Saccharomyces cerevisiae and application thereof

Abstract

The invention discloses saccharomyces cerevisiae and application thereof, and belongs to the technical field of fermentation. The Saccharomyces cerevisiae SAU-FWS is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.31659, the preservation time is August 12, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The saccharomyces cerevisiae is high in ethanol and ester production capacity, can tolerate strong acid and high-ethanol environments, can adapt to general fruit wine fermentation conditions, is used for fermenting pomegranate fruit wine, can increase volatile flavor substances of the pomegranate fruit wine, and is soft in taste and rich in fragrance.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically to a strain of brewer's yeast and its application in the fermentation process of pomegranate wine. Background Art

[0002] Saccharomyces cerevisiae Saccharomyces cerevisiae ) is also known as budding yeast or baker's yeast. It is an important representative species of the Fungi, Ascomycetes, Hemiascomycetes, Endosporales, Saccharomycetes, and Saccharomyces genus. In the past, fruit wine was brewed by natural fermentation. The complexity of the microorganisms involved in natural fermentation often led to uncertainty in the quality of fruit wine. The emergence of brewer's yeast plays a key role in determining the quality of fruit wine. It has been selected in fruit wine production and has been used for centuries. Because of its excellent characteristics of fruit wine fermentation, such as strong fermentation activity, low acetic acid production, and tolerance to low pH, ethanol and sulfur dioxide environments, it is widely used in the production of various fermented fruit wines such as wine, cider, pomegranate wine, etc., and has high commercial value.

[0003] Pomegranate( Punica granatum L.) is a small tree of the genus Punica in the family Punica. Its fruit is rich in nutrients. Different parts contain organic acids, alkaloids, flavonoids, steroids with special structures, and polyphenols. It is also rich in minerals such as potassium, phosphorus, calcium, and zinc. The pomegranate has a peculiar shape, bright color, sweet and sour taste, and good health care function, and is favored by consumers. The part of the pomegranate that can be eaten directly has extremely high content of polyphenols, vitamins, organic acids, and various essential trace elements. At present, the development and utilization of pomegranate resources are mainly pomegranate wine and pomegranate juice. In order to ensure the quality and taste of pomegranate wine, commercial brewing yeast is widely used in the brewing of pomegranate wine, which also causes the phenomenon of serious homogeneity of fruit wine, single style, and lack of local flavor. Therefore, selecting yeast strains that can adapt to adverse environments, have strong alcohol production ability, fast fermentation speed, and have the ability to enhance fragrance to ferment and improve the quality of fruit wine has become a current research hotspot. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 This is the single colony morphology of strain SAU-FWS;

[0005] Figure 2 Figure 2 is the cell morphology of the strain SAU-FWS under a 40×10 microscope. Figure a is the cell morphology of SAU-FWS stained with methylene blue, and Figure b is the cell morphology of SAU-FWS without staining.

[0006] Figure 3 The growth curve of strain SAU-FWS at different pH;

[0007] Figure 4 It is the growth curve of strain SAU-FWS under different alcohol concentrations; Summary of the invention

[0008] In view of the problems existing in the existing fermented fruit wine, the purpose of the present invention is to test and provide a brewer's yeast SAU-FWS that has strong environmental tolerance, ester production and flavor enhancement capabilities, and use the yeast to ferment pomegranate wine, thereby providing microbial resources for pomegranate wine brewing.

[0009] The strain provided by the present invention is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) SAU-FWS, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.31659 and the deposit date August 12, 2024.

[0010] The brewer's yeast SAU-FWS was isolated from the fruit surface, and was tested for fermentation performance and molecularly identified to have good ester production and flavor enhancement capabilities. Its storage conditions are as follows: a single colony was picked from a well-growing plate and transferred to a YPD medium, cultured at 28°C for 48 h, 600 μL of the culture solution was transferred to a preservation tube containing 600 μL of 60% glycerol, and stored in a -20°C refrigerator.

[0011] The YPD medium used was formulated as follows: 10 g yeast extract, 20 g anhydrous glucose, and 20 g peptone (if solids are required, add 20 g agar) mixed evenly in 1 L of distilled water and sterilized at 115°C for 30 min.

[0012] The above-mentioned Saccharomyces cerevisiae SAU-FWS culture characteristics: After the strain was inoculated into YPD liquid medium and grown for 24 hours, it was diluted and spread on the plate to obtain a single colony. It was observed that the bacteria had an oval shape, a protrusion in the middle, neat edges, a soft texture, a smooth and moist surface, opaque, and a milky white color. Figure 1 ; Under the objective lens 40×10, the bacteria are oval in shape, 3.0×7.5 μm in size, and reproduce by budding. Figure 2 . Specific implementation methods

[0013] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0014] In the following examples, the percentages are by mass unless otherwise specified.

[0015] The following examples further illustrate the specific implementation of the present invention, but are not limited to these examples.

[0016] Example 1: Identification of Saccharomyces cerevisiae SAU-FWS

[0017] Morphological identification: Saccharomyces cerevisiae SAU-FWS was inoculated into YPD liquid medium and grown for 24 h. 4 The bacteria were observed to be cheese-like in shape, with round colonies, regular edges, soft texture, smooth and moist surface, opaque, and milky white in color. Figure 1 .

[0018] Molecular identification: Extraction of the genome of Saccharomyces cerevisiae SAU-FWS: The strain SAU-FWS was inoculated into YPD liquid medium and cultured on a shaker at 28°C and 200 r / min for 48 h. The total genome of SAU-FWS was extracted according to the instructions of the genome kit. 5 μL of PCR product was separated by 1.2% (1×TAE) agarose gel electrophoresis, detected and imaged by UV gel imaging system, and the PCR amplification product was sent to Sangon Biotech Co., Ltd. for sequencing.

[0019] The sequence was imported into NCBI for BLAST homology comparison, and combined with morphological characteristics, the strain SAU-FWS was identified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ). It was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.31659 and the deposit date: August 12, 2024.

[0020] Example 2: Tolerance test of strain SAU-FWS

[0021] (1) Acidity (pH) tolerance test: The activated strain SAU-FWS seed solution was inoculated at a 2% inoculum into YPD liquid medium with pH values ​​of 1.0, 2.0, 3.0, 4.0, and 5.0, respectively, and cultured at 28°C for 24 h. Samples were taken every 2 h and the OD was measured. 600nm , draw the pH tolerance curve of yeast. Figure 3 As shown in the figure, the growth curve of strain SAU-FWS at pH 1.0 and 2.0 is approximately a straight line. This is because under strong acid conditions, the cells suffer certain damage, their physiological activity is affected, and the cells cannot grow; the growth curves at pH 3.0, 4.0, 5.0, and 6.0 all show a typical "S" curve model, and the cells can continue to grow. It can be seen that the strain has a tolerance range of 3.0-5.0 for acid, has a strong acid resistance, and meets the pH requirements of general fruit wine fermentation.

[0022] (2) Alcohol tolerance test: The activated strain SAU-FWS seed liquid was inoculated with 2% inoculum into YPD liquid culture medium with alcohol content (volume concentration) of 0% vol, 3% vol, 6% vol, 9% vol, 12% vol, and 15% vol, respectively. The culture was carried out at 28°C for 24 h. Samples were taken every 2 h and the OD was measured. 600nm , draw the alcohol tolerance curve of yeast. Figure 4 As shown in the figure, the strain SAU-FWS grew well when the concentration was lower than 9%vol, showing a typical "S" curve model, and the cells could continue to grow; at 12%vol and 15%vol, the growth curve of the strain tended to be a straight line, and the cell growth was inhibited. It can be seen that the strain has good ethanol tolerance during the fruit wine fermentation process, can meet the requirements of fruit wine fermentation and play an important role in the fermentation process.

[0023] Example 3: Determination of ethanol and ester production by strain SAU-FWS

[0024] The activated strain SAU-FWS was inoculated into bean sprout juice medium and fermented at a constant temperature of 28°C for 3 days. The ethanol content was determined by the second alcoholometer method in accordance with (GB5009.225-2016), and the ethanol content was 7.75%vol, which met the requirements for ethanol production in general fruit wine fermentation. The ester production capacity of the strain SAU-FWS was determined by the saponification reflux method in (GB / T10345-2022), and the total ester content in the fermentation broth was 4.865 g / L, indicating a strong ester production capacity.

[0025] Example 4: Determination of volatile flavor substances in pomegranate wine brewed by strain SAU-FWS

[0026] Fresh pomegranates were selected and squeezed to obtain pomegranate juice. The initial sugar content was adjusted to 24% with white sugar. The pH of the fermentation liquid was adjusted to 3.8 with food-grade sodium carbonate, and food-grade potassium metabisulfite was added at a dosage of 60 mg / kg. After standing for 1 day, the strain SAU-FWS was inoculated at a dosage of 200 mg / kg. The control group was not inoculated and fermented at 25°C until the alcohol content was about 12%vol. The fermentation was completed and samples were taken for the determination of volatile flavor substances.

[0027] Headspace solid phase micro extraction (SPME) conditions: 4 mL of wine sample, 1.5 g of sodium chloride, 16 μL of 2-octanol (1 g / L) were added to the headspace bottle, equilibrated at 45 °C for 30 min, and headspace adsorption at 45 °C for 40 min.

[0028] Gas chromatography conditions: Agilent VF-Waxms column (60 m×0.25 mm, 0.5 μm), helium (He) flow rate 1 mL / min; temperature program: splitless injection; initial temperature 40°C, hold for 3 min, increase to 60°C at 2°C / min, hold for 1 min, then increase to 120°C at 4°C / min, hold for 1 min, then increase to 240°C at 6°C / min, hold for 3 min; injection port temperature 260°C, detector temperature 260°C.

[0029] Mass spectrometry conditions: electron ionization (EI) source, interface temperature 250°C, ion source temperature 200°C, electron energy 70 eV, mass spectrometry scanning range 40.00-800.00 m / z, solvent delay time 2 min.

[0030] The results of volatile substance determination showed that 76 compounds were detected in the pomegranate wine fermented by the inoculated strain SAU-FWS, which was higher than the volatile substances in the naturally fermented pomegranate wine (60 kinds). There were 24 ester substances in the pomegranate wine fermented by the inoculated strain SAU-FWS, among which phenylethyl acetate had the highest content of 3.58%. There were only 14 ester volatile flavor substances in the naturally fermented pomegranate wine, among which ethyl octanoate had the highest content of 2.42%. Higher alcohols are the largest aromatic compounds in fruit wine in terms of quantity. There were 5 alcohol volatile substances in the pomegranate wine fermented by the inoculated strain SAU-FWS, among which phenylethyl alcohol had the highest content of 5.86%. Phenylethyl alcohol was produced through glycolysis and pentose phosphate pathway, mainly To provide the aroma of rose and honey to the fruit wine, the naturally fermented pomegranate wine contains more alcohol substances, 8 kinds, among which isopentanol has the highest content, accounting for 3.48%; SAU-FWS fermented pomegranate wine has a total of 5 ketone substances, among which 4-cyclopentene-1,3-dione has the highest content of 0.37%, and naturally fermented pomegranate wine has 3 ketone volatile flavor substances; the acid substances in the two fermented pomegranate wines are the least compared with other substances, SAU-FWS fermented pomegranate wine contains 2 kinds, and naturally fermented pomegranate wine contains only one. In summary, the types and contents of flavor substances in pomegranate wine fermented by the inoculated strain SAU-FWS are higher than those in naturally fermented pomegranate wine, and the pomegranate wine fermented by the inoculated strain SAU-FWS has better quality.

[0031] Example 5: Sensory evaluation of pomegranate wine brewed by strain SAU-FWS

[0032] A panel of 20 people (10 women and 10 men, aged between 20 and 40 years) who had undergone sensory training were selected to evaluate the sensory quality of the wine samples. Mouthwash was provided between the evaluation of different samples to avoid the residual effect of the aftertaste. The panelists gave the sensory quality scores of the pomegranate wine, including the scores of color, aroma, flavor, and acidity.

[0033] Table 1 Sensory evaluation table of pomegranate wine (total score 100 points)

[0034] Table 2 Sensory score table of pomegranate wine type acidity Color fragrance Flavor Total score Inoculation fermentation 18.7±1.641 17.3±1.847 26.2±1.980 27.1±2.162 89.3±2.054 Natural fermentation 17.2±1.246 18.1±1.502 24.1±2.126 25.5±1.936 84.9±1.755

[0035] The sensory scoring results showed that the pomegranate wine fermented by the inoculated strain SAU-FWS scored higher than those of the naturally fermented pomegranate wine in terms of acidity, aroma and flavor indicators, and the total sensory evaluation score was higher than that of the naturally fermented pomegranate wine. In summary, the pomegranate wine fermented by the inoculated strain SAU-FWS is more likely to be favored by consumers than the naturally fermented pomegranate wine.

Claims

1. A strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) SAU-FWS, characterized by, The brewer's yeast has been deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.31659. The deposit date is August 12, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The bacterial agent is characterized in that it is prepared from brewer's yeast SAU-FWS.

2. The bacterial agent according to claim 1 is a freeze-dried bacterial agent, and the preparation method of the freeze-dried bacterial agent comprises: The brewer's yeast SAU-FWS according to claim 1 is cultured to obtain bacterial bodies, and the bacterial bodies are freeze-dried to obtain a bacterial agent, which is used for fruit wine fermentation.

3. The use of the bacterial agent according to claim 2, wherein the saccharomyces cerevisiae SAU-FWS is used in the fermentation of pomegranate wine, wherein the saccharomyces cerevisiae SAU-FWS is inoculated into the fermented pomegranate juice and fermented for a certain period of time to obtain the pomegranate wine with a strong fruity aroma.

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