Saccharomyces cerevisiae for wine brewing and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to Saccharomyces cerevisiae J-1 and an application of the Saccharomyces cerevisiae J-1 in wine brewing. The saccharomyces cerevisiae J-1 which can be applied to wine brewing is screened out by researching the ethanol production capacity, the ester production capacity, the flocculability, the tolerance, the fermentation performance and the like of the saccharomyces cerevisiae. The screened excellent saccharomyces cerevisiae can tolerate ethanol with the pH value of 3.0 and the volume fraction of 14%, SO2 with the concentration of 300 mg / L and glucose with the concentration of 400 g / L, can increase the content of esters in wine fermentation, is rich in flower fragrance and fruit fragrance, is suitable for being used as an excellent strain for wine preparation, and has certain application potential in the wine industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of brewer's yeast J-1 and application thereof in wine brewing. Background Art

[0002] Yeast plays a very important role in the winemaking process. However, during the winemaking process, factors such as low pH, high ethanol, high concentration of SO2 and high sugar will affect the growth ability and fermentation rate of yeast strains, and even cause the fermentation process to fail to proceed normally. Therefore, excellent yeast strains can not only ensure the normal fermentation of wine, but also improve the quality of wine.

[0003] In recent decades, with the advancement of science and technology and the introduction of commercial yeast, winemakers have used commercial yeast for pure fermentation. Commercial yeast has many advantages, such as being able to withstand high alcohol content and high temperature, and having a strong ability to compete for nutrients, which ensures the flavor of the wine, but it has the problems of high cost and lack of prominent flavor characteristics. my country has a vast territory and rich biodiversity, among which there are many excellent Saccharomyces cerevisiae resources, so screening a yeast strain with strong stress resistance and local characteristics is crucial to enriching the flavor substances of wine and developing the desired wine style.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of saccharomyces cerevisiae used in wine brewing and application of the strain in wine fermentation.

[0006] One of the technical solutions provided by the present invention is a yeast strain, specifically Saccharomyces cerevisiae J-1, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 21, 2023, with the deposit number CGMCC No.27169.

[0007] The second technical solution provided by the present invention is the application of the above-mentioned Saccharomyces cerevisiae J-1 in wine production;

[0008] The above-mentioned brewer's yeast J-1 has good tolerance and ester production ability during wine fermentation.

[0009] The beneficial effects of the present invention are:

[0010] The present invention screens out a strain of saccharomyces cerevisiae J-1 that can be used for wine brewing by studying the ethanol production ability, ester production ability, flocculation, tolerance and fermentation performance of yeast. The excellent saccharomyces cerevisiae J-1 screened out by the present invention can tolerate ethanol with a pH value of 3.0 and a volume fraction of 14%, 300 mg / L SO2, and 400 g / L glucose, and can increase the content of ester substances in fermented wine, and has relatively strong flower and fruit aromas, is suitable as an excellent strain for wine preparation, and has certain application potential in the wine industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The tolerance of the yeast strain in Example 1 to different pH values.

[0012] Figure 2 The tolerance of the yeast strain in Example 1 to glucose (A), SO2 (B) and ethanol (C).

[0013] Figure 3 This is a scanning electron micrograph of yeast J-1 in Example 2.

[0014] Figure 4 This is the CO2 mass loss curve of the grape juice fermented by inoculating different yeast strains in Example 3.

[0015] Figure 5 This is a radar chart of sensory analysis of wine samples fermented by different yeast strains in Example 4. DETAILED DESCRIPTION

[0016] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the implementation cases described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0017] The technical solution of the present invention is further illustrated by the following examples.

[0018] Example 1 Isolation and screening of strains

[0019] 1. Isolation and purification of yeast strains

[0020] Weigh 1g of grape or soil sample collected from Helan Mountain, Ningxia, into 9mL of sterile saline, and culture for enrichment at 30℃ and 200r / min for 20min; draw 100μL of supernatant into 900μL of sterile saline, and dilute 10 times to 10 -3 , and then pipette 10 dilutions -2 , 10 -3100 μL of the bacterial solution was spread on YPD solid medium (containing 100 mg / L chloramphenicol) and cultured at 30°C for 24 h. Single colonies with typical yeast colony characteristics (such as smooth, moist, and sticky surface) and obvious morphological differences were selected and repeatedly isolated and purified by the plate streak method until pure colonies were obtained. The strains were stored at -80°C for later use.

[0021] The formula of the YPD solid culture medium is: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, and 20 g / L agar powder.

[0022] 2. Screening of excellent yeast

[0023] Screening of ethanol-producing yeast strains: The activated yeast strain was streaked and inoculated on the TTC lower culture medium, and commercial brewer's yeast F15 (commercial active dry yeast purchased from Laffort, France) was used as a control. After culturing at 30°C for 3 days, a layer of TTC upper culture medium was covered on the colonies under light-proof conditions and cultured at 30°C in the dark for 3 to 4 hours. The ethanol-producing ability of the yeast was judged by the color of the colonies.

[0024] The formula of the TTC upper layer medium is: TTC 0.5g / L, glucose 5g / L, agar 20g / L. TTC lower layer medium: glucose 10g / L, peptone 20g / L, yeast extract powder 1.5g / L, MgSO4 0.4g / L, KH2PO4 1.0g / L, agar 20g / L.

[0025] Table 1 shows the color development results of yeast strains on TTC medium. It can be seen from Table 1 that 6 yeast strains (J-1, J-3, J-4, J-6, J-8 and J-13) and commercial Saccharomyces cerevisiae F15 showed dark red on TTC medium, indicating that the ethanol production capacity was comparable. Therefore, yeast strains J-1, J-3, J-4, J-6, J-8 and J-13 were selected for subsequent research.

[0026] Table 1 Color development results of yeast strains on TTC medium

[0027]

[0028] Note: “+++” means dark red; “++” means red; “+” means light red.

[0029] Screening of ester-producing yeast strains: The yeast strains with strong ethanol production ability were streaked and inoculated on the ester-producing solid culture medium, and the commercial brewer's yeast F15 was used as the control. After culturing at 30°C for 3 days, the color of the colonies was observed.

[0030] The formula of the ester-producing solid culture medium is: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, 15 mL / L tributyrin, 0.04 g / L bromocresol purple, and 20 g / L agar.

[0031] The colors of commercial Saccharomyces cerevisiae F15 and 6 yeast strains on ester-producing medium are shown in Table 2. As shown in Table 2, the ester-producing abilities of different yeasts vary greatly, and the ester-producing abilities of two yeast strains (J-1 and J-3) are comparable to those of the control strain F15, showing yellow color on the ester-producing medium, so yeast strains J-1 and J-3 were selected for further study.

[0032] Table 2 Color of yeast strains on ester-producing medium

[0033]

[0034] Note: “++” indicates yellow; “+” indicates light yellow; “-” indicates white.

[0035] Determination of flocculation characteristics of yeast strains: The screened ester-producing yeast and commercial brewer's yeast F15 were inoculated in YPD liquid culture medium at an inoculation rate of 5% (V / V), cultured at 30°C for 24 hours, centrifuged at 7500r / min for 5 minutes, and the cells were collected. They were washed twice with 250mmol / L EDTA (ethylenediaminetetraacetic acid) and sterile water respectively, and the cells were suspended in 0.1mol / L sodium citrate buffer solution, and the absorbance value (A) was immediately measured at a wavelength of 600nm. The cell suspension was shaken and cultured at 30°C and 120r / min for 2 hours, and then taken out and cultured at room temperature for 30 minutes. The absorbance value (B) was measured at a wavelength of 600nm. The test was repeated three times, and the flocculation value (Flo) was calculated. The calculation formula is as follows:

[0036] Flocculation value = B / A × 100%

[0037] When Flo is between 70% and 100%, it is low flocculation; when Flo is between 30% and 70%, it is medium flocculation; when Flo is between 0% and 30%, it is high flocculation.

[0038] By measuring the flocculation properties of yeast strains F15, J-1 and J-3, it was found that the flocculation properties of the strains were significantly different (P < 0.05). The flocculation value of commercial brewer's yeast F15 was (57.9 ± 1.8)%, which was a medium flocculation strain. The flocculation values ​​of yeast strains J-1 and J-3 were (29.7 ± 1.6)% and (24.942.0)%, respectively, both of which were high flocculation strains. Therefore, yeast strains J-1 and J-3 were selected for subsequent research.

[0039] Tolerance assay of yeast strains

[0040] Acid tolerance: The activated yeast strain was inoculated at 5% (V / V) in YPD liquid medium with different pH values ​​(2.5, 3.0, 3.5, 4.0 and 4.5) and cultured at 30°C for 24 h. The OD 600nm value.

[0041] The acid tolerance of commercial Saccharomyces cerevisiae F15 and Saccharomyces cerevisiae strains J-1 and J-3 is shown in Figure 1 .Depend on Figure 1 It can be seen that, except at pH 3.5, the OD of strain J-1 600nm The value was slightly higher than that of commercial Saccharomyces cerevisiae F15. Under other pH conditions, the acid tolerance of strain J-1 was slightly worse than that of the control strain F15. However, under different pH conditions, the OD 600nm The values ​​were higher than those of yeast strain J-3, indicating that strain J-1 had better acid tolerance than strain J-3. When the pH dropped to 2.5, the OD600 of control strain F15 and yeast strains A1 and J-3 nm The values ​​were all less than 0.5, and the growth was obviously inhibited. The pH value of grape juice is usually between 3.0 and 4.0, indicating that yeast strains J-1 and J-3 can meet the pH environment of winemaking.

[0042] Tolerance to ethanol, SO2 and sugar: The activated yeast strain was inoculated into YPD liquid medium at a rate of 5% (V / V), cultured at 30°C for 24 h, and then sampled and diluted to 10% with physiological saline. -5 , respectively, the dilution was 10 -2 , 10 -3 , 10 -4 , 10 -5 The bacterial suspension was spread on YPD solid culture medium containing different volume fractions of ethanol (8%, 10%, 12%, 14% and 16%), different mass concentrations of SO2 (100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L and 300 mg / L), and glucose (100 g / L, 150 g / L, 200 g / L, 250 g / L and 300 g / L). After culturing at 30 °C for 2 days, the growth of the colonies was observed.

[0043] The tolerance of commercial Saccharomyces cerevisiae F15 and Saccharomyces cerevisiae strains J-1 and J-3 to glucose, SO2 and ethanol is shown in Figure 2 .Depend on Figure 2It can be seen that with the increase of glucose and SO2 mass concentrations, the growth of the control strain F15, strains J-1 and J-3 was not significantly inhibited, and they could all tolerate 400g / L glucose and 300mg / L SO2. In addition, after adding different volume fractions of ethanol to the culture medium, as the ethanol content increased, the growth of the control strain F15, strains J-1 and J-3 was gradually inhibited, but they could all tolerate 14% ethanol by volume, and strain J-1 had stronger tolerance to ethanol than strain J-3, but the two yeast strains basically met the conditions for winemaking. In summary, yeast strain J-1 has better tolerance to acid, glucose, SO2 and ethanol than strain J-3.

[0044] Example 2 Identification of yeast strains

[0045] The pure strain J-1 was selected and inoculated into YPD liquid medium, cultured at 28°C and 200r / min for 12h. After enrichment, it was washed with saline three times, fixed with gold spray and observed under a scanning electron microscope. The results are as follows: Figure 3 As shown, the yeast cells are morphologically complete and the bacteria are regular and full.

[0046] The pure strain J-1 was selected and inoculated into YPD liquid medium and cultured at 28°C for 24 hours for activation. An appropriate amount of activated bacterial liquid was taken, and the bacteria were collected by centrifugation. The genomic DNA of strain J-1 was obtained by oscillation and crushing. The strain sequence was amplified by PCR and sent to a sequencing company for sequencing analysis; the upstream primer NL1 sequence used for amplification was: 5'-GCATATCAATAAGCGGAGGAAAAG-3', and the downstream primer NL4 sequence was: 5'-GGTCCGTGTTTCAAGACGG-3'.

[0047] The PCR reaction conditions are: denaturation 95°C, 15s; annealing 55°C, 15s; extension 72°C, 15s; 30 cycles, extension at 72°C for 10min. By sequencing, the sequence of Saccharomyces cerevisiae J-1 is shown in SEQUENCE LISTING. The BLAST function of NCBI was used to perform sequence alignment in the GenBank database. The homology between the strain and Saccharomyces cerevisiae was greater than 99%, and it can be identified as Saccharomyces cerevisiae. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on April 21, 2023, with the deposit number CGMCC NO.27169, and the deposit address is the Chinese Institute of Microbiology, Beijing, China.

[0048] Example 3 Verification of wine fermentation effect

[0049] The isolated Saccharomyces cerevisiae J-1 was used for wine fermentation, and the changes of weight loss, glucose, fructose, glycerol and ethanol during the wine fermentation were determined.

[0050] Cabernet Sauvignon grapes were destemmed and crushed and then divided into triangular bottles. Each triangular bottle contained 150 mL of grape juice and was pasteurized (70°C, 15 min). Commercial brewer's yeast F15 was used as a control. The activated yeast strain was centrifuged (75000 r / min, 5 min) and the cells were collected. After washing twice with 0.9% saline, the cells were inoculated into triangular bottles at an inoculum of 2×106 CFU / mL and allowed to ferment at 25°C. The CO2 mass loss was measured every 12 h, and the fermentation curve was drawn to monitor the fermentation process. If the mass change was less than 0.2 g for two consecutive times, the fermentation was considered to be over. After the fermentation was completed, the supernatant was taken as a wine sample for subsequent analysis after centrifugation at 12000 r / min for 10 min. At the same time, commercial active dry yeast F15 fermentation was used as a control.

[0051] like Figure 4 The amount of carbon dioxide released during wine fermentation is shown in Figure 2. Figure 4 It can be seen that strains F15, J-1 and J-3 all completed fermentation within 96 hours. During the 24-60h fermentation period, the CO2 mass loss rate of strains J-1 and F15 was basically the same, while the CO2 mass loss rate of strain J-3 was slower, indicating that compared with strain J-3, strain J-1 had a faster fermentation speed, could quickly adapt to grape juice conditions, and shortened the alcohol fermentation cycle.

[0052] Determination of residual sugar, alcohol content, and total acid content: in accordance with GB / T 15038-2006 "General Analytical Methods for Grape and Fruit Wine"; determination of pH value: using pH meter method. Glycerol content was determined using high performance liquid chromatography, and the chromatographic conditions were: chromatographic column Bio-Rad HPX-87H, 300×7.8mm; detector differential refractive index detector (RID); mobile phase 5mmol / L sulfuric acid at a flow rate of 0.6mL / min; detector temperature 45℃, column temperature 65℃, each sample was measured for 23min, in order to maintain the accuracy of the data, each sample was injected twice and the average value was taken, the results are shown in Table 3.

[0053] Table 3 shows that after the alcohol fermentation, the residual sugar content in the wines fermented by commercial Saccharomyces cerevisiae F15 and strains J-1 and J-3 was 4.48-4.90 g / L, all greater than 4 g / L, meeting the requirements for semi-dry wines in GB / T 15037-2006 Wine (4.1-12.0 g / L). There was no significant difference in the total acid content and pH value of the three wine samples (P>0.05). The glycerol content in the wine samples fermented by strains F15, J-1, and J-3 was significantly different (P<0.05).

[0054] Table 3 Physical and chemical indicators and glycerol content of wine samples fermented by different yeast strains

[0055]

[0056]

[0057] Note: Different lowercase letters in the same row indicate significant differences (P<0.05).

[0058] Example 4 Aroma Analysis and Sensory Analysis in Wine

[0059] The aroma component analysis and sensory evaluation of the wine samples fermented by Saccharomyces cerevisiae J-1 in Example 3 are as follows:

[0060] (1) Determination of changes in volatile component content: The aroma components in wine samples were detected using headspace-solid phase microextraction-gas chromatography-mass spectrometer (HS-SPME-GC-MS) technology.

[0061] HS-SPME conditions: 5 mL of wine sample was placed in a 20 mL headspace bottle, 1.5 g NaCl and 5 μL 2.55 g / L 4-methyl-2-pentanol (internal standard) were added, equilibrated at 40 °C for 30 min, and extracted for 30 min.

[0062] GC-MS conditions: chromatographic column CP-WAX 57 CB (60m×0.25mn×0.4μm), extraction head desorbed at the injection port at 200℃ for 8min, injection in splitless mode, carrier gas was high-purity helium (He) (>99.99%), flow rate was 1.0mL / min, program temperature was 50℃ initial temperature, maintained for 1min, increased to 200℃ at 3℃ / min, maintained for 5min, total running time was 56min. Mass selective detector (MSD) transmission line was 250℃, ion source temperature was 230℃, connecting rod temperature was 150℃, and mass spectrometry was scanned in SCAN mode.

[0063] Qualitative and quantitative analysis: The mass spectrometry results were analyzed using the 14-spectral library of the National Institute of Standards and Technology (NIST) of the United States for the qualitative analysis of each volatile substance, and the internal standard method was used for quantitative analysis. The test results are shown in Table 4.

[0064] As shown in Table 4, a total of 67 aroma substances were detected, including 26 esters, 22 alcohols, 10 aldehydes and ketones, 6 terpenes, 2 acids and 1 phenol. Among them, 53 aroma substances were detected in the wine samples fermented by strain J-1, while 51 aroma substances were detected in the wine samples fermented by strains J-3 and F15.

[0065] Esters can provide wine with pleasant floral and fruity aromas and are an important component of wine aroma. As shown in Table 4, compared with the wine samples fermented by strain F15, the contents of ethyl acetate, ethyl hexanoate, ethyl decanoate and isoamyl acetate in the wine samples fermented by strain J-1 were higher, and these substances can bring fruit aromas such as green apples and bananas to wine.

[0066] Alcohols are secondary products of yeast metabolism. When the content of higher alcohols is less than 300 mg / L, the complexity of the wine will be improved. However, when the content is greater than 400 mg / L, it will bring bad flavors to the wine, which will lead to a decrease in the sensory quality of the wine. As shown in Table 4, the content of alcohols in each wine sample is 131.33 mg / L (J-3), 213.47 mg / L (J-1) and 223.48 mg / L (F15) from low to high, all of which are lower than 300 mg / L, which can have a positive impact on the aroma of the wine sample. Hexanol will bring green and plant flavors to the wine. The content of hexanol in the wine samples fermented by Saccharomyces cerevisiae J-1 and J-3 (5.10 mg / L, 3.67 mg / L) is lower than that in the wine sample fermented by the commercial Saccharomyces cerevisiae strain F15 (7.28 mg / L). Phenylethanol is produced by the metabolism of phenylalanine and can give the wine a unique aroma of violet, clove, fennel and rose. The phenylethanol content in the wine fermented by yeast strain J-1 (66.04 mg / L) is slightly higher than that in the wine fermented by commercial brewer's yeast strain F15 (65.21 mg / L).

[0067] Acids can increase the complexity of wine aroma and play a positive role in wine when close to their threshold, while when the concentration is too high (>20 mg / L), it will bring unpleasant aromas of fat and cheese to the wine. The content of acid in wine samples fermented by commercial brewer's yeast F15, yeast strains J-1 and J-3 was <20 mg / L.

[0068] Ketones are also an important component of wine aroma. As shown in Table 4, compared with the wine samples fermented by commercial Saccharomyces cerevisiae strain F15, geranyl acetone was detected in the wine samples fermented by strains J-1 and J-3, with contents of 360 μg / L and 295 μg / L, respectively, which can give wine a rich milky and fruity flavor.

[0069] In addition, 2,4-di-tert-butylphenol was detected in all fermented wine samples, which had a spicy and smoky aroma. The content of 2,4-di-tert-butylphenol in the wine samples fermented by strains J-1 and J-3 (1195.00 μg / L and 970.00 μg / L) was lower than that in the wine sample fermented by strain F15 (1260.54 μg / L).

[0070] Table 4 GC-MS analysis results of volatile flavor components of wine samples fermented by different yeast strains

[0071]

[0072]

[0073]

[0074]

[0075] Note: Different lowercase letters in the same row indicate significant differences (P<0.05); “ND” means not detected.

[0076] (2) Sensory evaluation: A professionally trained panel of 10 people (5 men and 5 women) was invited to conduct sensory evaluation of wine samples from six aspects: color, taste, fruity aroma, floral aroma, sourness and solvent aroma. The 10-point structure numerical quantification was used as the standard, with 0 to 10 points indicating gradually increasing intensity. Each sample was analyzed three times, and all results were averaged. The results are shown in the table below. Figure 5 .

[0077] Depend on Figure 5 It can be seen that the floral, fruity and sour scores of the wine fermented by Saccharomyces cerevisiae J-1 were higher than those of the wine fermented by commercial Saccharomyces cerevisiae F15. The taste score of the wine fermented by Saccharomyces cerevisiae J-1 was comparable to that of the wine fermented by commercial Saccharomyces cerevisiae F15, but the color score was slightly lower than that of the wine fermented by commercial Saccharomyces cerevisiae F15. However, the sensory score of the wine fermented by Saccharomyces cerevisiae J-3 was lower due to its lack of floral and fruity aroma. In summary, Saccharomyces cerevisiae J-1 is suitable as an excellent strain for wine preparation.

[0078] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, any modification, equivalent replacement, improvement, etc. made without departing from the technical concept of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Saccharomyces cerevisiae J-1, characterized in that: The accession number is CGMCC No.27169.

2. The Saccharomyces cerevisiae J-1 as claimed in claim 1 is screened out from grapes or soil in Helan Mountain, Ningxia.

3. Use of Saccharomyces cerevisiae J-1 as claimed in claim 1 or 2 in winemaking.

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