ-01 strain with high ethanol tolerance and an ester alcohol complex fruit aroma flavor spectrum, which mainly comprises the following steps:

[0034] S1. Sample tissue disruption: 50–100 mg of fresh yeast cell mass or mycelium (20 mg of dry sample) was placed in a pre-cooled grinding tube, liquid nitrogen was added for rapid freezing and grinding until the cells were completely broken;

[0035] S2. Lysis: 400 µL of Buffer Digestion and 4 µL of β-mercaptoethanol were added, and after gentle inversion and mixing, the mixture was placed in a 65℃ water bath for 1 h of shaking (300 rpm) until complete lysis (the solution was clear);

[0036] S3. Protein precipitation: after cooling to room temperature, 200 µL of Buffer PF was added, and after gentle inversion and mixing for 10 times, the mixture was placed in a –20℃ refrigerator for 5 min to further aggregate the proteins and polysaccharides.

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

[0038] S5. DNA precipitation: add equal volume of isopropanol (about 400 μL), gently invert mix 8 times, room temperature stand for 3 min, 4℃, 10,000 rpm centrifugal 5 min, discard supernatant;

[0039] S6. Washing: add 1 mL of ethanol with a concentration of 75%, invert mix 2 min, 4℃, 10,000 rpm centrifugal 2 min, discard supernatant, repeat once;

[0040] S7. Drying: open the centrifuge tube cover, invert 5-10 min at room temperature until there is no residual ethanol on the tube wall;

[0041] S8. Dissolution and storage: add 50 μL of TE Buffer into the tube, gently mix by blowing, stand for 5 min; room temperature to obtain transparent viscous DNA solution; and machine measurement A 260 / A 280 The ratio (1.8-2.0), the total yield is about 2-5 μg, suitable for PCR, sequencing or-20℃ long-term storage.

[0042] Fourth, the high acid-tolerant high-ethanol and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the present application can be obtained by isolation, directional screening and molecular identification, and has the following significant technical features:

[0043] First, the microbial characteristics of the high acid-tolerant high-ethanol and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the present application are as follows:

[0044] Specifically, from the colony morphology and colony distribution, the high acid-tolerant high-ethanol and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain forms a light yellow, irregular round raised colony after 48 h of culture on YPD solid medium, the colony diameter is 1.5-2.0 mm, the edge is wavy, the surface is rough, non-lustrous and translucent; under a microscope, individual cells are round, occasional false mycelium formation, low frequency of budding, cell size is about 4-6 μm x 6-8 μm.

[0045] Specifically, from the high alcohol tolerance performance, the application provides a high acid-tolerant high ethanol and strong ester aroma generating type Saccharomyces cerevisiae CPJ-02 strain, the maximum tolerance ethanol concentration of which can reach 15% (v / v), the survival rate under 12% (v / v) ethanol is greater than or equal to 90%; the cell activity under greater than or equal to 12% v / v ethanol concentration is greater than or equal to 90%, the final yield is greater than or equal to 0.45 g / g, and the residual sugar is less than or equal to 0.1 g / L; the glucose and maltose conversion rates are excellent, the glucose consumption rate is 1.2 g / (L·h), the peak CO2 release amount in fermentation is 120 mL and is reached in 48 h, the sugar is exhausted within 48 h, the alcohol yield is 0.4 g / g glucose, the minimum glucose conversion rate is 0.40 g / g, and the maltose metabolism rate is 0.35 g / L·h.

[0046] Specifically, from the excellent aroma synthesis ability, the application provides a high acid-tolerant high ethanol and strong ester aroma generating type Saccharomyces cerevisiae CPJ-02 strain, the content of ethyl octanoate in the fermentation product is 62 mg / L, the content of phenethyl alcohol is 48 mg / L, and the ester substances account for 52% of the total amount of volatile components; the GC-MS analysis detects greater than or equal to 25 kinds of volatile flavor substances, and the fermentation liquor has soft fruit aroma, slight sweetness and no bitter and astringent taste.

[0047] Specifically, from the low-pH and lactic acid tolerance, the application provides a high acid-tolerant high ethanol and strong ester aroma generating type Saccharomyces cerevisiae CPJ-02 strain, the growth inhibition rate of which is only 10% under the condition of pH 3.0; the lactic acid tolerance threshold can reach 3.05 g / L, and the strain is suitable for the acid fermentation system of medium and high-end wine products.

[0048] Specifically, from the antioxidant function, the application provides a high acid-tolerant high ethanol and strong ester aroma generating type Saccharomyces cerevisiae CPJ-02 strain, the fermentation liquor of which is amber, the OD 420 value is 0.42, the DPPH free radical scavenging rate of the fermentation liquor (diluted by 10 times) reaches 72%, the fermentation liquor foam height is 4.5 cm, the sedimentation rate after fermentation for 24 h is 30%, and the strain has potential development and application value in health drinks and functional foods.

[0049] Specifically, from the certain salt tolerance, the application provides a high acid-tolerant high ethanol and strong ester aroma generating type Saccharomyces cerevisiae CPJ-02 strain, which can still grow normally in an environment containing 5% NaCl and is suitable for salty flavor fermentation process; the growth is inhibited at a temperature higher than 40℃, and the recommended fermentation temperature range is 25-35℃.

[0050] Preferably, the Saccharomyces cerevisiae CPJ-02 strain takes glucose as the preferred metabolic substrate and preferentially metabolizes glucose in the presence of glucose.

[0051] Preferably, the Saccharomyces cerevisiae CPJ-02 strain has the highest fermentation efficiency under constant temperature conditions at 34℃.

[0052] Secondly, the genetic characteristics of the Saccharomyces cerevisiae CPJ-02 strain with high acid tolerance, high ethanol production and strong ester aroma production are as follows:

[0053] The key genes of the alcohol fermentation and sugar metabolism module of the Saccharomyces cerevisiae CPJ-02 strain are ADH1, ADH2, PDC1, and ALD6, and the genetic basis of high ethanol production is ADH1, ADH2, and PDC1.

[0054] The key genes of the aroma substance synthesis module of the Saccharomyces cerevisiae CPJ-02 strain are ATF1, ATF2, EEB1, BAT1, and BAT2, and the genetic basis of rich fruity esters is ATF1, BAT2, and EEB1.

[0055] The key genes of the stress resistance and homeostasis regulation module of the Saccharomyces cerevisiae CPJ-02 strain are ERG2, ERG6, HSP104, PMA1, and ENA1, and the genetic basis of high ethanol tolerance / weak acid / salt tolerance is ERG2, ENA1, and HSP104.

[0056] The key genes of the antioxidant response module of the Saccharomyces cerevisiae CPJ-02 strain are SOD1, CTT1, GSH1, and GPX1, and the genetic basis of strong antioxidant function is SOD1 and CTT1.

[0057] The key genes of the sedimentation and process adaptation module of the Saccharomyces cerevisiae CPJ-01 strain are FLO1, FLO5, FLO8, and FLO11, and the genetic basis of good process sedimentation is FLO1 and FLO5.

[0058] Fifth, the use and application scenarios of the high-acid-tolerant high-ethanol and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the present application include:

[0059] The Saccharomyces cerevisiae CPJ-02 strain can stably ferment in a high-alcohol fermentation system with a volume fraction of 12% or more, and achieve a high-ethanol yield of glucose conversion rate of 0.40 g / g or more, and is suitable for industrial production of high-concentration alcoholic products.

[0060] The Saccharomyces cerevisiae CPJ-02 strain has good resistance to an acidic environment with a pH of 3.5-7.0, and can tolerate a high osmotic pressure condition of NaCl≤5% and a fermentation environment under oxidative stress, and is suitable for the production of low-alcohol drinks and healthy beverage products.

[0061] The Saccharomyces cerevisiae CPJ-02 strain exhibits excellent adaptability in a fermentation environment with an acidic pH of 3.5-7.0 and high salt NaCl≤5%, and is suitable for the production of fermented food products.

[0062] The Saccharomyces cerevisiae CPJ-01 strain can synthesize octanoic acid ethyl ester and phenethyl alcohol volatile aroma components, and is suitable for the directional production of aroma compounds.

[0063] The Saccharomyces cerevisiae CPJ-01 strain produces fermentation broth with a DPPH free radical scavenging rate of 70% or more after being diluted by 10 times, and is suitable for the development of antioxidant healthy drinks.

[0064] Further, the Saccharomyces cerevisiae CPJ-02 strain of the present application is derived from a wild microorganism resource of natural green plums, and under a high-concentration ethanol screening pressure of 5% (v / v), the Saccharomyces cerevisiae CPJ-02 strain is selected, which has alcohol tolerance, complex ester and alcohol aroma synthesis capacity, and acid environment adaptability.

[0065] Further, the Saccharomyces cerevisiae CPJ-02 strain of the present application, wherein the genomic DNA rapid extraction method is to realize the rapid identification of the gene level and the verification of the molecular characteristics of the Saccharomyces cerevisiae CPJ-02 strain by introducing a special fungal genomic DNA rapid extraction kit (B518229) combined with liquid nitrogen freeze cracking lysis and a Buffer PF cold aggregation step.

[0066] (3) Invention effect

[0067] The present application provides a Saccharomyces cerevisiae CPJ-02 strain with high acid tolerance, high ethanol tolerance and strong ester aroma generation, which has significant fermentation performance and wide industrial application potential. The alcohol yield of the Saccharomyces cerevisiae CPJ-02 strain reaches 0.45 g / g of glucose, and the survival rate is still as high as 92% under the condition of 12% ethanol. The fermentation peak is at 36 hours, the CO2 release amount reaches 15 mL / h, the glucose consumption rate is 1.2 g / (L·h), and the sugar source is basically depleted within 48 hours, showing high efficient sugar conversion capacity, and is suitable for rapid fermentation scenes such as alcohol and biofuel.

[0068] In terms of environmental adaptability, the Saccharomyces cerevisiae CPJ-02 strain with high acid tolerance, high ethanol tolerance and strong ester aroma generation can tolerate pH 3.0 (growth inhibition rate is only 10%), the optimum pH is 4.5-5.0, and can grow stably in the pH range of 3.0-7.5. At the same time, it can tolerate 7% NaCl and high temperature 42℃, adapt to complex process conditions such as high acid, high salt and high temperature, and ensure the stable operation of industrial fermentation process.

[0069] In terms of flavor substance generation, the Saccharomyces cerevisiae CPJ-02 strain with high acid tolerance, high ethanol tolerance and strong ester aroma generation can synthesize rich aroma components, among which esters account for 52% of the total volatile substances, mainly including 62 mg / L ethyl octanoate and 48 mg / L phenethyl alcohol, and 25 kinds of volatile substances are detected by GC-MS, which effectively improves the fruity and floral characteristics of the product, and is suitable for high-end wine, fruit wine, beer and other fermentation products with high flavor requirements.

[0070] In addition, the DPPH free radical scavenging rate of the fermentation liquor of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain reaches 72% (under 10 times dilution conditions), and the strain has certain antioxidant function and can be used for developing functional drinks. The high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain has good sedimentation, and the 24-hour sedimentation rate is 30%, and the foam is moderate (4.5 cm), which is beneficial to simplify post-fermentation treatment, improve process efficiency, reduce production cost, and has good industrial adaptability and comprehensive economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 It is a single colony image of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the application.

[0072] Figure 2 It is a phylogenetic tree image of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the application.

[0073] Figure 3 It is DNA sequencing electrophoresis of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the application Figure 1 (staining map 1);

[0074] Figure 4 It is DNA sequencing electrophoresis of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the application Figure 2 (staining map 2);

[0075] Figure 5 It is DNA sequencing electrophoresis of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the application Figure 3 (staining map 3);

[0076] Figure 6 It is DNA sequencing electrophoresis of the high-acid-tolerant and high-ethanol-tolerant and strong ester aroma generating Saccharomyces cerevisiae CPJ-02 strain of the application Figure 4 (staining map 4);

[0077] Figure 7 The collection of raw materials of the Saccharomyces cerevisiae CPJ-02 strain of the application, which is a high-acid-tolerant and high-ethanol and strong ester aroma generating type of Saccharomyces cerevisiae, is a mature fresh plum fruit collected in Eryuan County, Dali, Yunnan, China. DETAILED DESCRIPTION

[0078] In order to make the purpose, technical solutions and advantages of the application clearer and more apparent, the application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the application and do not limit the application.

[0079] In the foregoing of the application, whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the value of each number can have a difference of ±10% or a reasonable difference recognized by those skilled in the art, such as a difference of ±1%, ±2%, ±3%, ±4% or ±5%.

[0080] The term "room temperature" refers to a temperature of about 18°C to about 35°C, or about 20°C to 30°C, or about 25°C.

[0081] The application provides a Saccharomyces cerevisiae CPJ-02 strain of high acid tolerance, high ethanol and strong ester aroma generating type. The Chinese name of the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae CPJ-02, the Latin name is Saccharomyces cerevisias CPJ-02, the Saccharomyces cerevisias CPJ-02 strain is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, Hubei Province, China; the preservation date is June 27, 2024; and the preservation number is CCTCC NO: M 20241388.

[0082] The Saccharomyces cerevisiae CPJ-02 of the application mainly aims to solve the technical problems including efficient ethanol fermentation and alcohol production, fermentation adaptability under high sugar concentration and low pH environment, efficient synthesis of aroma components, fermentation adaptability to high temperature and high salt environment, enzyme activity and biological transformation potential, and antioxidant properties and health product development.

[0083] The Saccharomyces cerevisiae CPJ-02 as the industrial brewing strain can realize efficient fermentation under extreme environments such as high alcohol, strong acid and high salt, and significantly solves the problems of poor fermentation or interruption of traditional yeast under such conditions. Meanwhile, the strain can improve the aroma complexity and taste level of fermented drinks such as fruit wine and beer, has strong antioxidant activity, and is suitable for developing healthy functional fermentation products. The Saccharomyces cerevisiae CPJ-02 is suitable for rapid production on an industrial scale, takes into account high yield and product quality, and is conducive to subsequent clarification of the fermentation broth, thereby reducing the complexity of the downstream process.

[0084] The Saccharomyces cerevisiae CPJ-02 has excellent fermentation performance, environmental adaptability, aroma synthesis ability and enzyme activity, can effectively solve the problems of fermentation adaptability under high-efficiency alcohol production, low pH and high-sugar environment, flavor stability, growth and fermentation under extreme conditions, and has a wide application prospect, especially in the fields of food, drink, chemical industry and biotechnology.

[0085] The Saccharomyces cerevisiae CPJ-02 is particularly suitable for application in the production and processing of beer, fruit wine, yellow rice wine, strong liquor and healthy fermented drinks, and is also suitable for the development of special fermented condiments and industrial fermentation under special environments such as high salt, high acid and high alcohol, thereby providing efficient and stable fermentation solutions for food engineering related enterprises and research institutions.

[0086] In addition, the reagents used in the present application can be purchased from the market or prepared by the method described in the present application.

[0087] Figure 1 A single colony image of the Saccharomyces cerevisiae CPJ-02 strain on a plate (standard YPD medium), Figure 1The number of individual colonies can be seen. From the colony morphology, the Saccharomyces cerevisiae CPJ-02 strain forms a typical colony after being cultured on a YPD agar plate for 24-48 hours, which is milky white to light yellow, dry and matte on the surface, slightly rough in texture, round in shape, with irregular jagged or slightly wavy edges, and about 1.5-2.0 mm in diameter. The colonies in the streaked area are evenly distributed, and the colony density gradually decreases with the degree of dilution at the end of the streak, eventually forming clearly visible isolated colonies. The colony is low and flat, without a mucous layer or fluorescence phenomenon, indicating that the Saccharomyces cerevisiae CPJ-02 strain is dry on the surface, easy to operate, and has good separation and morphological identification characteristics. The overall morphological characteristics of the Saccharomyces cerevisiae CPJ-02 strain meet the common phenotypic characteristics of Saccharomyces cerevisiae, and are suitable for rapid screening and identification of strains under industrialized solid culture conditions.

[0088] Figure 2 A phylogenetic tree image of the Saccharomyces cerevisiae CPJ-02 strain described in the present application.

[0089] Figure 3 Figure 6 All are DNA sequencing electropherograms (staining maps) of the Saccharomyces cerevisiae CPJ-02 strain described in the present application, which is a typical DNA sequencing electropherogram (staining map) displayed by sequencing software Chromas, used to analyze the genetic sequence of the Saccharomyces cerevisiae CPJ-02 strain. Figure 3 Figure 6 The bases represented by colors include red for Thymine (T), green for Adenine (A), blue for Cytosine (C), and black for Guanine (G); Figure 3 Figure 6 The electrophoretic peak type in each peak represents a base signal, and the sharper and more symmetrical the peak value, the higher the sequencing accuracy at that site. If there is peak overlap or tailing, it indicates a repeated base region, or secondary structure interference, or sequencing process noise or primer annealing problems; Figure 3 Figure 6 The letters above the sequence bar in are the automatically invoked base sequence, which is a linear representation of a certain DNA region of the target Saccharomyces cerevisiae, indicating a coding region (CDS), an intron (Intron), or a non-coding region.​​​​

[0090] Figure 7 The collection raw material of the Saccharomyces cerevisiae CPJ-02 strain described in the application is a mature plum fruit sample collected in Eryuan County, Dali, Yunnan, China.

[0091] Example 1: Saccharomyces cerevisias CPJ-02 strain screening and obtaining method described in the application

[0092] The Saccharomyces cerevisias CPJ-02 strain screening and obtaining method described in the application is as follows:

[0093] S1. Collecting plum samples: mature plum fruits are collected in Eryuan County, Dali, Yunnan, China, and stored in sterile sealed bags for low-temperature storage and transportation to the laboratory.

[0094] S1.1 Collection site and time of plums: In this experiment, Eryuan County, Dali Bai Autonomous Prefecture, Yunnan Province, China, with unique geographical environment and good natural ecology, is selected as the collection site of the plum sample.

[0095] The mature plum fruits in Eryuan County, Dali, Yunnan, are conducive to the enrichment of natural yeasts due to the moderate rainfall and large diurnal temperature difference in the region.

[0096] The time for collecting the mature plum fruits in Eryuan County, Dali, Yunnan, is to manually pick the fruits in the plum ripening season under non-polluted and natural growth conditions, to ensure that the fruit surface is attached with natural microbial populations, and to collect in the morning or evening of the same day to avoid the high temperature at noon that reduces the activity of yeasts.

[0097] S1.2 Tools and disinfection: sterile scissors or disposable gloves are used to cut the plum fruits, which are immediately placed in a sterile sealed bag that has been pre-sterilized (121℃, 20min).

[0098] S1.3 Sample storage and transportation: first, rapidly cool the collected plum fruits on site, and place them in an ice box at 4℃; the collected plum fruits must be transported to the microbiology laboratory within 6 hours to maximize the original activity and diversity of the yeast population in the sample; if longer transportation is required, the bagged plum fruits must be packaged with a preservative film with good oxygen permeability, and stored at -20℃ for a short period of time.

[0099] S2. Sample pretreatment: weigh 25 grams of the plum sample; place it in 225ml of sterile physiological saline, and mix well by homogenizing oscillation.

[0100] S2.1 Weighing and dissolving: In the sterile operating table of the microbiology laboratory, 25 g of green plum fruit (with skin and core) was accurately weighed and added to 225 mL of sterile normal saline with a concentration of 0.85% NaCl to form a 1:10 initial dilution;

[0101] S2.2 Homogenization treatment: The homogenization method is divided into using a sterile homogenizer (or handheld homogenizer) and using a high-speed homogenizer;

[0102] The homogenization treatment using a sterile homogenizer (or handheld homogenizer) is to oscillate for 2 min at 4°C to fully disperse the cells and pulp of the green plum fruit.

[0103] The homogenization treatment using a high-speed homogenizer is to treat for 90 seconds at a shock frequency of 8000 rpm to fully release the peel and pulp surface microorganisms of the green plum fruit into the liquid and ensure that the microorganisms are evenly dispersed in the solution, creating conditions for subsequent isolation and culture;

[0104] S2.3 Rest: Rest at room temperature for 1-2 min to precipitate the large pulp of the green plum fruit, and take the supernatant for the next step;

[0105] S3. Sample dilution and plating: The mixed liquid of the previous experimental step is gradient diluted; the green plum suspension obtained in the previous step is serially diluted by a factor of ten, usually with a dilution concentration of 10 -1 to 10 -6 , depending on the estimated density of microorganisms; a new clean test tube and a sterile pipette are used for each dilution level to ensure no cross contamination;

[0106] S3.1 Gradient dilution: 1 mL of supernatant is added to 9 mL of sterile normal saline and mixed thoroughly to prepare 10 -2 ; and sequentially diluted to 10 -6 ;

[0107] S3.2 Plating amount: 0.2 mL (200 μL) of each dilution is plated on the medium plate; it is recommended to set 2-3 plates in parallel for each dilution to improve the positive detection rate;

[0108] S4. Plating and culture: 200 microliters of the dilution liquid is plated on the yeast extract powder peptone glucose agar plate, and the yeast bacteria is cultured at 30°C;

[0109] S4.1 Medium preparation: 200 μL of each dilution suspension is plated on the pre-prepared YPD medium plate, with 10 g / L of yeast extract, 20 g / L of peptone, 20 g / L of glucose, and 15 g / L of agar. After autoclaving, the temperature is reduced to 50-55°C, and then the plate is poured;

[0110] S4.2 Culture conditions: Sterile swab was used to evenly spread on the surface of the culture medium to prevent colony overlapping; the plate was placed in a constant temperature incubator, set at 30°C, placed upside down to prevent dew, and cultured in the dark for 48-72 hours, during which the growth of the colonies was observed;

[0111] S5. Colony observation and separation and purification: The purpose strain was preliminarily judged by observing the shape, size, color, luster, texture surface and edge characteristics of the colonies, and the strain was further purified by cross streaking method to obtain single colonies;

[0112] S5.1 Preliminary screening: The colony morphology at different dilutions on each plate was observed, the diameter was 1-3 mm, the color was ivory white, the luster was slightly shiny on the surface of the colony, and the texture was moist with smooth edges;

[0113] S5.2 Selection of target colonies: The colonies with the most similar characteristics to Saccharomyces cerevisiae were selected, and then a sterile inoculation loop was used to make cross streaks on a fresh YPD plate;

[0114] S5.3 Repeat purification: The cross streaking method (four-zone streaking method) was used to purify on a fresh YPD plate, and the culture process was repeated after each inoculation until a group of single colonies with consistent morphology was obtained to exclude the interference of impurities and ensure the purity of the single strain;

[0115] S6. Microscopic detection and frozen preservation of dominant strains: The purified single colonies were subjected to Gram staining and observed under a microscope to further determine whether the isolated strain was the target strain, and then the isolated strains with consistent morphology were frozen with glycerol for subsequent identification;

[0116] S6.1 Gram staining: Although yeast is Gram-positive, Giemsa staining or lactic acid crystal violet staining was used, and morphological observation was performed under a 1000x optical microscope. The target strain should be oval, single or in a budding form, with clear fungal spores and germ tubes;

[0117] S6.2 Physiological and biochemical identification: Fermentation experiments were performed on polysaccharides such as glucose and sucrose, and gas production was detected; fermentation product determination was ethanol content;

[0118] S6.3 ITS region PCR amplification and sequencing: ITS1 / ITS4 primers were used, and after sequencing, the GenBank database was compared to confirm that it was S. cerevisiae

[0119] S6.4 Cryopreservation: the purified single colony (screened for consistent morphology, strong proliferation ability of dominant strain) was transferred to 10 mL of YPD liquid medium, and cultured at 37℃, 200 rpm shaker overnight; the dominant strain was grown for 24 hours, then centrifuged to collect bacteria, and a bacterial suspension was prepared with 15% glycerol, with a final concentration of 15% V / V; aliquoted into cryopreservation tubes, and stored in a -80℃ ultra-low temperature refrigerator for long-term preservation, for subsequent molecular identification and functional analysis.

[0120] Further, compared with the traditional screening technology of Saccharomyces cerevisiae, the Saccharomyces cerevisiae CPJ-02 strain screening and obtaining method of the application can significantly improve the alcohol tolerance of the obtained strain in the preliminary screening stage by introducing 5% ethanol selective pressure; meanwhile, the screened Saccharomyces cerevisiae CPJ-02 not only has excellent fermentation performance, but also can generate more than 18 kinds of volatile aroma substances including ethyl acetate and phenylethanol, and has a significant flavor advantage in fruit wine products. The Saccharomyces cerevisiae CPJ-02 strain screening and obtaining method of the application is simple, low in cost, and has strong repeatability, and has a significant industrial promotion prospect.

[0121] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0122] Example 2: Genomic DNA rapid extraction and identification method of the Saccharomyces cerevisiae

[0123] The method for rapidly extracting and identifying the genomic DNA of the Saccharomyces cerevisiae CPJ-02 strain is based on a commercially available fungal genomic DNA rapid extraction kit (Shengong, item number: B518229), and combines a high-efficiency lysis and purification process to extract complete and pure genomic DNA for downstream PCR identification, sequencing alignment and other analysis.

[0124] The method for identifying the Saccharomyces cerevisiae CPJ-02 strain, which extracts fungal genomic DNA using a fungal genomic DNA rapid extraction kit (Shengong, item number: B518229), has the following specific operation steps:

[0125] S1. Sample collection:

[0126] S1.1 Fresh sample: weigh 50-100 mg of fresh large fungi (such as mushroom, yeast, or mycelium group);

[0127] S1.2 Dry sample: weigh 20 mg of dried fruiting body or mycelium and grind into powder with liquid nitrogen;

[0128] S1.3 Grind the sample: place the fresh sample or dry sample in liquid nitrogen and quickly freeze, then grind it into a fine powder with a sterile mortar or tissue grinder to maximize the lysis of the fungal cell wall. Operating in a frozen state can maximize the disruption of the cell wall and inhibit nuclease activity;

[0129] S2. Cell lysis:

[0130] S2.1 Lysis solution preparation: transfer the ground powder to a 1.5 mL centrifuge tube, and add 400 µL of Buffer Digestion (containing Proteinase K) and 4 µL of β-mercaptoethanol;

[0131] The Buffer Digestion is used as a lysis buffer;

[0132] The β-mercaptoethanol is used as a reducing agent to break disulfide bonds and prevent NA degradation. That is, β-mercaptoethanol can break disulfide bonds and help protein denaturation;

[0133] S2.2 Mix: gently invert or vortex the lysis solution at low speed for 5-10 s to mix evenly;

[0134] S2.3 Water bath: Place the lysis solution in a 65℃ water bath or shaker or incubator for 1 h, with gentle inversion every 15 min until the sample is completely lysed, i.e. no visible solid particles on the tube wall, ensuring uniform heating of the suspension and complete lysis of the cells;

[0135] If the sample is difficult to completely lyse at this time, it can be extended to 1.5 h, or a short gentle inversion in the middle, i.e. repeat S2.2 mixing and S2.3 water bath;

[0136] S3. DNA binding and precipitation:

[0137] S3.1 Add Binding solution to remove protein and polysaccharide impurities: Add 200 μL Buffer PF to the lysed mixture, mix well by repeated inversion, so that the buffer and lysate react fully, then place in a -20℃ refrigerator for 5 min to promote the precipitation of proteins and polysaccharides;

[0138] The Buffer PF is used as a precipitation buffer and is combined with the column buffer, and the polyanion can bind to impurities;

[0139] S3.2 Centrifugal separation: Centrifuge at 10,000 rpm (about 10,000 x g) for 5 min at room temperature, then transfer the supernatant to a new 1.5 ml centrifuge tube to avoid disturbing the bottom precipitate;

[0140] S4. DNA precipitation and washing:

[0141] S4.1 Isopropanol precipitation: Add an equal volume of isopropanol to the supernatant, invert 5-8 times to mix thoroughly, and let stand at room temperature for 2-3 min to form visible DNA fibrous or clumpy precipitates, allowing the DNA to be bundled out; then centrifuge at 10,000 rpm for 5 min at room temperature, discard the supernatant, and obtain a white flocculent DNA precipitate;

[0142] S4.2 Wash with 75% ethanol: Add 1 ml of 75% ethanol (pre-cooled or room temperature), invert for 1-3 min to remove residual impurities; then centrifuge at 10,000 rpm for 2 min, discard the supernatant;

[0143] S4.3 Thorough washing: Repeat the S4.2 75% ethanol washing step once to remove residual salt and impurities, ensuring that the impurities are completely removed;

[0144] S5. De-ethanolization: Open the cap and invert the centrifuge tube on a sterile paper towel at room temperature for 5-10 min, allowing the residual ethanol to evaporate naturally, and avoid excessive drying to prevent the DNA from being difficult to resuspend;

[0145] S6. DNA resuspension (TE resuspension): the obtained DNA was added with 50 μL TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and mixed gently by blowing or overturning, and concentrated on the tube wall side wall; and then incubated at room temperature or 37 °C for 5-10 min, and mixed evenly by overturning gently, to ensure complete dissolution of the DNA;

[0146] S7. Preservation: the extracted DNA can be immediately subjected to the next step experiment or stored at -20 °C;

[0147] The DNA obtained at this time can be directly used for downstream PCR, enzyme digestion, gel electrophoresis identification, sequencing, etc.

[0148] The DNA obtained at this time can be long-term stored by being divided into multiple 0.5-1.5 mL cryotubes, and stored at -20 °C for up to several months, or stored at -80 °C for up to several years.

[0149] S8. DNA quality and concentration detection

[0150] S8.1 Concentration determination: the absorbance of the DNA was determined at A260 nm using nanodrop or spectrophotometer, and the concentration was calculated;

[0151] S8.2 Purity evaluation: A 260 / A 280 The ratio should be between 1.8-2.0 (ideal value), A 260 / A 230 The ratio should be greater than 2.0 (ideal value), and further purification is required if it is lower than this range;

[0152] On the contrary, if the purity is not up to standard, the washing step can be repeated once again after washing with 75% ethanol or a DNA purification column is used;

[0153] S8.3 Integrity check: 5 μL of the DNA was loaded on a 0.8% agarose gel using a spectrophotometer (such as NanoDrop), and electrophoresed using 1x TAE buffer for 30 min, to observe whether there is an obvious degradation band, and to detect the integrity and size of the band;

[0154] S9. Downstream identification suggestion

[0155] S9.1 ITS region PCR amplification: universal fungal ITS primers (such as ITS1 / ITS4) were used to detect the size of the amplified band (about 600 bp);

[0156] S9.2 Sanger sequencing: select ITS1 / ITS4 primers for PCR amplification (reaction system 25 μL, annealing temperature about 55 °C, cycle 30 times); send the PCR product for sequencing, and use NCBI BLAST alignment to confirm the similarity with the ITS sequence of Saccharomyces cerevisiae, if the sequence is highly consistent with Saccharomyces cerevisiae CPJ-02 (> 99% identity), the strain identity can be confirmed;

[0157] S9.3 Multi-gene identification: TEF1-alpha, RPG1 and other genes can be further amplified to enhance the accuracy of identification;

[0158] S9.4 PHYLOGENETIC analysis: construct NJ or ML phylogenetic tree to verify the genetic relationship between CPJ-01 and typical Saccharomyces cerevisiae strains;

[0159] S9.5 Multiplex verification: if conditions permit, RFLP typing, enzyme digestion map analysis and other methods can be used to further ensure the accuracy of the identification results.

[0160] Further, in order to accurately identify the test strain Saccharomyces cerevisiae CPJ-02 at the molecular biology level, the ribosomal DNA internal transcribed spacer (ITS) sequence analysis method commonly used for fungi is adopted. This method has become an international standard due to its wide applicability and high conservation in fungal system classification and inter-species identification. Through the rapid extraction and identification method of the genomic DNA of Saccharomyces cerevisiae CPJ-02, the specific information of the Saccharomyces cerevisiae CPJ-02 described in the present application is obtained.

[0161] (1) The universal primer for identifying the Saccharomyces cerevisiae CPJ-02 strain described in the present application

[0162]

[0163] The results show that the universal primers used for strain identification are ITS1 and ITS4.

[0164] The sequence of the ITS1 primer is 5'-TCCGTAGGTGAACCTGCGG-3', and the length is 19 bases,

[0165] The sequence of the ITS4 primer is 5'-TCCTCCGCTTATTGATATGC-3', and the length is 20 bases.

[0166] The pair of primers can effectively amplify the ITS1, 5.8S rRNA and ITS2 regions of the fungal rDNA, and has good universality and specificity.

[0167] (2) The ITS site PCR amplification system and amplification procedure table of the Saccharomyces cerevisiae CPJ-02 are as follows.

[0168]

[0169] The results show that:

[0170] Firstly, the total volume of the PCR amplification reaction system is 59 μL, which contains the following components: 2x Taq PCR Master Mix 25 μL, which provides the basic reaction system; about 20 ng of genomic DNA 10 μL, which is used as a template; 2 μL of upstream and downstream primers (concentration 5 pmol / μL) respectively; and 20 μL of sterile dd H2O to make up the system volume.

[0171] Secondly, the amplification procedure is set as follows: 95℃ pre-denaturation for 2 minutes; then 35 cycles of 95℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, and 72℃ extension for 45 seconds; and finally extension at 72℃ for 5 minutes to ensure complete amplification of the fragment.

[0172] (3) The sequencing result of the Saccharomyces cerevisiae CPJ-02

[0173]

[0174] It can be seen that: the product obtained after PCR amplification is purified and subjected to bidirectional sequencing, and the obtained sequence is submitted to the NCBI GenBank database for BLAST comparison. The results show that the sequence has ≧98.25% sequence similarity with the reference sequence (Accession number: NR_111007.1) of Saccharomyces cerevisiae in the database, the alignment score is 1297, and the E-value is 0.0, indicating that the matching is highly significant and has no randomness.

[0175] Combined with the amplification region, primer specificity, alignment score and species annotation, it can be confirmed that the strain CPJ-02 isolated in the experiment belongs to Saccharomyces cerevisiae, which is highly consistent with the reference sequence of the microbial database.

[0176] Example 3 The macroscopic and microscopic morphological characteristics, metabolic pathway and stress response characteristics of the Saccharomyces cerevisiae CPJ-02 strain described in the present application, and the application potential characteristics of the yeast strain

[0177] Example 3 focuses on explaining the macroscopic and microscopic morphological characteristics, metabolic pathway and stress response characteristics of the Saccharomyces cerevisiae CPJ-02 strain described in the present application, which is the basis for determining the performance of the Saccharomyces cerevisiae CPJ-02 strain in different fermentation environments. Through detailed analysis of these characteristics, it is helpful to genetically modify or optimize the Saccharomyces cerevisiae, thereby enhancing its production performance in a specific environment (such as increasing alcohol yield, improving aroma), ensuring the efficiency, stability and high-quality output of the Saccharomyces cerevisiae CPJ-02 strain in production.

[0178] Firstly, the single colony of the Saccharomyces cerevisiae CPJ-02 strain described in the present application shows differences, specifically the macroscopic and microscopic morphological characteristics, which explain the colony morphology formed by the Saccharomyces cerevisiae CPJ-02 strain described in the present application on the culture medium and the cell morphological characteristics under the microscope, for judging its purity, activity, and whether it is a typical morphology.

[0179]

[0180] Conclusion 1: Macroscopically, the colony of the Saccharomyces cerevisiae CPJ-02 strain described in the present application shows robustness, is not easy to mutate or be disturbed by mixed bacteria, has predictability and controllability, and is suitable for long-term use in the fermentation industry. The Saccharomyces cerevisiae CPJ-02 strain described in the present application shows relatively stable, moderate but slightly special morphology, especially the jagged edge and abnormal color, which characterizes the difference in genetic background or metabolic pathway of the Saccharomyces cerevisiae CPJ-02 strain. Microscopically, the Saccharomyces cerevisiae CPJ-02 strain described in the present application maintains the standard characteristics of Saccharomyces cerevisiae, with regular cell morphology, suitable for large-scale fermentation control and cell recovery. Its low budding frequency can prolong the fermentation period and improve the delicacy of flavor.

[0181] In summary, the macroscopic features (circular, jagged edge, dry and matte surface, light yellow) and microscopic features (circular cells, low budding frequency, moderate cells) of the Saccharomyces cerevisiae CPJ-02 strain of the present application together reflect its excellent biological adaptability and industrial potential. These features help the Saccharomyces cerevisiae CPJ-02 strain of the present application to maintain high activity and purity in fermentation production, while facilitating colony identification, isolation and quality control. Low budding frequency helps to reduce unnecessary growth energy consumption, improve fermentation efficiency and product consistency, and is an important phenotypic basis for excellent fermentation yeast.

[0182] Secondly, the microphysiological differences of the Saccharomyces cerevisiae CPJ-02 strain of the present application, specifically the metabolic pathways and stress response characteristics, indicate the behavior of the Saccharomyces cerevisiae CPJ-02 strain of the present application under specific metabolic pathways, time axis and stress environment.

[0183]

[0184] Conclusion 2:

[0185] In fruit wine, fruit juice or mixed sugar source fermentation, the Saccharomyces cerevisiae CPJ-02 strain of the present application can avoid the secondary delay problem of "glucose preferential depletion-stasis-refermentation", achieve a smooth fermentation curve, and is conducive to industrial assembly line production; the Saccharomyces cerevisiae CPJ-02 of the present application has a "atypical sugar inhibition type" carbon metabolism regulation mode, and is an excellent strain for low-temperature slow fermentation or complex sugar source fermentation.

[0186] The Saccharomyces cerevisiae CPJ-02 strain of the present application is a mild and low-intensity fermentation yeast, suitable for flavor-sensitive products (such as rice wine, medicinal wine, honey wine) or time-delay fermentation process scenarios, such as some hand-crafted craft beer, slow-fermentation bread and low-foam fermentation systems. At the same time, the later peak can leave more time for flavor fermentation, which is conducive to the generation of flavor substances and mellowing; but it is not suitable for high-alcohol industrial fermentation.

[0187] The acid resistance index of the Saccharomyces cerevisiae CPJ-02 strain described in the application is very outstanding, which is one of the highlights of its industrial potential, and has obvious application value in acid drinks (such as sour fruit wine, certain fermented tea beverages) or low pH conditions, biological preservation scenes, can reduce the sterilization cost and improve the product safety.

[0188] The Saccharomyces cerevisiae CPJ-02 strain described in the application has slow and stable fermentation characteristics, which makes it easier to control flavor release during fermentation, reduces the common problem of secondary fermentation stagnation, and is especially suitable for products with high flavor requirements such as craft beer, slow-fermented bread, fruit wine and fruit-flavored sparkling beverages. The high acid resistance of the Saccharomyces cerevisiae CPJ-02 strain described in the application gives it strong process adaptability, and it still maintains good growth activity in an acidic environment with a pH of 3.0, which helps to reduce the dependence on pH regulation during production, prolongs the shelf life of the product, and is suitable for the production of vinegar, fruit vinegar beverages and other acidified fermented products. In addition, the Saccharomyces cerevisiae CPJ-02 strain described in the application has good carbon source utilization ability, can smoothly metabolize glucose and fructose at the same time, and eliminate metabolic lag caused by "glucose effect", and is especially suitable for mixed sugar source fermentation systems such as fruit juice, sugar syrup, fruit vinegar, and other special process scenarios such as yeast leaching.

[0189] Thirdly, the Saccharomyces cerevisiae CPJ-02 strain described in the application has behavior ability and product performance in the industrial fermentation process, specifically the comprehensive performance description of the fermentation behavior and environmental adaptability, which illustrates the practical application value of the Saccharomyces cerevisiae CPJ-02 strain described in the application in "alcohol tolerance", "aroma expression", "acid-base adaptation", "metabolic efficiency", "antioxidant capacity" and other multidimensional performances, mainly used for strain selection, process optimization and product flavor positioning.

[0190]

[0191] Conclusion 3: The Saccharomyces cerevisiae CPJ-02 strain of the application can undertake the fermentation task of medium-high concentration alcohol drinks, is a typical representative of high-yield and stable fermentation yeast, has strong ethanol tolerance, and can undertake the fermentation demand of medium-high concentration alcohol drinks. The Saccharomyces cerevisiae CPJ-02 strain of the application has obvious ester metabolism advantages, can efficiently synthesize octanoic acid ethyl ester, phenylethanol and other key aroma substances, endows the wine body with rich fruity and floral aroma, and is suitable for the brewing of aroma-dominant wine products and is very suitable for the development of aroma-dominant wine products. The Saccharomyces cerevisiae CPJ-02 strain of the application has a wide pH adaptation range and good organic acid tolerance, can adapt to various fermentation substrates and process environments, and has strong fermentation process stability. The Saccharomyces cerevisiae CPJ-02 strain of the application has rapid sugar metabolism and high alcohol conversion rate, is an important advantage strain for improving production capacity and reducing raw material waste in industrial scale fermentation, and is helpful to improve production capacity and reduce raw material loss. The Saccharomyces cerevisiae CPJ-02 strain of the application has a wide pH adaptation range and good organic acid tolerance, is widely adaptable and strongly acid-resistant, can adapt to various fermentation substrates and process environments, and has strong fermentation process stability. The Saccharomyces cerevisiae CPJ-02 strain of the application has moderate foam generation and general subsidence, and is more suitable for dynamic fermentation process, and is suitable for use in combination with mechanical clarification or filtration and other post-processing technologies. In addition, the fermentation broth of the Saccharomyces cerevisiae CPJ-02 strain of the application is amber (OD 420 = 0.42), the DPPH free radical scavenging rate reaches 72% after being diluted by 10 times, the fermentation product has significant antioxidant function, also has functional drink development potential, and is suitable for the research and development of healthy wine or nutritional brewing products.

[0192] Fourthly, the Saccharomyces cerevisiae CPJ-02 strain of the application has yeast strain characteristics and application potential, which shows that the Saccharomyces cerevisiae CPJ-02 strain of the application meets the morphological standard of typical Saccharomyces cerevisiae, has high genetic stability and purity, and has good flavor shaping ability, and the overall flavor performance is natural and refreshing in sensory, and is suitable for brewing fruity wine, low-alcohol fermented drinks or functional drinks.

[0193]

[0194] Conclusion 4: The Saccharomyces cerevisiae CPJ-02 strain described in the present application is a Saccharomyces cerevisiae strain with multiple advantages, which has significant application value in the direction of flavor-type fermented wine, fruit wine, and drinkable functional fermentation liquor. If used for industrial development, it is recommended to focus on its temperature stability and aroma component regulation mechanism. Firstly, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has strong tolerance and can adapt to high temperature (42℃) and high salt environment (NaCl is 7%), so it can grow stably in complex environment. Secondly, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has high fermentation capacity and can quickly consume sugar and produce alcohol in a short time. Thirdly, the Saccharomyces cerevisiae CPJ-02 strain described in the present application contains rich aroma components, especially ester aroma, which is very suitable for fruit wine, floral drink and other fermented products that require special aroma. Fourthly, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has good sugar source utilization, so it can perform well in different fermentation substrates. Therefore, the Saccharomyces cerevisiae CPJ-02 strain described in the present application is not only suitable for efficient brewing and fermented drink production, but also can be applied to the field of functional food, which has great industrial application potential.

[0195] From the aspects of efficient alcohol production and diversified fermentation application, since the Saccharomyces cerevisiae CPJ-02 strain described in the present application has high alcohol yield (10.2% v / v) and fast fermentation rate (CO2 release peak is reached within 36 hours), the Saccharomyces cerevisiae CPJ-02 strain described in the present application can be widely used in the production of high-alcohol drinks, such as liquor, fruit wine and beer. This high-efficiency brewing capacity makes it have a competitive advantage in industrial large-scale production, which can greatly improve production efficiency and reduce cost.

[0196] From the aspect of special drinks and flavor customization, the Saccharomyces cerevisiae CPJ-02 strain of the present application exhibits rich aroma components, especially ester aroma, which makes it an ideal choice for developing special flavor drinks; the Saccharomyces cerevisiae CPJ-02 strain of the present application is suitable for the fermentation of floral and fruity drinks, such as the development of flower-fruit wine and low-alcohol flavored beverages, to meet the market demand for low-alcohol, rich-taste and unique-flavor drinks; meanwhile, the Saccharomyces cerevisiae CPJ-02 strain of the present application can be combined with other flavoring agents to further develop personalized drinks that meet the taste of consumers, such as special fruit-flavored beer and wine mixed drinks.

[0197] From the aspect of functional food development, the Saccharomyces cerevisiae CPJ-02 strain of the present application has potential in the field of functional foods due to its antioxidant activity (DPPH free radical scavenging rate of 72%). Its fermentation broth may contribute to anti-aging, anti-inflammatory, immune regulation, etc. With the continuous development of the functional food market, the Saccharomyces cerevisiae CPJ-02 strain of the present application can be applied to the development of nutritionally enhanced drinks, such as fermented products with antioxidant and intestinal flora regulating functions; and the development of functional yeast products, such as dietary supplements.

[0198] From the aspect of application expansion in high-temperature and high-salt environments, the Saccharomyces cerevisiae CPJ-02 strain of the present application has tolerance to high salt (NaCl 7%) and high temperature (42℃) environments, which provides a wide range of possibilities for its application in extreme environment fermentation. This makes the Saccharomyces cerevisiae CPJ-02 strain of the present application have great application prospects in marine, industrial wastewater and extreme climate fermentation, such as the production of marine fermented foods (such as seaweed drinks, seawater fermented products, etc.).

[0199] Example 4 Molecular functional structure of the Saccharomyces cerevisiae CPJ-02 strain of the present application

[0200] Example 4 provides a (residue) sequence list of the Saccharomyces cerevisiae CPJ-02 strain, which can be used for molecular identification and phylogenetic analysis, confirming the species attribution of the Saccharomyces cerevisiae CPJ-02 strain and the genetic 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-02 strain in fermentation metabolism, aroma synthesis, stress resistance, etc. can be preliminarily revealed, and important basis for subsequent molecular marker development, strain improvement and molecular detection method design is provided.

[0201] Specifically, the sequence list of the Saccharomyces cerevisiae CPJ-02 strain of the present application is as follows.

[0202]

[0203] Further, the sequence function positioning and BLAST analysis of the Saccharomyces cerevisiae CPJ-01 strain of the present application are as follows:

[0204] First, the sequence list of the Saccharomyces cerevisiae CPJ-02 strain of the present application is as shown in the following table through NCBI BLAST (or local blastn) comparison results.

[0205]

[0206] Conclusion 1: The sequence list of the Saccharomyces cerevisiae CPJ-02 strain of the present application, after NCBI BLASTn comparison of the Saccharomyces cerevisiae CPJ-02 nucleic acid sequence listed in Table 8, the Top hit is the model strain S. cerevisiae S288c (GenBank: NC_001133.9), the comparison length is 861 bp, the sequence similarity is 99.30%, the E-value is 0.0, which indicates that the sequence has clear source, clear species attribution, and high homology based on the characteristics of microvariation, and has the potential of strain molecular identification. The sequence can be used as the genetic marker sequence of the Saccharomyces cerevisiae CPJ-02 strain of the present application, for its molecular identification, traceability protection and commercial application support.

[0207] Secondly, the sequence table, sequence annotation and characteristic interpretation of the Saccharomyces cerevisiae CPJ-02 strain of the present application are as follows.

[0208]

[0209] Conclusion 2:

[0210] First, from the species accuracy confirmation, the Saccharomyces cerevisiae CPJ-02 strain of the present application is definitely a member of the Saccharomyces cerevisiae species; that is, the nucleic acid sequence is located in the D1 / D2 region of the 26S rDNA, the D1 / D2 region is the "gold standard" marker region in the classification of eukaryotic microbial species, and through NCBI BLASTn comparison, it has a homology of up to 98.7% with the standard Saccharomyces cerevisiae strain (such as S288C, CBS1171), fully meeting the molecular standard for yeast species confirmation, and it is definitely confirmed that the CPJ-02 strain is a member of the Saccharomyces cerevisiae population. In microbial taxonomy, the sequence difference of 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-02 strain of the present application matches the Saccharomyces cerevisiae reference strain (such as S288C) by 99.19%, which falls within the "same species" interval. Therefore, the sequence of the Saccharomyces cerevisiae CPJ-02 strain of the present application can exclude other fungi (yeast or mold), bacteria and other miscellaneous bacteria, ensuring the accuracy and reliability of the strain identification of the Saccharomyces cerevisiae CPJ-01 strain of the present application.

[0211] Second, from the phylogenetic characteristics, the Saccharomyces cerevisiae CPJ-02 strain of the present application has species specificity and high intra-species consistency, that is, the sequence evolution rate in the D1 / D2 region is moderate, the inter-species difference is significant, and the intra-species difference is extremely small, so the sequence of the Saccharomyces cerevisiae CPJ-02 strain of the present application has extremely high resolution and is suitable for phylogenetic and strain tracing research. Further, the D1 / D2 region sequence of the Saccharomyces cerevisiae CPJ-02 strain of the present application is highly consistent with the existing industrial yeast strains, indicating that it has a strong genetic relationship with the mainstream strains widely used in industrial fermentation at present.

[0212] Third, the sequence of the Saccharomyces cerevisiae CPJ-02 strain described in the present application can be screened for SNP sites, primer binding sites, etc., for the development of specific detection primers for CPJ-01 (for strain tracking, purity control, etc. in industrial production processes). In the D1 / D2 region of 600-700 bp, find nucleotide polymorphisms (SNP) or small insertions / deletions (Indel) that are different from the Saccharomyces cerevisiae reference strain; based on these specific sites, design a pair of PCR primers that can only amplify the sequence of the Saccharomyces cerevisiae CPJ-02 strain described in the present application, and not amplify other strains. In addition, in qPCR or conventional PCR detection, as long as the specific amplification product is detected, the presence of the Saccharomyces cerevisiae CPJ-02 strain described in the present application can be confirmed, and real-time monitoring of whether the fermenter is invaded by other strains or contaminated bacteria can be performed.

[0213] Fourth, in terms of molecular marker value, the Saccharomyces cerevisiae CPJ-02 strain described in the present application can serve as an "identity card" for industrial yeast tracking and contamination monitoring. The sequence of the Saccharomyces cerevisiae CPJ-02 strain provided in the present application can serve as a specific molecular marker thereof. Once a database is constructed, rapid discrimination and tracking of strains in an industrial environment can be achieved through PCR and sequence alignment. In food, alcohol, enzyme preparation, and other industrial fermentation scenarios, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has traceability and manageability, which helps to improve the biological safety and process stability of the production process. The sequence can serve as a specific molecular marker of the Saccharomyces cerevisiae CPJ-02 strain described in the present application.

[0214] Fifth, from the strain source characteristics, high consistency implies that the Saccharomyces cerevisiae CPJ-02 strain described in the application is an industrial preferred or improved strain, that is, in view of its high similarity with the industrial strain, rather than the wild type sequence, it is explained that the Saccharomyces cerevisiae CPJ-02 strain described in the application is an advantageous mutant strain screened from the industrial production process; It is also a functional strain artificially optimized and bred, which has excellent fermentation performance, stress resistance or specificity of metabolic products; This endows the Saccharomyces cerevisiae CPJ-02 strain described in the application with higher industrial application potential.

[0215] Sixth, the sequence of the Saccharomyces cerevisiae CPJ-02 strain described in the application does not encode enzymes or metabolic proteins, and belongs to part of the untranslated structural RNA (rRNA) gene. The 26S D1 / D2 region is essentially part of the structural rRNA, which is used to assemble the large subunit of ribosomes, and never produces any enzyme or metabolic protein; 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 that there is little variation within the same species, ensuring that the results are stable and reliable; variability is enough to distinguish between species, and different species will not be confused. Therefore, with the help of this D1 / D2 sequence, both the species attribution of the Saccharomyces cerevisiae CPJ-02 strain described in the application can be explained at the basic taxonomy level, and a rapid and specific detection and monitoring system can be established at the industrial application level, ensuring production safety and product consistency, and becoming the preferred molecular marker for strain monitoring, purity control and pollution investigation in the industrialization process.

[0216] Example 5: Phylogeny and species attribution of the Saccharomyces cerevisiae CPJ-02 strain described in the application

[0217] As shown in Figure 2 , this is the development tree image of the Saccharomyces cerevisiae CPJ-02 strain described in the application, and example 5 focuses on the phylogenetic relationship, species attribution, close relationship and possible industrial application source of the Saccharomyces cerevisiae CPJ-02 strain described in the application.

[0218] First, the Saccharomyces cerevisiae CPJ-02 strain described in the present application is classified as Saccharomyces cerevisiae in the phylogenetic tree, rather than other closely related species; as shown in the figure, this is the conclusion from two levels of branch clustering and branch length, sequence similarity comparison. Figure 2

[0219] From the aspects of branch clustering and branch length, including cluster clustering and obvious differentiation from closely related species.

[0220] First, in terms of cluster clustering, in the phylogenetic tree constructed after multiple sequence alignment, the Saccharomyces cerevisiae CPJ-02 strain described in the present application and the typical Saccharomyces cerevisiae reference strain (such as S288C, CBS1171 and other strains) fall into the same monophyletic clade; and the divergence between all nodes in the cluster is small, with a branch distance close to 0, which indicates that the sequences of these strains in the D1 / D2 extension region are almost identical; and other closely related species, such as S. paradoxus, S. bayanus, S. kudriavzevii, etc., are obviously divided into different branches.

[0221] Conclusion 1: The Saccharomyces cerevisiae CPJ-02 strain described in the present application is clearly classified as Saccharomyces cerevisiae; the branch of the Saccharomyces cerevisiae CPJ-02 strain described in the present application is in the same cluster (Cluster) as multiple standard Saccharomyces cerevisiae (S. cerevisiae) strains, and the branch length is extremely short (distance close to 0), indicating that these strains are highly homologous in the D1 / D2 region; the D1 / D2 sequence of the Saccharomyces cerevisiae CPJ-02 strain described in the present application has homology with the authentic strain of S. cerevisiae, and does not fall into the branch of any other Saccharomyces species.

[0222] ​Secondly, the obvious differentiation from the close species further supports the conclusion of BLAST in Example 4 that the Saccharomyces cerevisiae CPJ-02 strain described in the present application is the standard Saccharomyces cerevisiae species, rather than other close species, such as Saccharomyces paradoxus (heterogeneous Saccharomyces cerevisiae), Saccharomyces uvarum (mountain grape yeast), Kluyveromyces lactis (lactose yeast) or wild-type yeast population.

[0223] As shown in Figure 2 the following table illustrates the position of the close species of the Saccharomyces cerevisiae CPJ-02 strain described in the present application.

[0224]

[0225] As shown in Figure 2 the following table illustrates the sequence similarity comparison of the Saccharomyces cerevisiae CPJ-02 strain described in the present application.

[0226]

[0227] As can be seen, in mycology, the difference between the same species in 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 the present application and Saccharomyces cerevisiae reach 99.19% (the difference is only 0.81%), which is much lower than the difference level with other close species (such as Saccharomyces bayanus (low-temperature yeast), Saccharomyces pastorianus (beer yeast hybrid), Kluyveromyces lactis (lactose yeast) or wild-type yeast population), which meets the "intraspecific variation" category.

[0228] Conclusion 1: According to Figure 2As shown in the phylogenetic tree, the Saccharomyces cerevisiae CPJ-02 strain of the present application is clearly classified as Saccharomyces cerevisiae species in systematic taxonomy, rather than other species closely related thereto. From the structure of the phylogenetic tree, the CPJ-02 strain has a highly consistent branch clustering relationship with the standard S. cerevisiae reference strain, with a very short branch length, indicating that the two have minimal differences at the genomic level and have high similarity. At the same time, through sequence alignment of the ribosomal ITS region and the 26S rDNA D1 / D2 domain (BLAST analysis as described in Example 4), it is further confirmed that the sequence similarity between the CPJ-02 strain and the standard S. cerevisiae is as high as 99% or more, and no mutation of key conserved sites occurs. This result is clearly reflected in the phylogenetic tree, with the branch of CPJ-02 being clearly distinguished from other closely related species such as S. paradoxus, S. bayanus, S. kudriavzevii, S. pastorianus, etc., showing a clear differentiation trend.

[0229] Conclusion 2: From the strain identification, the Saccharomyces cerevisiae CPJ-02 strain of the present application can be determined to not contain any S. bayanus, S. pastorianus or other hybrid / wild background, avoiding deviations in process applicability or flavor expectations.

[0230] Conclusion 3: From the controllable fermentation, the Saccharomyces cerevisiae CPJ-02 strain of the present application is highly consistent with the industrial standard strain, and the fermentation performance and genetic background can be referred to the known strain, facilitating batch-to-batch stability comparison and parameter optimization. CPJ-02 belongs to typical Saccharomyces cerevisiae, rather than the above-mentioned closely related species. This phylogenetic result provides a molecular systematic basis for the species attribution of the strain of the present application, and also lays a clear classification foundation for subsequent research and application of the strain in fermentation characteristics, biosynthesis ability, etc.

[0231] Example 6: Explanation of the genetic basis of the Saccharomyces cerevisiae CPJ-02 strain of the present application with industrial potential

[0232] Example 6 will be from the existing performance of the Saccharomyces cerevisiae CPJ-02 strain described in the application, combined with the key functional gene sequence type of Saccharomyces cerevisiae species, to illustrate the genetic basis of the industrialization potential of the Saccharomyces cerevisiae CPJ-02 strain described in the application.

[0233] First, the industrial traits overview of the Saccharomyces cerevisiae CPJ-02 strain described in the application and the target gene analysis requirements are as follows.

[0234]

[0235] Second, the core industrial functional module gene analysis of the Saccharomyces cerevisiae CPJ-01 strain described in the application.

[0236] First, the key genes of the alcohol fermentation and sugar metabolism module of the Saccharomyces cerevisiae CPJ-01 strain described in the application are ADH1, ADH2, PDC1, ALD6, which are specifically described in the following table.

[0237]

[0238] Conclusion 1: The Saccharomyces cerevisiae CPJ-02 strain described in the application has a complete enzyme system at the "acetaldehyde→ethanol→acetic acid" three-phase node, ADH1, ADH2 (alcohol dehydrogenase) and PDC1 (pyruvate decarboxylase) act on the terminal reaction of glycolysis, efficiently catalyze pyruvic acid to acetaldehyde, and further to ethanol, improve the alcohol conversion efficiency; ALD6 (acetaldehyde dehydrogenase) further controls the conversion of acetaldehyde, affecting the ethanol yield and fermentation efficiency.

[0239] Second, the key genes of the aroma substance synthesis module of the Saccharomyces cerevisiae CPJ-02 strain described in the application are ATF1, ATF2, EEB1, BAT1, BAT2, which are specifically described in the following table.

[0240]

[0241] Conclusion 2: The Saccharomyces cerevisiae CPJ-02 strain described in the application exhibits excellent aroma synthesis capacity under the synergistic effect of key genes ATF1, ATF2, EEB1, BAT1 and BAT2, especially in the high yield of ethyl caprylate and phenethyl alcohol, and has a natural expression trend of up-regulating synthesis enzymes and down-regulating degradation enzymes, which is beneficial to aroma enrichment, and is an ideal "flavor-tuning fermentation yeast"; the gene optimization and industrial application of the Saccharomyces cerevisiae CPJ-02 strain described in the application will provide important theoretical basis and technical support for aroma-oriented wine products, and has excellent industrial development potential and broad market prospect.

[0242] Thirdly, the key genes of the stress resistance and homeostatic regulation module of the Saccharomyces cerevisiae CPJ-02 strain described in the application are ERG2, ERG6, HSP104, TPS1, PMA1 and ENA1, which are specifically described in the following table.

[0243]

[0244] Conclusion 3: The Saccharomyces cerevisiae CPJ-02 strain described in the application exhibits excellent ability in stress resistance and homeostatic regulation, especially in the growth stability under adverse environments such as high alcohol, low pH and high salt. By optimizing the expression of key genes such as ERG2, ERG6, HSP104, TPS1, PMA1 and ENA1, the performance of the Saccharomyces cerevisiae CPJ-02 strain described in the application in the industrial fermentation process can be further improved, the adaptability of the Saccharomyces cerevisiae CPJ-02 strain described in the application in complex environments can be increased, and more efficient and stable production can be promoted.

[0245] Fourthly, the key genes of the antioxidant response module of the Saccharomyces cerevisiae CPJ-02 strain described in the application are SOD1, CTT1, GSH1 and GPX1, which are specifically described in the following table.

[0246]

[0247] Conclusion 4: The Saccharomyces cerevisiae CPJ-02 strain described in the present application has both enzymatic and non-enzymatic antioxidant system dual pathway activity, and is an important yeast resource for the development of functional health drinks. In the antioxidant response module, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has high expression of key genes such as SOD1, CTT1, GSH1, GPX1, etc., so that the Saccharomyces cerevisiae CPJ-02 strain described in the present application exhibits strong antioxidant capacity under oxidative stress environments such as high oxygen, high temperature, and hydrogen peroxide. These genes not only enhance the survival ability of the Saccharomyces cerevisiae CPJ-02 strain described in the present application, but also improve the cell activity and stability during fermentation; by further optimizing the expression of these genes, the antioxidant capacity of the strain in industrial fermentation can be improved, the product quality can be improved, the yield can be increased, and it is especially suitable for brewing processes under harsh environments such as high oxygen or high temperature.

[0248] Fifth, the key genes of the Saccharomyces cerevisiae CPJ-01 strain described in the present application for the sedimentation and process adaptation module are FLO1, FLO5, FLO8, FLO11, which are specifically explained in the following table.

[0249]

[0250] Conclusion 5: The Saccharomyces cerevisiae CPJ-02 strain described in the present application has significant potential in terms of sedimentation and process adaptation, especially in the separation process after fermentation. The Saccharomyces cerevisiae CPJ-02 strain described in the present application can further improve the sedimentation and cell aggregation of the yeast, improve the separation efficiency of the fermentation broth and solid materials, and reduce the energy consumption and time cost in the industrial fermentation process, by optimizing the expression of key genes such as FLO1, FLO5, FLO8, FLO11, etc. Genetic engineering for sedimentation optimization can make the Saccharomyces cerevisiae CPJ-02 strain described in the present application more efficient and stable in large-scale industrial fermentation, and adapt to various fermentation process requirements.

[0251] Third, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has a genetic basis and phenotype support for industrialization potential, which is specifically explained in the following table.

[0252]

[0253] Conclusion 6: The Saccharomyces cerevisiae CPJ-02 strain has strong industrial potential of alcohol production, aroma synthesis, stress resistance, and sedimentation, and the genetic basis and phenotype support provide a solid foundation for its wide application in industrial fermentation. Through optimization and engineering of related key genes, the Saccharomyces cerevisiae CPJ-02 strain can exhibit efficient and stable production performance in various industrial fermentation environments, has great market application potential, and is particularly suitable for high-alcohol concentration, brewing, and fruit wine production.

[0254] From the aspects of high alcohol tolerance and fermentation efficiency, the alcohol yield of the Saccharomyces cerevisiae CPJ-02 strain is 0.45 g / g, and the alcohol concentration can reach 8.5% (v / v), which shows that the Saccharomyces cerevisiae CPJ-02 strain has extremely high alcohol tolerance and efficient alcohol fermentation capacity.

[0255] From the synthesis capacity of aroma substances, the ester aroma substances of the Saccharomyces cerevisiae CPJ-02 strain account for 52% of the total aroma, ethyl caprylate is 62 mg / L, and phenethyl alcohol is 48 mg / L, which indicates that the Saccharomyces cerevisiae CPJ-02 strain has high potential in the synthesis of aroma substances and is suitable for the production of high-quality alcohol and fruit wine.

[0256] From the aspects of antioxidant capacity and stress adaptability, in high-oxygen, high-temperature, and hydrogen peroxide environments, the Saccharomyces cerevisiae CPJ-02 strain can still grow normally, showing significant antioxidant and stress resistance capacity; this ensures the stability and production efficiency of the Saccharomyces cerevisiae CPJ-02 strain in adverse environments.

[0257] From good sedimentation and process adaptability, the Saccharomyces cerevisiae CPJ-02 strain has good sedimentation, and the 24-hour sedimentation rate reaches 30%; the Saccharomyces cerevisiae CPJ-02 strain has a faster sedimentation speed and flocculation, so that in the large-scale industrial fermentation process, the separation efficiency is improved, and the energy consumption in the separation process is reduced.

[0258] Example 7 The Saccharomyces cerevisiae CPJ-02 strain in the food engineering application of the present application

[0259] Example 7 focuses on the reproducibility, practicability and popularization value of the Saccharomyces cerevisiae CPJ-02 strain, and the purpose is to fully reflect the implementability, application value and industrial potential of the Saccharomyces cerevisiae CPJ-02 strain and related technical solutions. The reproducibility ensures that the strain and its screening method have a standardized operation process and can be repeatedly obtained by those skilled in the art; the practicability highlights the actual application effect and performance advantage of the Saccharomyces cerevisiae CPJ-02 strain in the fermentation of fruit wine and other products, and proves that the Saccharomyces cerevisiae CPJ-02 strain has a clear technical use; the popularization value shows that the Saccharomyces cerevisiae CPJ-02 strain has universality, universality and industrial adaptability, and supports the implementation in different scenarios and different subjects in food engineering applications.

[0260] (1) The reproducibility of the Saccharomyces cerevisiae CPJ-02 strain of the present application

[0261] In order to verify the reproducibility of the Saccharomyces cerevisiae CPJ-02 strain of the application, different batches (n=5) of Yunnan Dali Eryuan county plum samples were selected, and the independent repeated separation experiment was carried out according to the steps S1-S6. The experimental results show that the Saccharomyces cerevisiae CPJ-02 strain with consistent morphology and identified by ITS sequence is successfully separated from the five batches of samples, and the strain fermentation performance and microscopic morphology remain consistent, indicating that the Saccharomyces cerevisiae CPJ-02 strain of the application can be stably obtained under the conditions of different sample sources and different operators, and has good reproducibility and popularization and application value.

[0262] The Saccharomyces cerevisiae CPJ-02 strain of the application is obtained by the Saccharomyces cerevisias CPJ-02 strain screening and obtaining method of the application. The method makes clear technical provisions for each key link in the whole process of strain separation, including plum sample collection, preservation, treatment, dilution, separation, purification and final molecular identification. The parameters such as temperature, time, volume and reagent dosage involved in each operation step are specific and clear, which ensures that other technical personnel can completely reproduce according to the method, and there is no missing key technical node.

[0263] The reproducibility of the Saccharomyces cerevisiae CPJ-02 strain of the application refers to that the same type of yeast strain with similar characteristics can be stably obtained by different experimenters and different batches of operation according to the screening and obtaining method, and its physiological, biochemical, molecular and functional characteristics are highly consistent with the original report, which verifies the reliability and standardization of the method. Through the above standardized operation process, the Saccharomyces cerevisiae CPJ-02 strain of the application with the same characteristics as CPJ-02 is repeatedly obtained from multiple batches of plum samples, and the same type of target strain can also be successfully separated and obtained by different operators using the method at different time points. Through functional detection and molecular analysis, the obtained Saccharomyces cerevisiae CPJ-02 strain has no substantial difference in morphological characteristics, physiological activity and gene sequence from the original strain, which proves that the strain screening and obtaining method of the application has the technical advantages of strong stability, high reproducibility and popularization and application.

[0264] To ensure the accuracy and operability of the screening results, the present application further specifies the criteria for the target yeast strain (the Saccharomyces cerevisiae CPJ-02 strain described in the present application): the colony morphology includes characterization indicators such as color, diameter, edge morphology, and glossiness; microscopic observation includes quantitative and qualitative description of characteristics such as budding mode, cell size, and morphology; in terms of physiological and biochemical and molecular identification, functional indicators such as sugar source utilization and ethanol yield are combined with molecular means such as ITS sequence alignment to ensure that the isolated strain is a true Saccharomyces cerevisiae and is highly consistent with the Saccharomyces cerevisiae CPJ-02 strain described in the present application. Specifically, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has clear target strain discrimination criteria, and the colony morphology characteristics of the Saccharomyces cerevisiae CPJ-02 strain described in the present application have clear standards (color, size, gloss, texture, etc.); under a microscope, the morphology and budding mode are quantitatively or qualitatively described; the physiological and biochemical and molecular identification steps are detailed, such as ITS sequencing alignment, to ensure that the isolated strain is Saccharomyces cerevisiae.

[0265] Further, an external third-party experimental institution successfully reproduced a Saccharomyces cerevisiae strain consistent with CPJ-02 according to the method disclosed in the present application, and the verification results showed that the target strain remained consistent in morphology, biochemical characteristics, and ITS sequence, and the reproduction rate reached 100%, further supporting the scientificity and repeatability of the screening method of the present application.

[0266] In summary, the reproducibility of the Saccharomyces cerevisiae CPJ-02 strain described in the present application is reflected in the stable isolation of yeast strains with similar characteristics under different experimental personnel and different sample batches, and the physiological, biochemical, molecular, and functional indicators are consistent with the original strain, fully verifying the reliability of the present application in terms of controllability, consistency, and standardization.

[0267] (2) Practicality of the Saccharomyces cerevisiae CPJ-02 strain described in the present application

[0268] The Saccharomyces cerevisiae CPJ-02 strain of the present application has a wide industrial application potential, especially in alcohol fermentation, aroma substance synthesis, antioxidant capacity, stress adaptability and sedimentation, and has a significant advantage.

[0269] From the aspects of alcohol fermentation and high-efficiency alcohol production, the Saccharomyces cerevisiae CPJ-02 strain of the present application has the characteristics of high alcohol yield and strong high-alcohol tolerance. In terms of high alcohol yield, the Saccharomyces cerevisiae CPJ-02 strain of the present application exhibits a high alcohol yield (0.45 g / g) during alcohol fermentation, and can generate high-concentration alcohol in a short time. Therefore, the Saccharomyces cerevisiae CPJ-02 strain of the present application can significantly improve the efficiency of alcohol production, and is suitable for various types of alcohol production processes, including traditional alcohol fermentation, industrial alcohol production and biofuel production. In terms of strong high-alcohol tolerance, the Saccharomyces cerevisiae CPJ-02 strain of the present application has strong alcohol tolerance, can continue to ferment under high-alcohol concentration (for example, 8.5% v / v), and exhibits excellent alcohol tolerance characteristics. Therefore, the Saccharomyces cerevisiae CPJ-02 strain of the present application maintains high growth activity under high-alcohol concentration environment, can cope with the inhibitory effect of traditional yeast in high-alcohol environment, and is suitable for fermentation processes with high alcohol concentration.

[0270] From the aroma substance synthesis ability, the Saccharomyces cerevisiae CPJ-02 strain has the synthesis ability of aroma substances and the value of improving product quality. In terms of synthesis of aroma substances, the Saccharomyces cerevisiae CPJ-02 strain has a significant advantage in the synthesis of aroma substances, and the ester aroma substance accounts for 52% of the total aroma, octanoic acid ethyl ester 62 mg / L, phenethyl alcohol 48 mg / L. Therefore, the Saccharomyces cerevisiae CPJ-02 strain is suitable for fruit wine, grape wine, beer and other fermentation products that require specific aroma. The efficient synthesis of aroma substances can improve the flavor and aroma of the fermentation product, and has a wide application prospect in the brewing industry. In terms of improving product quality, by optimizing the synthesis of aroma substances, the Saccharomyces cerevisiae CPJ-02 strain can bring richer and more layered aroma to wine products, improving the sensory experience of consumers. Therefore, the Saccharomyces cerevisiae CPJ-02 strain is suitable for the production of wine and other fermented products (such as yogurt, vinegar), which can improve the market competitiveness of products and meet the needs of consumers for high-quality aroma.

[0271] From the stress resistance and environmental adaptability, the Saccharomyces cerevisiae CPJ-02 strain has antioxidant capacity and stress resistance and homeostatic regulation capacity. In terms of antioxidant capacity, the Saccharomyces cerevisiae CPJ-02 strain exhibits strong antioxidant capacity and can grow well in high-oxygen, hydrogen peroxide and other oxidative stress environments, indicating that it has a good antioxidant response mechanism. Therefore, in a high-oxygen environment and a long fermentation process, the Saccharomyces cerevisiae CPJ-02 strain can maintain high activity and stability, adapt to more harsh fermentation conditions, and improve the stability of large-scale industrial fermentation. In terms of stress resistance and homeostatic regulation, the Saccharomyces cerevisiae CPJ-02 strain has strong stress resistance and can grow well in high-alcohol, low-pH, salt stress and other environments, showing excellent homeostatic regulation mechanism. Therefore, the Saccharomyces cerevisiae CPJ-02 strain is suitable for various fermentation processes with large environmental changes, and can maintain stable performance in complex and changing industrial fermentation processes to ensure product quality consistency and production stability.

[0272] From the sedimentation and process adaptability, the Saccharomyces cerevisiae CPJ-02 strain has the characteristics of excellent sedimentation performance and strong process adaptability. In terms of excellent sedimentation performance, the Saccharomyces cerevisiae CPJ-02 strain has good sedimentation performance, and the 24-hour sedimentation rate reaches 30%, showing high sedimentation speed and effective cell aggregation. Therefore, the Saccharomyces cerevisiae CPJ-02 strain has excellent sedimentation performance, which can realize rapid separation in the post-processing of wine and other fermented products, reduce the time and energy consumption of solid-liquid separation. In terms of process adaptability, the Saccharomyces cerevisiae CPJ-02 strain can adapt to various industrial fermentation processes, including large-scale alcohol fermentation, aroma substance synthesis and other complex fermentation processes. Therefore, in actual industrial application, the Saccharomyces cerevisiae CPJ-02 strain can improve production efficiency, reduce energy consumption, optimize process flow, and has good adaptability in different types of fermentation environment.

[0273] (3) The fermentation performance and adaptability of the Saccharomyces cerevisiae CPJ-02 strain

[0274] The Saccharomyces cerevisiae CPJ-02 provided by the present application is an excellent fermentation industrial strain obtained by screening, which has significant fermentation performance advantages and environmental adaptability in various fermentation environments, and has industrial application value. The Saccharomyces cerevisiae CPJ-02 strain has stable physiological and biochemical characteristics, clear genetic background, high sugar fermentation capacity, high-concentration ethanol tolerance, high-osmotic pressure tolerance, high-aroma-substance yield, and is suitable for beer, yellow rice wine, fruit wine, grape wine, distilled liquor and other types of brewing scenes.

[0275] Compared with the control experiment, the Saccharomyces cerevisiae CPJ-02 strain provided by the application has excellent fermentation performance, mainly embodied in the following aspects: first, the Saccharomyces cerevisiae CPJ-02 strain provided by the application has a high sugar conversion efficiency, and in the wort or grape juice substrate with an initial sugar concentration of 30-35 °Bx, the sugar content can be reduced to 1-2 g / L within 48-72 hours, the sugar conversion rate is increased by 15%-22% compared with the control industrial yeast, and the fermentation period can be significantly shortened; secondly, under the conventional fermentation conditions (28-30 °C, pH 4.0-4.5, static or slight aeration), the final ethanol yield of the Saccharomyces cerevisiae CPJ-02 strain provided by the application is stable at 13.5-14.8% (v / v), the ethanol yield reaches more than 91% of the theoretical conversion rate, and the Saccharomyces cerevisiae CPJ-02 strain provided by the application has the advantages of high ethanol yield; in addition, the Saccharomyces cerevisiae CPJ-02 strain provided by the application can synthesize and enrich a variety of volatile aroma substances in the metabolic process, including esters (such as ethyl acetate and ethyl lactate) and higher alcohols (such as isoamyl alcohol and phenethyl alcohol), and the total aroma component content of the Saccharomyces cerevisiae CPJ-02 strain provided by the application is increased by 30%-45% compared with the control strain, which significantly improves the flavor quality and sensory characteristics of the fermentation product, and reflects the excellent brewing applicability and industrial promotion value.

[0276] The Saccharomyces cerevisiae CPJ-02 strain provided by the application has excellent environmental adaptability, and the cell survival rate is still higher than 80% under the condition of 40 °Bx high osmotic pressure, and the sugar metabolism activity is good; when the ethanol concentration in the fermentation system is increased to 16% (v / v), the cell membrane integrity is not significantly damaged, the physiological activity is stable, and the fermentation process continues; the suitable growth and fermentation temperature is 25-35 °C, and the upper limit of heat tolerance can reach 38 °C, and no performance degradation is found. At the same time, the Saccharomyces cerevisiae CPJ-02 strain provided by the application can maintain active metabolism in a wide range of pH 3.0-6.0, and adapt to the acid-base change of various natural yeast fermentation systems.

[0277] The Saccharomyces cerevisiae CPJ-02 strain provided by the present application has excellent industrial adaptability and genetic stability, and after continuous subculture for more than 10 generations, the fermentation capacity, ethanol yield and aroma metabolism characteristics of the Saccharomyces cerevisiae CPJ-02 strain do not appear significant variation, the RAPD and ISSR molecular fingerprint analysis results show that the genotype is highly consistent, and the Saccharomyces cerevisiae CPJ-02 strain belongs to an industrial strain with good genetic stability. In terms of process adaptability, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application can be widely applied to various brewing modes such as continuous fermentation, semi-continuous fermentation and traditional batch fermentation, and can be used in cooperation with pectinase, saccharifying enzyme, esterifying enzyme and other enzyme preparations, and shows good process compatibility. In addition, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application has high adaptability to various fermentation substrates, and can effectively ferment malt juice, fruit and vegetable juice, glutinous rice slurry, sweet sorghum juice and molasses hydrolysate and other carbon source substrates, and has good industrialization promotion prospect and application flexibility.

[0278] (4) The promotion value of the Saccharomyces cerevisiae CPJ-02 strain described in the present application is explained.

[0279] The Saccharomyces cerevisiae CPJ-02 strain provided by the present application is a Saccharomyces cerevisiae with excellent fermentation performance, wide environmental adaptability and excellent industrial application potential, and has significant industrialization promotion value. The Saccharomyces cerevisiae CPJ-02 strain provided by the present application not only can grow and ferment stably under multiple stress conditions such as high sugar, high osmotic pressure and high ethanol concentration, but also is suitable for various complex or extreme fermentation environments, and shows good adaptability and process flexibility in multiple traditional and novel brewing processes, and can be widely used in the preparation of beer, fruit wine, yellow rice wine, liquor, fermented beverage and functional wine and other fermented products.

[0280] The Saccharomyces cerevisiae CPJ-02 strain provided by the present application has fast sugar conversion rate, high alcohol yield and strong flavor generation capacity, can effectively improve product quality and shorten production cycle, and is helpful for enterprises to reduce comprehensive cost, improve production efficiency and product added value. At the same time, the Saccharomyces cerevisiae CPJ-02 strain provided by the present application has stable genetic background and long-term fermentation consistency, reduces the risk of strain degradation and process fluctuation in industrial application, and improves the controllability and product consistency of the brewing process.

[0281] In combination with the above advantages, the Saccharomyces cerevisiae CPJ-02 strain provided by the application meets the core requirements of modern fermentation industry for high-efficiency, stable, safe and flavor excellent strains, has good market application prospect and technical popularization value, and is suitable for being used as a basic fermentation core strain in the fields of food, wine, biological engineering and the like for large-scale deployment and long-term application.

[0282] Example 1 Comparison of high alcohol tolerance and fermentation performance of the Saccharomyces cerevisiae CPJ-02 strain of the application

[0283] In order to illustrate the fermentation stability of the Saccharomyces cerevisiae CPJ-02 strain of the application under high acidity and high alcohol concentration conditions and the unique advantages thereof in aroma generation, Example 1 is to compare the batch fermentation performance of the Saccharomyces cerevisiae CPJ-02 strain of the application and the commonly used control strains Saccharomyces cerevisiae Lalvin EC1118, Saccharomyces cerevisiae Angel YZ-3, Saccharomyces cerevisiae Red Star CEC01 and Saccharomyces cerevisiae VIN13 (abbreviated as EC1118, Angel YZ-3, Red Star CEC01 and VIN13) under the same fermentation conditions (30℃, initial sugar concentration of 200g / L, fermentation period of 48h).

[0284] Firstly, the three strains (Saccharomyces cerevisiae Lalvin EC1118, Saccharomyces cerevisiae Angel YZ-3, Saccharomyces cerevisiae Red Star CEC01 and Saccharomyces cerevisiae VIN13) used for comparison in Comparative Example 1 are described.

[0285]

[0286] Then, in order to objectively and systematically evaluate the performance of the strain under high alcohol fermentation conditions, the multi-index comprehensive evaluation system was used in Comparative Example 1, and the process significance of each index was analyzed. Unlike the traditional experiment which only uses a single index of "fermentation yield", the multi-index comprehensive evaluation system quantitatively evaluates the alcohol tolerance, cell activity, metabolic efficiency, and fermentation thoroughness, which can more truly reflect the comprehensive performance of the strain under industrial production conditions.

[0287]

[0288] As shown in Table 2, through the systematic determination and comparison of the above indexes, not only can the advantages of the strain be proved from a single dimension, but also the industrial application potential of the strain can be comprehensively evaluated from multiple angles such as tolerance, activity maintenance, conversion efficiency, and fermentation rate. The evaluation system can convert experimental data from simple numerical values into technical basis that can directly reflect the actual process value and industrialization advantages, and can intuitively understand the real industrial value of the Saccharomyces cerevisiae CPJ-02 strain.

[0289] Finally, the four strains (Saccharomyces cerevisiae Lalvin EC1118, Saccharomyces cerevisiae Angel YZ-3, Saccharomyces cerevisiae Red Star CEC01, and Saccharomyces cerevisiae VIN13) used for comparison in Comparative Example 1 were compared with the Saccharomyces cerevisiae CPJ-02 strain in terms of high alcohol tolerance and fermentation performance, and the results are shown in the following table.

[0290]

[0291] From Table 3, the following conclusions can be drawn:

[0292] From the maximum tolerance of ethanol concentration, the tolerance of ethanol concentration of the Saccharomyces cerevisiae CPJ-02 strain reaches 15.0%, which is 3.0% higher than that of the EC1118 yeast strain, 4.0% higher than that of the Angel YZ-3 yeast strain and the VIN13 yeast strain, and 3.5% higher than that of the Red Star CEC0 yeast strain; from the relative growth rate, the Saccharomyces cerevisiae CPJ-02 strain is 25.0% higher than that of the EC1118 yeast strain, 36.4% higher than that of the Angel YZ-3 yeast strain, 30.4% higher than that of the Red Star CEC01 yeast strain, and 36.4% higher than that of the VIN13 yeast.

[0293] Specifically, compared with EC1118 yeast strain (12.0%), EC1118 yeast strain as an industrial classic Saccharomyces cerevisiae has good ethanol tolerance, but under high ethanol stress (>12%), the cell membrane permeability increases, the intracellular balance is disturbed, and the fermentation power decreases; and the Saccharomyces cerevisiae CPJ-02 strain in the application is 3.0% higher, which indicates that the cell membrane lipid composition or membrane protein regulation mechanism of the Saccharomyces cerevisiae CPJ-02 strain is better than that of EC1118, and the cell membrane structure can be better stabilized, and the ethanol stress damage can be reduced. Specifically, compared with Angel YZ-3 yeast strain (11.0%) or VIN13 yeast strain (11.0%), the maximum ethanol tolerance of the two yeast strains is only 11.0%, which indicates that the Angel YZ-3 yeast strain and the VIN13 yeast strain have weak ethanol stress response mechanism, and are prone to membrane damage, protein denaturation and intracellular metabolic disorder; and the tolerance level of the Saccharomyces cerevisiae CPJ-02 strain in the application is 4.0% higher than that of the Angel YZ-3 yeast strain and the VIN13 yeast strain, which is very obvious, which means that under the high ethanol concentration fermentation environment (>13%), the Angel YZ-3 yeast strain and the VIN13 yeast strain are prone to premature aging and inactivation, while the Saccharomyces cerevisiae CPJ-02 strain in the application still has strong activity. Specifically, compared with Red Star CEC01 yeast strain (11.5%), the ethanol tolerance of the Red Star CEC01 yeast strain is 11.5%, which is slightly better than that of the Angel YZ-3 yeast strain and the VIN13 yeast strain, but still significantly lower than that of the Saccharomyces cerevisiae CPJ-02 strain in the application. The Saccharomyces cerevisiae CPJ-02 strain in the application is 3.5% higher, which indicates that the tolerance threshold of the Saccharomyces cerevisiae CPJ-02 strain to ethanol stimulation is significantly improved, and the Saccharomyces cerevisiae CPJ-02 strain has stronger intracellular protection and stress regulation capacity, such as heat shock protein expression, glycerol regulation and active oxygen scavenging mechanism.

[0294] Conclusion 1: The ethanol tolerance of the Saccharomyces cerevisiae CPJ-02 strain is as high as 15.0 % (v / v), which is significantly better than that of the control strain (11.0 % - 12.0 %). As an important metabolic product produced during the fermentation process, ethanol has an inhibitory effect on microorganisms. A too high ethanol concentration can interfere with the structure of the cell membrane and the metabolic function, affecting the survival of the yeast and the fermentation kinetics. The high ethanol tolerance of the Saccharomyces cerevisiae CPJ-02 strain indicates that the cell membrane structure of the Saccharomyces cerevisiae CPJ-02 strain is more stable, and the intracellular osmotic regulation mechanism and stress response system are more perfect, which can adapt to a high ethanol stress environment and is suitable for high-concentration ethanol fermentation process and alcohol production in a high-sugar substrate environment.

[0295] From the 24-hour survival rate, the 24-hour survival rate (92.0%) of the Saccharomyces cerevisiae CPJ-02 strain described in the application is obviously better than all the control strains, especially compared with the commonly used industrial strains EC1118 (78.5%), Red Star CEC01 (74.2%) and VIN13 (69.5%), the survival advantage is 17.2%-32.4%, which shows that the Saccharomyces cerevisiae CPJ-02 strain described in the application has strong adaptation and stress resistance ability in the starting stage of high-sugar and high-osmotic environment. From the cell membrane and osmotic pressure regulation, the Saccharomyces cerevisiae CPJ-02 strain described in the application has a higher proportion of unsaturated fatty acids or a more optimized membrane lipid composition, and the cell membrane has better stability to high-sugar and high-osmotic pressure environment, thereby resisting external osmotic stress and maintaining cell integrity. From the stress response system, the Saccharomyces cerevisiae CPJ-02 strain described in the application is obviously superior in the expression level or activity of stress proteins (Hsp104, Gpd1 / 2), and the cell in high-osmotic or high-ethanol environment will activate heat shock proteins (HSP), osmotic pressure protective proteins (such as glycerol synthase), quickly repair damaged proteins and maintain protein folding. From the active oxygen (ROS) removal, the Saccharomyces cerevisiae CPJ-02 strain described in the application has stronger antioxidant enzyme system (SOD, CAT, GPX) activity, high osmotic pressure and high ethanol will produce excess active oxygen, reduce oxidative damage, thereby improving the survival rate. From the metabolic network optimization, the Saccharomyces cerevisiae CPJ-02 strain described in the application is more efficient in glycolytic flow, pentose phosphate shunt and respiratory chain coupling, and the high survival rate in the early fermentation stage also depends on whether sufficient energy supply and metabolic balance can be maintained to ensure sufficient ATP and support cell stress resistance.

[0296] Conclusion 2: Compared with Angel YZ-3 yeast strain (90.0%), the Saccharomyces cerevisiae CPJ-02 strain of the present application still has a small advantage (+2.2%), which shows that in the same high-activity strain, the Saccharomyces cerevisiae CPJ-02 strain of the present application has better resistance to early environmental fluctuations. The high survival rate of the Saccharomyces cerevisiae CPJ-02 strain of the present application can effectively shorten the lag phase, improve the initial cell quantity and metabolic activity of fermentation, which means that the Saccharomyces cerevisiae CPJ-02 strain of the present application can rapidly adapt and maintain a high activity in the early stage of fermentation, which is very beneficial to the continuous fermentation process, especially in the continuous fermentation process with high alcohol concentration, which lays a foundation for subsequent high-efficiency ethanol production, and is an important characteristic for the development of industrial large-scale continuous fermentation or high-concentration fermentation process.

[0297] From the final ethanol yield, the Saccharomyces cerevisiae CPJ-02 strain of the present application reaches 0.45 g / g, while the best control (EC1118) is only 0.40 g / g, which is absolutely improved by 0.05 g / g; equivalent to 50 kg of ethanol produced per ton of glucose consumed.

[0298] From the relative promotion amplitude, the Saccharomyces cerevisiae CPJ-02 strain in the application is promoted by about 11.1% compared with the EC1118 yeast strain, is promoted by about 13.3% compared with the Angel YZ-3 yeast strain or the VIN13 yeast strain, and is promoted by about 15.6% compared with the Red Star CEC01 yeast strain. This two-digit gain can significantly reduce the raw material cost and improve the single batch yield on an industrial scale. From the metabolic pathway optimization, the Saccharomyces cerevisiae CPJ-02 strain in the application is superior to the control in the expression level or activity of key enzymes (such as PDC and ADH) in glycolysis and alcohol fermentation, reduces the accumulation of intermediate metabolites, and improves the proportion of carbon flow to ethanol. From the stress resistance ability, in industrial fermentation, temperature, ethanol inhibition, pH fluctuation and the like can affect the yield; if the Saccharomyces cerevisiae CPJ-02 strain in the application has stronger heat resistance or ethanol inhibition resistance, it can still maintain high efficiency under high concentration conditions. From the substrate utilization rate, in addition to glucose, the Saccharomyces cerevisiae CPJ-02 strain in the application can more comprehensively utilize various sugars in the fermentation substrate, and also brings about the promotion of the overall yield.

[0299] From the residual sugar at the 48-hour fermentation endpoint, the 48-hour fermentation endpoint residual sugar of the Saccharomyces cerevisiae CPJ-02 strain described in the present application is 3.2 g / L, which is the lowest in the table, and is significantly lower than other strains (EC1118 is 6.2 g / L, Angel YZ-3 is 7.7 g / L, Red Star CEC01 is 7.8 g / L, and VIN13 is 8.4 g / L). In terms of sugar consumption efficiency and fermentation speed, the Saccharomyces cerevisiae CPJ-02 strain described in the present application only has 3.2 g / L left after 48 hours, which is much lower than all the control strains (the lowest is 6.2, and the highest is 8.4 g / L), indicating that the lowest residual sugar level of the Saccharomyces cerevisiae CPJ-02 strain described in the present application is relatively low, and it can more fully convert sugar into products under the same fermentation conditions. In terms of fermentation kinetics, the Saccharomyces cerevisiae CPJ-02 strain described in the present application still maintains a high metabolic activity in the later stage, and the lower the residual sugar, the more active ethanol production in the fermentation terminal stage, while the control strains tend to be stable or inhibited at about 30-40 h. In terms of rate advantage, if the sugar consumption rate is estimated, the average consumption rate of the Saccharomyces cerevisiae CPJ-02 strain described in the present application is significantly higher than that of other strains in 0-48 hours, and the sugar emptying is more thorough in the fermentation period.

[0300] Conclusion 3: The Saccharomyces cerevisiae CPJ-02 strain described in the present application has a higher metabolic efficiency, and can generate more ethanol under the same substrate load, indicating that the Saccharomyces cerevisiae CPJ-02 strain described in the present application has a higher carbon source conversion efficiency, and is suitable for industrial large-scale fermentation production; the Saccharomyces cerevisiae CPJ-02 strain described in the present application is the best choice in the current comparison in terms of 48-hour fermentation residual sugar control. This is not a small advantage, but a key indicator that reflects its fermentation potential and application breadth.

[0301] The low residual sugar of the Saccharomyces cerevisiae CPJ-02 strain described in the present application means higher overall product recovery rate, which is complementary to the high yield of 0.45 g / g before, reducing the waste of unconverted sugar. In terms of production cost reduction, the Saccharomyces cerevisiae CPJ-02 strain described in the present application can reduce the input of recovery or re-fermentation part, because when the residual sugar is high, additional post-processing (such as recovery or re-fermentation) is required, increasing the complexity and cost of the process. In terms of more accurate determination of fermentation endpoint, the fermentation endpoint of traditional strains is difficult to determine (residual sugar cannot be reduced), while the Saccharomyces cerevisiae CPJ-02 strain described in the present application shows a clear baseline of endpoint residual sugar, which is convenient for online monitoring and intelligent control, that is, it can effectively reduce the risk of secondary fermentation of finished beverages in the bottle, improve the safety of preservation and shelf life.

[0302] In terms of physiological and molecular mechanisms, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has better expression or affinity of main sugar transporters (such as HXT family), quickly transports exogenous sugar into cells, and has high-efficiency sugar transport; the Saccharomyces cerevisiae CPJ-02 strain described in the present application has lower accumulation of intermediate metabolites, which reduces bypass loss (such as sugar alcohol or organic acid), increases the proportion of carbon flow to ethanol, and reduces bypass inhibition. In terms of enhanced inhibition resistance, the Saccharomyces cerevisiae CPJ-02 strain described in the present application has better alcohol tolerance, and its metabolic enzyme system can still maintain activity, thereby continuing to consume residual sugar.

[0303] Conclusion 4: The Saccharomyces cerevisiae CPJ-02 strain described in the present application shows very high efficiency in sugar conversion process, which can almost completely consume the substrate sugar within 48 hours, reducing the residual sugar level after fermentation; and the low residual sugar characteristic makes the fermentation period shorter and the raw material utilization rate higher, which can improve the production efficiency and reduce energy consumption.

[0304] Comparative Example 2 Comparison of acid production performance of the Saccharomyces cerevisiae CPJ-02 strain described in the present application and industrial yeast

[0305] Comparative Example 2 illustrates the acid production performance of the Saccharomyces cerevisiae CPJ-02 strain of the present application compared with industrial yeasts. The comparison between the Saccharomyces cerevisiae CPJ-02 strain and other industrial yeasts was analyzed through multiple key indicators, such as acid production capacity, pH change, acid tolerance, and the like.

[0306] (1) The acid production performance comparison between the Saccharomyces cerevisiae CPJ-02 and industrial yeasts

[0307] In the comparative example of the Saccharomyces cerevisiae CPJ-02 and industrial yeasts of the present application, the control strains of the Saccharomyces cerevisiae CPJ-02 were common industrial yeast strains, including Saccharomyces cerevisiae Lalvin EC1118, Saccharomyces cerevisiae Angel YZ-3, and Saccharomyces cerevisiae Red Star CEC01. The experimental period of the example was 24 hours, and the pH change and acid production of the Saccharomyces cerevisiae CPJ-02 and the control strains were measured. The fermentation conditions of the experiment were designed as follows: temperature 30℃, pH 5.0, and initial glucose concentration 200g / L. The results of the acid production performance comparison between the Saccharomyces cerevisiae CPJ-02 and industrial yeasts of the present application are shown in the following table.

[0308]

[0309] Conclusion 1: The Saccharomyces cerevisiae CPJ-02 of the present application exhibits the strongest acid production capacity and the most significant pH drop (pH from 5.0 to 3.2) within 24 hours, which indicates that the Saccharomyces cerevisiae CPJ-02 of the present application can efficiently generate organic acids and maintain a low pH value of the fermentation environment. Compared with other strains, the acid production rate of the Saccharomyces cerevisiae CPJ-02 of the present application is higher (0.19 g / L / h), which makes it have stronger fermentation potential and adaptability in an acidic environment; on the contrary, the acidity of the control strains EC1118, Angel YZ-3 and Red Star CEC01 changes more slowly, and the final pH is higher, which shows that the acid production capacity of these control strains is relatively weak. The Saccharomyces cerevisiae CPJ-02 of the present application can generate more acidic by-products in the industrial fermentation process, thereby being beneficial to strengthening the acidification environment in the brewing process and optimizing the taste and flavor of certain products.

[0310] (2) Comparison of acid production performance of the Saccharomyces cerevisiae CPJ-02 of the present application and industrial yeasts under different fermentation conditions

[0311] In the comparative example of the Saccharomyces cerevisiae CPJ-02 of the present application and industrial yeasts, the control strains of the Saccharomyces cerevisiae CPJ-02 are common industrial yeast strains, including Saccharomyces cerevisiae Lalvin EC1118, Saccharomyces cerevisiae Angel YZ-3, Saccharomyces cerevisiae Red Star CEC01; the fermentation time of the example is 48 hours, and the pH value change and acid production of the Saccharomyces cerevisiae CPJ-02 of the present application and the control strains are measured; the fermentation conditions designed in the experiment are temperature 30℃, and the initial pH value is set to 4.5, 5.0 and 5.5, and the initial glucose concentration is 200 g / L.

[0312] Firstly, the acid production performance of the Saccharomyces cerevisiae CPJ-02 of the present application and industrial yeasts under the condition of initial pH value of 4.5 is shown in the following table.

[0313]

[0314] Secondly, the acid production performance of the Saccharomyces cerevisiae CPJ-02 and the industrial yeast under the condition of initial pH value of 5.0 is shown in the following table.

[0315]

[0316] Thirdly, the acid production performance of the Saccharomyces cerevisiae CPJ-02 and the industrial yeast under the condition of initial pH value of 5.5 is shown in the following table.

[0317]

[0318] Conclusion 2:

[0319] From the data trend, the Saccharomyces cerevisiae CPJ-02 always shows the maximum acid production and the fastest acid generation rate under the three groups of pH; and with the increase of initial pH (decrease of acidity), the maximum acid production and the acidification rate of the four strains are slightly decreased, but the decrease of the Saccharomyces cerevisiae CPJ-02 is the smallest, and the performance is the most stable; compared with the other strains, the EC1118 in the control strain has the weakest acidification ability under all conditions, and is more sensitive to the change of initial pH; the Angel YZ-3 and the Red Star CEC01 in the control strain are in the middle, and the adaptability is better than the EC1118 but lower than the Saccharomyces cerevisiae CPJ-02.

[0320] When the pH value is 4.5, the Saccharomyces cerevisiae CPJ-02 shows the strongest performance, the pH value can be reduced to 3.0 in 48 hours, the maximum acid production is as high as 10.2 g / L, and the acid generation rate is 0.21 g / L / h, which is significantly better than other strains, which shows that the Saccharomyces cerevisiae CPJ-02 is suitable for high-acid environment and is conducive to the production of high-acid fruit wine, seasoning fermented products and the like; and the control strain EC1118 has weak pH reduction and acid production capacity, and is not suitable for extreme low-pH environment.

[0321] When the pH value is 5.0, the Saccharomyces cerevisiae CPJ-02 still has the maximum acid production capacity, the pH value is reduced to 3.2 in 48 hours, and the maximum acid production is 9.6 g / L; the acid production and the pH reduction range of the other control strains are similar to or slightly lower than those of the group of 4.5, and still not as good as CPJ-02.

[0322] When the pH value is 5.5, the maximum acid production of the Saccharomyces cerevisiae CPJ-02 is 8.4 g / L, the acid production rate is 0.15 g / L / h, the pH is reduced to 3.5 after 48 hours, and the acid production capacity is still better than that of the control group. The end point pH and acid production of the other control strains are significantly higher, and the reduction range is further reduced, indicating that the high initial pH condition has a greater impact on traditional strains (control strains), and the Saccharomyces cerevisiae CPJ-02 has strong adaptability.

[0323] In summary, the pH change of the Saccharomyces cerevisiae CPJ-02 in 48 hours is the most significant, the final pH is reduced to 3.2, and the acid production is the highest, indicating that it can efficiently produce acidic substances under different initial pH conditions and maintain a low pH value, thereby creating a more acidic and demand- meeting environment during fermentation. The pH change, acid production rate and acid production of the control strains EC1118, Angel YZ-3 and Red Star CEC01 are significantly lower than those of the Saccharomyces cerevisiae CPJ-02; and the control strains EC1118, Angel YZ-3 and Red Star CEC01 are sensitive to high pH conditions and have significantly lower acid production capacity.

[0324] Therefore, the Saccharomyces cerevisiae CPJ-02 can continuously exhibit high acid production capacity and environmental acidification capacity under both low and high initial pH conditions, and the data show that its acid production performance is not sensitive to initial pH and has strong stability; the acid production performance of the Saccharomyces cerevisiae CPJ-02 is better than that of traditional industrial yeast, making it more competitive in the production of fermentation products that require a low pH environment, and it is particularly suitable for ideal strains of various acidity requirements (such as high-acid fruit wine, sour fermented beverages, low-pH food, etc.), and it can also reduce the risk of contamination by other bacteria and ensure product flavor and fermentation safety.

[0325] (3) Comparative example of acid tolerance of Saccharomyces cerevisiae CPJ-02 and common industrial yeast

[0326] In the comparative example of Saccharomyces cerevisiae CPJ-02 and industrial yeast, the control strains of Saccharomyces cerevisiae CPJ-02 are common industrial yeast strains, including Saccharomyces cerevisiae Lalvin EC1118, Saccharomyces cerevisiae Angel YZ-3, Saccharomyces cerevisiae Red Star CEC01. The fermentation time of the example is 48 hours, the pH value change and lactic acid consumption of Saccharomyces cerevisiae CPJ-02 and the control strains are measured; the fermentation conditions of the experiment design are temperature 30℃, adding different concentrations of lactic acid (0 g / L, 2 g / L, 4 g / L), and the acid tolerance of each strain is evaluated.

[0327] Firstly, the acid production performance of Saccharomyces cerevisiae CPJ-02 and industrial yeast under the condition of 0 g / L of lactic acid addition is shown in the following table.

[0328]

[0329] Secondly, the acid production performance of Saccharomyces cerevisiae CPJ-02 and industrial yeast under the condition of 2 g / L of lactic acid addition is shown in the following table.

[0330]

[0331] Thirdly, the acid production performance of Saccharomyces cerevisiae CPJ-02 and industrial yeast under the condition of 4 g / L of lactic acid addition is shown in the following table.

[0332]

[0333] Conclusion 3:

[0334] From the data trend, the pH change shows that the more lactic acid is added, the higher the final pH of each strain is, but the Saccharomyces cerevisiae CPJ-02 still can reduce to the lowest pH; the lactic acid consumption rate and consumption amount show that the higher the lactic acid addition is, the absolute consumption amount is also improved, but the Saccharomyces cerevisiae CPJ-02 is always the highest; the survival rate shows that with the increase of acidity (lactic acid increase), the activity of each strain decreases, but the Saccharomyces cerevisiae CPJ-02 has the smallest decrease and the strongest tolerance. The control strain is more sensitive to high lactic acid, which is manifested as that the pH does not decrease obviously, the lactic acid consumption amount is low, and the survival rate decreases.

[0335] Under the condition of no lactic acid addition (0 g / L), each group of strains can well complete sugar fermentation, and the pH decreases, the Saccharomyces cerevisiae CPJ-02 can reduce to 3.1, and the rest of the control strains are 3.4-3.7, and the survival rate difference is not large. Therefore, it is shown that under no additional acid stress, all strains have good fermentation capacity.

[0336] Under the condition of medium lactic acid addition (2 g / L), the final pH of the Saccharomyces cerevisiae CPJ-02 is still the lowest (3.2), the lactic acid consumption rate and consumption amount are the highest, and the survival rate is as high as 97%, which is better than the rest of the control strains; especially the control strains Angel YZ-3 and Red Star CEC01 perform in the middle, the decrease of EC1118 is the largest, and the survival rate is only 91%. Therefore, it is shown that the Saccharomyces cerevisiae CPJ-02 has strong adaptability and metabolic activity in the medium acid environment, and is suitable for medium-high acid products.

[0337] Under the condition of high lactic acid addition (4 g / L), the final pH of the Saccharomyces cerevisiae CPJ-02 can still reduce to 3.3, the lactic acid consumption amount is the largest (4.5 g / L), and the activity remains 94%; while the control strains Angel YZ-3 and Red Star CEC01 are 3.7 and 3.9 respectively, and EC1118 is 4.1, which performs the worst, and the activity decreases to 80%, which shows that in the high lactic acid environment, the tolerance and activity of ordinary industrial yeast decrease obviously, the acid production capacity is weak, and the fermentation is incomplete; while the Saccharomyces cerevisiae CPJ-02 performs stably, and shows unique industrial acid tolerance advantage.

[0338] In summary, the Saccharomyces cerevisiae CPJ-02 of the present application can effectively adapt to acidic environments under different concentrations of lactic acid, maintain high fermentation activity and lactic acid consumption capacity, has the lowest end pH, the largest lactic acid consumption rate, and the smallest survival rate reduction, and is significantly superior to common industrial yeasts in acid resistance. Further, the Saccharomyces cerevisiae CPJ-02 of the present application is particularly suitable for application in high-acid fermentation environments, such as high-acid fruit wine, sour fermented drinks, and special flavor wine, and can also reduce the risk of contamination and improve the flavor stability and fermentation safety of the product. In contrast, the fermentation performance of traditional industrial yeasts such as EC1118 and Red Star CEC01 is significantly attenuated under high-acid stress, making it difficult to meet the needs of high-acid processes.

[0339] Therefore, the Saccharomyces cerevisiae CPJ-02 of the present application exhibits the strongest acid resistance, can maintain a low pH value under the addition of lactic acid, and has a high lactic acid consumption rate, indicating that it is highly adaptable to acidic environments and can effectively ferment under high-acid conditions. In contrast, EC1118 and Red Star CEC01 have a lower lactic acid consumption rate and are more fragile in acidic environments, limiting their application in high-acid conditions. The acid resistance of the Saccharomyces cerevisiae CPJ-02 of the present application makes it particularly suitable for fermentation in high-acid environments, such as certain flavor wines, acidic fruit wines, and special fermented products that require acidification.

[0340] Application Example 1: Application of the Saccharomyces cerevisiae CPJ-02 of the present application for high-concentration alcohol production

[0341] Application Example 1 focuses on the Saccharomyces cerevisiae CPJ-02 of the present application, which is a yeast with excellent performance, and its application in high-concentration alcohol production, such as spirits and fruit wine. The Saccharomyces cerevisiae CPJ-02 of the present application has high ethanol tolerance (up to 15% ethanol concentration), high ethanol yield (0.45 g / g glucose), and strong aroma generation capacity, making it suitable for efficient fermentation under high-alcohol conditions, which can improve production efficiency, shorten fermentation cycles, and reduce production costs.

[0342] The objective of application example 1 is to perform high concentration alcohol (wine) fermentation experiments under laboratory conditions using Saccharomyces cerevisiae CPJ-02 strain, compare its fermentation performance and alcohol yield under high alcohol concentration, and evaluate its application potential in industrial scale production.

[0343] The experimental conditions of application example 1 are as follows: the strain used is Saccharomyces cerevisiae CPJ-02; the substrate of the experiment is glucose (200 g / L) and fructose (200 g / L); the initial pH of the fermentation broth is 5.0, and the temperature is 30℃; the fermentation substrate is grape juice mixed with fruit mixture (such as grape, apple and citrus); the fermentation vessel is a 5L fermenter; the fermentation period is 48 hours; the ethanol concentration is controlled by gradually adding alcohol to the fermentation broth, and the ethanol concentration is set to 10% v / v, 12% v / v and 14% v / v, respectively.

[0344] The application of Saccharomyces cerevisiae CPJ-02 in high concentration alcohol production is as follows:

[0345] S1. Inoculation preparation: Saccharomyces cerevisiae CPJ-02 is cultured in a liquid medium containing glucose, and pre-cultured to the logarithmic growth phase. Then the pre-cultured yeast is inoculated into the fermenter, and the inoculation amount is 1×10 7 CFU / mL;

[0346] S2. Start fermentation: the initial glucose concentration is set to 200 g / L, the fermentation substrate includes mixed grape juice and fructose solution, the pH is adjusted to 5.0, and the temperature is set to 30℃; and during the fermentation process, the CO2 release amount, pH change, ethanol concentration and glucose consumption rate are monitored;

[0347] S3. Ethanol concentration control and data recording: after 24 hours, alcohol is gradually added to the fermentation broth to increase the ethanol concentration to 10% v / v, 12% v / v and 14% v / v, the changes of the fermentation broth are monitored, and the ethanol yield and glucose consumption are recorded; and the CO2 release amount, pH change, alcohol concentration and residual sugar amount are measured every 12 hours.

[0348] The experimental results of Saccharomyces cerevisiae CPJ-02 in the application of high concentration alcohol (such as liquor, fruit wine, etc.) production are as follows.

[0349]

[0350] From the table, it can be seen that from the alcohol tolerance, even at an ethanol concentration of 14% (v / v), Saccharomyces cerevisiae CPJ-02 still maintains a high activity, and the alcohol yield is 0.44 g / g of glucose, indicating that Saccharomyces cerevisiae CPJ-02 has strong alcohol tolerance. From the fermentation efficiency, at a higher alcohol concentration, Saccharomyces cerevisiae CPJ-02 has good fermentation efficiency, and the fermentation can be completed within 48 hours, and the residual sugar amount is low, indicating that Saccharomyces cerevisiae CPJ-02 can quickly and completely convert glucose into ethanol, improving the production efficiency. From the aroma and flavor, Saccharomyces cerevisiae CPJ-02 can maintain strong fruity, ester and floral aroma at different ethanol concentrations, and the content of ester substances increases with the increase of ethanol concentration, which helps to improve the flavor of the wine body. From the fermentation period, under the condition of higher ethanol concentration, Saccharomyces cerevisiae CPJ-02 can complete fermentation faster, and the fermentation period of Saccharomyces cerevisiae CPJ-02 is shorter than that of conventional Saccharomyces cerevisiae, which has obvious advantages for industrial production.

[0351] Conclusion: Saccharomyces cerevisiae CPJ-02 has significant advantages in high-concentration alcohol production, especially in the production of strong liquor, fortified fruit wine and other high-alcohol wine; and the high alcohol tolerance, high fermentation efficiency, rich aroma generation capacity and short fermentation period of Saccharomyces cerevisiae CPJ-02 make Saccharomyces cerevisiae CPJ-02 an ideal choice for the production of high-concentration ethanol drinks, functional alcohol and biological medical alcohol products. Therefore, Saccharomyces cerevisiae CPJ-02 not only can improve the ethanol yield and production efficiency, but also can improve the flavor and quality of the wine body, and has strong industrial application potential.

[0352] Application Example 2: Application of Saccharomyces cerevisiae CPJ-02 based on the present application to low-alcohol healthy fermented beverages

[0353] The purpose of application example 2 is to produce a low-alcohol healthy fermented beverage by using Saccharomyces cerevisiae CPJ-02, and to evaluate its fermentation performance, flavor, alcohol yield and antioxidant capacity.

[0354] With the increasing demand of consumers for healthy drinks, low-alcohol beverages (alcohol concentration less than 5%) are becoming a new favorite in the market as a light drinking option. The Saccharomyces cerevisiae CPJ-02 described in the present application has strong fermentation performance, rich aroma generation capacity and high antioxidant capacity, and is suitable for application in the production of low-alcohol healthy fermented beverages. The Saccharomyces cerevisiae CPJ-02 described in the present application not only can efficiently convert sugar into alcohol, but also can generate a variety of beneficial volatile components and antioxidant substances, adding flavor and nutrition to low-alcohol healthy drinks.

[0355] The purpose of application example 2 is to verify the application effect of the Saccharomyces cerevisiae CPJ-02 described in the present application in the production of low-alcohol healthy fermented beverages through fermentation tests under laboratory conditions, and to evaluate the performance of the Saccharomyces cerevisiae CPJ-02 in terms of low alcohol concentration, rich antioxidant components and fruit aroma generation.

[0356] The experimental materials and conditions of application example 2 are as follows: the strain used is Saccharomyces cerevisiae CPJ-02; the substrate is glucose, fructose, honey, fruit juice (such as lemon juice, orange juice or apple juice); the initial pH of the fermentation broth is 4.0, and the temperature is 30℃; the fermentation container is a 2L fermentation bottle; the target alcohol concentration is less than 5% (v / v); the fermentation period is 72 hours; other additives are a small amount of vitamin C and mineral additives (such as calcium and magnesium).

[0357] The application of the Saccharomyces cerevisiae CPJ-02 described in the present application to low-alcohol healthy fermented beverages is as follows:

[0358] S1. Inoculation preparation: inoculate CPJ-02 Saccharomyces cerevisiae in a glucose liquid medium, and pre-culture to the logarithmic growth phase; then inoculate the pre-cultured yeast into a fermentation bottle, and the inoculation amount is 1×10 7 CFU / mL;

[0359] S2. Fermentation start: The substrate in the fermentation broth is configured as a mixed sugar source, the concentrations of glucose and fructose are set to 100 g / L, honey (5%) is added as an additional sugar source, the initial pH is adjusted to 4.0, and the temperature is set to 30℃; an appropriate amount of lemon juice and orange juice is added to make the final fermentation broth contain 10% fruit juice as a flavor source; during the fermentation process, the CO2 release amount, pH change, alcohol concentration, and residual sugar amount are regularly monitored;

[0360] S3. Flavor and nutrient component analysis during fermentation: sample analysis of alcohol concentration, residual sugar amount, volatile components (aroma components), and DPPH free radical scavenging rate in the fermentation broth every 12 hours; and at the end of the experiment (72 hours), sensory evaluation (aroma, taste, acidity) and antioxidant activity testing are performed.

[0361] The experimental results of the application of Saccharomyces cerevisiae CPJ-02 in the production of low-alcohol healthy fermented beverages are as follows.

[0362]

[0363] As shown in the table, in terms of alcohol concentration, by using Saccharomyces cerevisiae CPJ-02, the alcohol concentration of the low-alcohol fermented beverage is successfully controlled at 4.8% (v / v), which meets the standard of healthy fermented beverages; this indicates that Saccharomyces cerevisiae CPJ-02 can effectively convert sugar into ethanol while maintaining a low alcohol concentration. In terms of residual sugar amount, the residual sugar amount in the fermentation broth after 72 hours is 2.1 g / L, which is much lower than that of traditional fermentation yeast, indicating that Saccharomyces cerevisiae CPJ-02 is very efficient in sugar conversion and the fermentation process is almost complete. In terms of aroma components, Saccharomyces cerevisiae CPJ-02 generates main aroma components such as ethyl octanoate and phenethyl alcohol during fermentation, which enhances the flavor of the beverage and makes it have a rich fruity aroma and a slight ester aroma, meeting the flavor requirements of low-alcohol healthy beverages. In terms of antioxidant activity, the DPPH free radical scavenging rate is 65%, indicating that Saccharomyces cerevisiae CPJ-02 generates components with antioxidant function during fermentation, which helps to enhance the health attributes of the beverage. In terms of fermentation rate, the CO2 release amount is relatively low (12 mL / h), but the fermentation rate remains stable, indicating that the fermentation process is mild and controllable, suitable for producing low-alcohol alcoholic beverages.

[0364] Conclusion: The Saccharomyces cerevisiae CPJ-02 described in the present application has shown significant advantages in the production of low-alcohol health fermented beverages. The Saccharomyces cerevisiae CPJ-02 described in the present application has good effects in controlling low alcohol content, providing rich aroma components, and improving antioxidant activity, and is suitable for the production of functional drinks, low-alcohol fruit wine, and fermented beverages. The high-efficiency fermentation capacity, low residual sugar characteristics, and strong aroma generation capacity of the Saccharomyces cerevisiae CPJ-02 described in the present application make it an ideal health beverage fermentation yeast, which can meet the needs of modern consumers for healthy, low-alcohol, and high-flavor beverages. Application Example 2 demonstrates the application potential of the Saccharomyces cerevisiae CPJ-02 described in the present application in low-alcohol health fermented beverages, especially for the production of fermented beverages that require low-alcohol content, rich antioxidant components, and excellent flavor.

[0365] Application Example 3 Application of the Saccharomyces cerevisiae CPJ-02 described in the present application to fermented food (bread)

[0366] The purpose of Application Example 3 is to demonstrate the application of the Saccharomyces cerevisiae CPJ-02 described in the present application to fermented bread, and to evaluate the effects of the Saccharomyces cerevisiae CPJ-02 described in the present application on the fermentation performance, flavor, texture, nutritional components, and antioxidant activity of bread.

[0367] With the increasing attention to healthy diet and natural fermented food, more and more fermented foods have become an important part of modern diet. The Saccharomyces cerevisiae CPJ-02 described in the present application has excellent fermentation performance, antioxidant capacity, and diverse aroma generation characteristics, so it has great potential in the application of fermented food; especially in sour fermented food (such as yogurt, fermented bread, and fermented vegetables), the Saccharomyces cerevisiae CPJ-02 described in the present application not only can provide high-quality flavor, but also can effectively improve the nutritional components and the health attributes of food.

[0368] The objective of application example 3 is to verify the fermentation effect of Saccharomyces cerevisiae CPJ-02 on bread through fermentation bread production under laboratory conditions, analyze the influence of Saccharomyces cerevisiae CPJ-02 on the texture, flavor and nutritional components of bread, and evaluate the application prospect of Saccharomyces cerevisiae CPJ-02 in the production of fermented food.

[0369] The experimental materials and conditions of application example 3 are as follows: the strain used is Saccharomyces cerevisiae CPJ-02; the ratio of bread raw materials is high-gluten flour (500 g), water (300 mL), yeast (1.0 g), salt (5 g), honey (10 g), and olive oil (10 mL); the initial pH value of the fermentation liquid is 4.5, and the temperature is 30°C; the fermentation container is a fermentation basin and a baking oven; the fermentation cycle is 2 hours of fermentation time and 30 minutes of baking time; and the experimental control is traditional Saccharomyces cerevisiae.

[0370] The application of Saccharomyces cerevisiae CPJ-02 to fermented food (bread) is described as follows:

[0371] S1. Dough preparation: mix high-gluten flour, salt, honey and olive oil evenly; then use Saccharomyces cerevisiae CPJ-02 (or use traditional Saccharomyces cerevisiae as a control yeast) as a fermenting agent, add an appropriate amount of water to dissolve, and add the yeast solution to the flour, stirring evenly to form a dough;

[0372] S2. Fermentation process: place the dough in a fermentation basin, cover it with a damp cloth, and perform secondary fermentation; then ferment for 2 hours at 30°C, and observe the degree of dough expansion;

[0373] S3. Baking process: after fermentation is complete, place the dough in a preheated oven at 180°C for baking, and the baking time is 30 minutes; then remove the bread after baking is complete and cool it to room temperature;

[0374] S4. Data recording and analysis of bread results: compare the fermentation effect, flavor, taste, and antioxidant activity of the bread; then determine the antioxidant activity (such as DPPH free radical scavenging rate) of the bread, and perform sensory evaluation.

[0375] The experimental results of the application of Saccharomyces cerevisiae CPJ-02 in the production of fermented food (bread) are as follows.

[0376]

[0377] The table shows that the Saccharomyces cerevisiae CPJ-02 strain described in the application is not suitable for the field of high-efficiency sugar conversion fermentation, but has obvious advantages for the preparation of slow fermentation bread.

[0378] From the fermentation effect, the dough using the Saccharomyces cerevisiae CPJ-02 described in the application has a faster swelling speed during the fermentation process, and the final volume swelling rate is 3.2 times, which is significantly higher than that of the traditional Saccharomyces cerevisiae (2.5 times); this shows that the Saccharomyces cerevisiae CPJ-02 described in the application has stronger fermentation capacity, can efficiently convert sugar in a shorter time, generate CO2 and promote the swelling of the dough. From the taste and flavor, the bread fermented by the Saccharomyces cerevisiae CPJ-02 described in the application has uniform color, golden yellow crust with a slight luster, soft interior and moderate pores; compared with the traditional Saccharomyces cerevisiae, the bread fermented by the Saccharomyces cerevisiae CPJ-02 described in the application has rich fruity and ester aroma, rich flavor levels, and softer and more delicate taste. From the antioxidant property, the antioxidant substances (such as ester substances and alcohol compounds) obtained by the Saccharomyces cerevisiae CPJ-02 described in the application in the bread fermentation help to improve the antioxidant property of the bread, and the DPPH free radical scavenging rate is 68%, which is significantly higher than that of the bread fermented by the traditional Saccharomyces cerevisiae (53%), which makes the bread fermented by the Saccharomyces cerevisiae CPJ-02 described in the application have higher health value. From the aroma components, through GC-MS analysis, the content of ethyl octanoate in the bread fermented by the Saccharomyces cerevisiae CPJ-02 described in the application is 55 mg / L, which is significantly higher than that of ethyl acetate generated by the traditional Saccharomyces cerevisiae (40 mg / L); the generation of ethyl octanoate helps to improve the fruity and ester aroma of the bread, making the bread more attractive.

[0379] Conclusion: Application Example 3 demonstrates the potential of the Saccharomyces cerevisiae CPJ-02 described in the present application in fermented food, especially in the production of healthy bread, which has a broad market prospect. The application of the Saccharomyces cerevisiae CPJ-02 described in the present application in fermented food has significant advantages, especially in the production of fermented bread; the Saccharomyces cerevisiae CPJ-02 described in the present application can improve the fermentation effect, taste, flavor and antioxidant properties of bread, and improve the health attributes of bread, making it an ideal healthy fermented food production yeast; the Saccharomyces cerevisiae CPJ-02 described in the present application has high fermentation capacity and rich aroma generation characteristics, making it particularly suitable for the production of healthy bread and fermented food products, which helps to improve the flavor, nutritional ingredients and consumer acceptance of food.

[0380] Application Example 4 Application of the Saccharomyces cerevisiae CPJ-02 described in the present application for the targeted production of aroma compounds

[0381] Application Example 4 focuses on the application of the Saccharomyces cerevisiae CPJ-02 described in the present application in the targeted production of aroma compounds (ester substances), aiming to improve the yield of aroma components and optimize their flavor effect in food and beverages. The purpose of Application Example 4 is to demonstrate the application of the Saccharomyces cerevisiae CPJ-02 described in the present application for the targeted production of aroma compounds (such as ethyl octanoate and phenethyl alcohol), to evaluate the influence of the Saccharomyces cerevisiae CPJ-02 described in the present application on the generation of aroma substances, and to explore its application prospects in the aroma compound industry.

[0382] Aroma compounds, especially esters, alcohols and acids, play a crucial role in the flavor formation of food and beverages. The Saccharomyces cerevisiae CPJ-02 described in the present application generates rich aroma components (such as ethyl octanoate and phenethyl alcohol) during fermentation, which has great application potential in the targeted production of aroma compounds.

[0383] The objective of application example 4 is to evaluate the influence of the Saccharomyces cerevisiae CPJ-02 on the generation of aroma substances by using the Saccharomyces cerevisiae CPJ-02 to direct production of specific aroma compounds (such as ethyl octanoate and phenethyl alcohol) under controlled conditions, and to explore the application prospect of the Saccharomyces cerevisiae CPJ-02 in the aroma compound industry.

[0384] The experimental materials of application example 4 are as follows: the strain used is Saccharomyces cerevisiae CPJ-02; the experimental substrates are glucose, ethanol, acetic acid, and amino acids (such as phenylalanine); the initial pH value of the fermentation broth is 4.5, and the temperature is 30°C; the fermentation vessel is a 5L fermenter; the fermentation period is 72 hours; and the target aroma compounds are ethyl octanoate and phenethyl alcohol.

[0385] The fermentation conditions of application example 4 are as follows:

[0386] Experiment 1: addition of acetic acid and phenylalanine to investigate the generation of ester aroma;

[0387] Experiment 2: change of ethanol concentration to study its influence on the generation of aroma substances;

[0388] Experiment 3: optimization of pH and temperature conditions to optimize the generation of aroma components.

[0389] The application of the Saccharomyces cerevisiae CPJ-02 to the directed production of aroma compounds, and the specific preparation method, are as follows:

[0390] S1. Inoculation preparation: inoculate the Saccharomyces cerevisiae CPJ-02 into a liquid medium containing glucose, and pre-culture to the logarithmic growth phase; then inoculate the pre-cultured yeast into a fermenter, and the inoculation amount is 1×10 7 CFU / mL;

[0391] S2. Aroma generation regulation:

[0392] Experiment 1: add acetic acid (10 g / L) and phenylalanine (2 g / L) into the fermentation broth respectively, which can be used as precursor substances of aroma compounds, to investigate their influence on the generation of aroma substances (such as ethyl octanoate and phenethyl alcohol);

[0393] Experiment 2: adjust the ethanol concentration in the fermentation broth to explore the effect of ethanol on the generation of ester aroma substances (such as ethyl octanoate).

[0394] Experiment 3: Adjusting pH (4.0, 4.5, 5.0) and fermentation temperature (25°C, 30°C, 35°C) to optimize the generation of aroma components, and measuring the generation amount of aroma substances under each condition respectively.

[0395] S3. Data recording and analysis: sampling and analyzing the concentration of ethyl octanoate and phenethyl alcohol in the fermentation broth every 12 hours; then detecting and quantitatively analyzing the aroma components in the fermentation broth by GC-MS (gas chromatography-mass spectrometry); finally evaluating the differences in aroma generation under different conditions and performing sensory evaluation to analyze the flavor characteristics.

[0396] The experimental results of the application of the Saccharomyces cerevisiae CPJ-02 in the targeted production of aroma compounds are as follows.

[0397]

[0398] The table shows that:

[0399] From the generation of ethyl octanoate, in experiment 1, by adding acetic acid and phenylalanine, the generation amount of ethyl octanoate reached 65 mg / L, which was significantly increased compared to the generation amount without adding, and acetic acid and phenylalanine as aroma precursors could effectively promote the synthesis of ethyl octanoate; in experiment 2, with the increase of ethanol concentration, the generation amount of ethyl octanoate decreased, which indicated that the concentration of ethanol had a certain inhibitory effect on the generation of ester aroma substances; experiment 3 showed that by optimizing the pH (4.5) and temperature (30℃), the generation amount of ethyl octanoate was further improved, which indicated that under suitable fermentation conditions, the Saccharomyces cerevisiae CPJ-02 of the application could more efficiently generate ester aroma substances.

[0400] From the generation of phenethyl alcohol, under all experimental conditions, the generation amount of phenethyl alcohol was relatively stable, reaching 60 mg / L in experiment 3 (pH 4.5, 30℃), showing good stability and high conversion capacity; the generation of phenethyl alcohol was mainly affected by the addition amount of phenylalanine, and by optimizing the addition amount, the yield of phenethyl alcohol could be further improved.

[0401] From the aroma characteristics and sensory evaluation, the Saccharomyces cerevisiae CPJ-02 generates rich fruit aroma and ester aroma under the optimized fermentation conditions, especially in experiment 3, the aroma of bread and fruit is particularly prominent, and the sensory score is 9.0 / 10, which proves that the Saccharomyces cerevisiae CPJ-02 has great potential in aroma generation.

[0402] Conclusion: Application Example 4 demonstrates the potential of the Saccharomyces cerevisiae CPJ-02 in aroma compound production, suitable for application in the fields of aroma essential oil, food flavor, etc., especially in the high-end beverage and food flavor enhancement, which has a wide market application prospect. The application of the Saccharomyces cerevisiae CPJ-02 in the production of aroma compounds has significant advantages. By optimizing the fermentation conditions (such as acetic acid, phenylalanine addition, pH and temperature adjustment), the Saccharomyces cerevisiae CPJ-02 can efficiently generate aroma components, especially key aroma compounds such as ethyl caprylate and phenylethanol, which can improve the richness of aroma and flavor, and meet the production needs of high-quality aroma products. The characteristics of the Saccharomyces cerevisiae CPJ-02 make it an ideal choice for the production of directional aroma substances in the food, beverage, aroma chemical and other industries.

Claims

1. A high acid-tolerant and high ethanol-tolerant and strong ester-aroma-producing Saccharomyces cerevisiae strain CPJ-02, characterized in that, The Chinese name of the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae CPJ-02, and the Latin name is Saccharomyces cerevisias CPJ-02. The Saccharomyces cerevisiae CPJ-02 strain is preserved in the China Center for Type Culture Collection, located in Wuhan University, Wuhan, Hubei Province, and the preservation date is June 27, 2024, with the preservation number CCTCC NO: M 20241388.

2. The high acid-tolerant and high ethanol-tolerant and strong ester-aroma-producing Saccharomyces cerevisiae CPJ-02 of claim 1, characterized in that, The Saccharomyces cerevisiae CPJ-02 strain forms a light yellow, irregular round raised colony on YPD solid medium after 48 hours of culture, with a colony diameter of 1.5-2.0 mm, a wavy edge, a rough, matte and translucent surface; The Saccharomyces cerevisiae CPJ-02 strain has a single cell under a microscope, occasional false mycelium formation, low bud frequency, and a cell size of about 4-6 μm x 6-8 μm; The Saccharomyces cerevisiae CPJ-02 strain has a maltose metabolism rate of 0.35 g / L・h; The Saccharomyces cerevisiae CPJ-02 strain can tolerate an ethanol concentration of up to 15% v / v, with a survival rate of ≥92% at 12% ethanol; The Saccharomyces cerevisiae CPJ-02 strain maintains ≥90% cell activity at ≥12% v / v ethanol concentration, with a final yield of ≥0.45 g / g and residual sugar of ≤0.1 g / L. The Saccharomyces cerevisiae CPJ-02 strain can grow normally at a pH of 3.5-7.0, with a lactic acid tolerance threshold of 3.0 g / L; The Saccharomyces cerevisiae CPJ-02 strain produces volatile aroma components during fermentation, with an ethyl octanoate content of 62 mg / L, a phenethyl alcohol content of 48 mg / L, and esters accounting for 52% of the total volatile components; The Saccharomyces cerevisiae CPJ-02 strain has a glucose consumption rate of 1.2 g / L・h, achieves sugar depletion within 48 hours, an alcohol yield of 0.45 g / g glucose, and a minimum glucose conversion rate of 0.40 g / g; The Saccharomyces cerevisiae CPJ-01 strain has a growth inhibition rate of 10% at a pH of 3.0, and a lactic acid tolerance threshold of 3.0 g / L; The Saccharomyces cerevisiae CPJ-02 strain has a fermentation liquid foam height of 4.5 cm, and a sedimentation rate of 30% after 24 hours of fermentation. The Saccharomyces cerevisiae CPJ-01 strain, the fermentation liquor is amber, OD 420 value is 0.42, and the removal rate of the fermentation liquor diluted 10 times on DPPH free radicals reaches 72%. The Saccharomyces cerevisiae CPJ-01 strain can maintain normal growth in an environment containing a NaCl concentration of 7%.

3. The high acid-tolerant and high ethanol-tolerant and strong ester-aroma-producing Saccharomyces cerevisiae strain CPJ-02 according to claim 1 or 2, characterized in that, The Saccharomyces cerevisiae CPJ-02 strain has glucose as the preferred metabolic substrate and preferentially metabolizes glucose in the presence of glucose.

4. The high acid-tolerant and high ethanol-tolerant and strong ester-aroma-producing Saccharomyces cerevisiae strain CPJ-02 according to claim 1 or 2, characterized in that, The Saccharomyces cerevisiae CPJ-02 strain has the highest fermentation efficiency under constant temperature conditions of 34°C.

5. The Saccharomyces cerevisiae CPJ-02 of claim 1 or 2, which is characterized by high acid tolerance, high ethanol tolerance, and strong ester aroma production. The key genes of the alcohol fermentation and sugar metabolism module of the Saccharomyces cerevisiae CPJ-02 strain are ADH1, ADH2, PDC1, and ALD6, and the genetic basis for high ethanol yield is ADH1, ADH2, and PDC1. The key genes of the aroma substance synthesis module of the Saccharomyces cerevisiae CPJ-02 strain are ATF1, ATF2, EEB1, BAT1, and BAT2, and the genetic basis for rich fruit esters is ATF1, BAT2, and EEB1. The key genes of the stress resistance and homeostasis regulation module of the Saccharomyces cerevisiae CPJ-02 strain are ERG2, ERG6, HSP104, PMA1, and ENA1, and the genetic basis for high ethanol tolerance / weak acid / salt tolerance is ERG2, ENA1, and HSP104. The key genes of the antioxidant response module of the Saccharomyces cerevisiae CPJ-02 strain are SOD1, CTT1, GSH1, and GPX1, and the genetic basis for strong antioxidant function is SOD1 and CTT1. The key genes of the sedimentation and process adaptation module of the Saccharomyces cerevisiae CPJ-01 strain are FLO1, FLO5, FLO8, and FLO11, and the genetic basis for good process sedimentation is FLO1 and FLO5.

6. The Saccharomyces cerevisiae CPJ-02 strain of claim 1 or 2, which is highly acid-tolerant and high-ethanol, and has strong ester aroma generation. The Saccharomyces cerevisiae CPJ-02 strain can stably ferment in a high-alcohol fermentation system with a volume fraction of ≥12% v / v and achieve a high ethanol yield of ≥0.40 g / g of glucose conversion rate, and is suitable for industrial production of high-concentration alcoholic beverages. The Saccharomyces cerevisiae CPJ-02 strain has good tolerance to an acidic environment with pH 3.5-7.0, can tolerate high osmotic pressure conditions with NaCl≤5% and fermentation environment under oxidative stress, and is suitable for the production of low-alcohol drinks and healthy beverage products; The Saccharomyces cerevisiae CPJ-02 strain shows excellent adaptability in the fermentation environment with pH 3.5-7.0 and high salt NaCl≤5%, and is suitable for the production of fermented food products; The Saccharomyces cerevisiae CPJ-01 strain can synthesize octanoic acid ethyl ester and phenethyl alcohol volatile aroma components, and is suitable for the targeted production of aroma compounds; The fermentation broth produced by the Saccharomyces cerevisiae CPJ-01 strain has a DPPH free radical scavenging rate of ≥70% after being diluted 10 times, and is suitable for the development of antioxidant healthy drinks.

7. The method for screening and obtaining Saccharomyces cerevisiae CPJ-02 for high acid resistance, high ethanol and strong ester flavor production according to claims 1-6, characterized in that, A wild microbial resource derived from natural green plums is used, and under the selection pressure of 5% v / v high-concentration ethanol, a yeast strain Saccharomyces cerevisiae CPJ-02 with alcohol tolerance, complex ester alcohol aroma synthesis capacity and strong acid resistance is selected.

8. The molecular biology method for identifying the Saccharomyces cerevisiae CPJ-02 strain of high ethanol tolerance and ester alcohol aroma profile lineage according to claim 7, characterized by, The BLAST alignment result has similarity ≥98.25% to the ITS region of the reference sequence Accession NR_111007.1, a score ≥1297 and an E-value=0.0, confirming that the identified strain is the Saccharomyces cerevisiae CPJ-02 strain.

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