CN107937293B - Fruity saccharomyces cerevisiae and application thereof in brewing of wine in Beijing area - Google Patents
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- CN107937293B CN107937293B CN201810029367.4A CN201810029367A CN107937293B CN 107937293 B CN107937293 B CN 107937293B CN 201810029367 A CN201810029367 A CN 201810029367A CN 107937293 B CN107937293 B CN 107937293B
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Abstract
The invention discloses a fruity saccharomyces cerevisiae and application thereof in brewing wine in Beijing area. The fruity saccharomyces cerevisiae is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation name of LH39 and the preservation number of CGMCC No. 14592. In the native grape mash of Beijing area, LH39 shows excellent fermentation capacity, especially outstanding fruit flavor, balanced taste and high quality. Compared with imported commercial saccharomyces cerevisiae, LH39 can better present the characteristics of wine in Beijing area, thereby helping the wine industry in Beijing area to form own native characteristics and promoting the development of the wine industry in the whole production area.
Description
Technical Field
The invention relates to the technical field of microorganisms. The invention particularly relates to a fruity saccharomyces cerevisiae and application thereof in brewing of wine in Beijing area.
Background
The brewing of the wine can not be separated from the saccharomyces cerevisiae, the saccharomyces cerevisiae not only converts sugar in the grape mash into alcohol through the fermentation effect, but also can influence the flavor, aroma and even structure of the wine through a series of metabolic effects. However, almost all wine production enterprises in China currently select imported commercial saccharomyces cerevisiae during brewing, the brands of the commercial saccharomyces cerevisiae selected by each winery are concentrated, two different commercial yeasts are selected only by distinguishing red wine from white wine, and the consciousness of selecting different saccharomyces cerevisiae according to the characteristics of different production places, different wine grape varieties, different final products, target products, aroma, taste and the like is weak, so that the quality of the wine in China is relatively single, and the wine is lack of characteristics, especially the wind and soil characteristics. The local yeast resources with various categories, pertinence, production areas and variety characteristics in China are developed in time, so that the local characteristics of the wine production areas in China are formed, and the diversity of the local saccharomyces cerevisiae resources in China is protected.
The history of Beijing's wine production was long, and could be traced back to 1910 (two years of Xuan Tong) at the earliest, the French saint Tian Master professor implies the Cumput repairman in the Shangyi wine factory created in Beijing. Although the production history is long, the wine industry in Beijing is very slow, and hundreds of wine villages in the areas of Liang shan, Yanqing, Miyun, Tongzhou and the like in Beijing have been developed in recent ten years along with the rapid development of the wine industry in China. However, research work on the indigenous saccharomyces cerevisiae in the Beijing area has not been carried out yet. The method screens out native high-quality saccharomyces cerevisiae in the Beijing area in time, is beneficial to the formation of regional characteristics of wine in the Beijing area, and has important significance for promoting the good and fast development of the wine industry in the Beijing area. At present, no related patent of saccharomyces cerevisiae screened from Beijing exists.
Disclosure of Invention
Therefore, the invention aims to provide the native saccharomyces cerevisiae screened from Beijing area with good fermentation performance, and make up for the shortage of native saccharomyces cerevisiae selection in our market.
The invention also aims to provide the application of the saccharomyces cerevisiae with good fermentation performance in brewing the dry red wine.
Aiming at the purposes, the technical scheme provided by the invention is as follows:
the invention provides Saccharomyces cerevisiae (Saccharomyces cerevisiae) named as LH39, belonging to Saccharomyces sp, which is separated from a vineyard of Gongborberg in mountain area of Beijing to pick ripe red wine grape variety- "Cabernet Sauvignon" grape fruits. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2017 at 9 and 4 months, and the preservation address is as follows: the microorganism research institute of China academy of sciences No. 3, GmbH No.14592, Tokyo province, hoja, has a collection number of CGMCC No.14592, and is suggested to be classified and named as: saccharomyces cerevisiae (Saccharomyces cerevisiae).
Unless otherwise indicated, Saccharomyces cerevisiae LH39CGMCC No.14592, herein abbreviated Saccharomyces cerevisiae LH 39.
The saccharomyces cerevisiae LH39 provided by the invention can be used for brewing wine. The wine brewed by using the saccharomyces cerevisiae LH39 has the advantages that: in the native grape mash of Beijing area, LH39 shows better fruit flavor, and the wine brewed by the LH39 has outstanding fruit flavor, balanced mouthfeel and high quality. Therefore, the wine yeast LH39 is inoculated to the indigenous wine brewing grape raw materials in Beijing, so that better fruit aroma can be obtained, and compared with the imported commercial wine yeast, the wine yeast can better present the characteristics of the wine in Beijing, thereby helping the wine industry in Beijing to form the indigenous characteristics of the wine industry in Beijing and promoting the development of the wine industry in the whole production area.
Drawings
FIG. 1 colony morphology of strain LH39 on WL medium.
FIG. 2 pilot Saccharomyces cerevisiae F15 pilot test fermentation record.
FIG. 3 record of pilot-plant fermentation tests of Saccharomyces cerevisiae LH39 of the present invention.
Detailed Description
For a better understanding and appreciation of the invention, reference will now be made in detail to the following examples and accompanying drawings; the examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The methods used in the following examples are conventional methods unless otherwise specified.
Unless otherwise indicated, the reagents used in the following examples are all of analytical grade and are commercially available from a regular channel.
Unless otherwise indicated, the quantitative tests in the following examples were set up in triplicate and the results were statistically averaged.
The media formulations referred to in the examples are as follows:
YPD solid Medium (per 1L): 10g of yeast powder, 20g of peptone, 20g of glucose and 20g of agar.
Wine yeast WLN differentiation medium (per 1L): 4g of yeast powder, 5g of tryptone, 50g of glucose and KH2PO40.55g，KCl 0.425g，CaCl2 0.125g，MgSO4 0.125g，FeCl3 2.5mg，MnSO42.5mg, agar 20g, bromocresol green 22 mg.
Example 1 isolation, purification and characterization of Saccharomyces cerevisiae LH39
The invention relates to a saccharomyces cerevisiae with good brewing performance, which is obtained from cabernet sauvignon grape varieties in a vineyard of the bauhinia wine village in Beijing, and the screening process is as follows:
selecting and picking a bunch of grape samples which are complete and mature and have no rotten fruits, arranging three parallel samples, wherein each grape sample is about 1kg, storing the grape samples in an aseptic bag, processing the grapes collected on the same day, removing stalks of the grape samples under aseptic conditions, selecting complete, mature and mildew-free fruit grains, collecting the fruit grains in a 500mL triangular flask, crushing the grapes in the triangular flask by using a medicine spoon, adding 1mL/L sulfurous acid into about 400mL of grape mash in each bottle after the grapes are crushed, sealing the grape mash by using an aseptic sealing film after uniformly stirring, and naturally fermenting the grape mash at 25 ℃.
Sampling every 24h in aseptic condition from the 0 th day (grape crushing time) of natural fermentation, taking 1mL of grape juice every time, and diluting with sterile water to 10-7Selecting appropriate 3 dilution concentrations, respectively 0.1mL, coating the dilution on a WLN identification medium plate, and carrying out inverted culture at 28 ℃ for 72 h. Then, samples were taken again at the later stage of fermentation where the dynamic and chemical composition changes of the grape mash flora were gradual, at days 10, 12 and 20. Selecting a flat plate with 30-300 colonies, classifying and numbering the colonies with different characteristics according to a method for classifying and identifying the yeasts by using a WLN identification culture medium, recording the characteristics and the number of various colonies, and photographing and archiving. 2-3 strains of yeast of different colony morphologies and appearance periods were selected and stored in YPD slant medium at 4 ℃. After the primary screening of the yeast is finished, the bacterial colony is prevented from being impure, and the bacterial strain is purified. Selecting single bacterial colony, identifying the purified bacterial strain on a WLN identification medium plate by using a plate marking method, carrying out inverted culture for 72h at the temperature of 28 ℃, recording the characteristics of the bacterial colony after each purification, photographing and archiving, observing the purity of the bacterial strain by combining a microscope, and purifying each bacterial strain for 2-3 times. The purified yeast was frozen using 40% glycerol at-80 ℃ in tubesAnd storing under the condition of condition for molecular identification.
Activating the yeast to be tested in advance, inoculating the yeast to be tested in YPD liquid culture medium, carrying out constant temperature shaking culture at 28 ℃ and 150rpm for 12h, centrifuging and taking yeast cells for later use. Extracting the genomic DNA of the yeast to be detected by using a rhizopus yeast genomic DNA extraction kit. The 5.8S ITS region of yeast rDNA gene is PCR amplified by using primer pair ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 52 deg.C for 2min, extension at 72 deg.C for 2min, and circulating for 35 times; extension was supplemented at 72 ℃ for 10 min. And (3) PCR reaction system: 10 XPCR buffer (Taq buffer with KCl) 5. mu.L, 25mmol/L MgCl2mu.L of 6. mu.L, 10mmol/L dNTPs 1. mu.L, 2.5. mu.L of each 10. mu. mol/L primer, 2.0U of Taq enzyme and 1. mu.L of template DNA, and adding double distilled water to the volume of 50. mu.L. mu.L of the amplification product was detected by electrophoresis on a 1% agarose gel.
The sequencing of PCR products is completed by Beijing Huada gene and Beijing Optimus department New Biotechnology Co.
Searching a target sequence of a strain to be detected by a BLAST tool in a National Center for Biotechnology Information (NCBI) website (https:// blast.ncbi.nlm.nih.gov/blast.cgi), searching a similar sequence, preliminarily determining the species position of the LH39 strain according to the similarity of the homologous sequence, and simultaneously correcting a sequencing map. Comparing the similarity degree of LH39 with the corresponding sequence of known yeast, the similarity degree is over 99%, and finally identifying the genus status of the strain to be detected through the colony morphology and cell microscopic morphology of LH39 on WLN identification culture medium.
Wherein, the sequencing result of 5.8S ITS rDNA of the saccharomyces cerevisiae LH39 (preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.14592) is shown as SEQ ID No. 1.
Similarity to related model strain saccharomyces cerevisiae YJM 271: 99.30 percent of the total weight of the yeast, and is identified as Saccharomyces cerevisiae.
Example 2 Saccharomyces cerevisiae LH39 comparative Small Saccharomyces cerevisiae test with commercial Saccharomyces cerevisiae
The method comprises the steps of removing stems of mature Meile variety wine grapes, crushing, using a 1L glass jar as a wine fermentation container, and respectively arranging 3 commercial wine yeasts used by LH39 and a wine village in two groups in parallel. After the yeast was activated, the yeast was inoculated into YPD liquid medium and cultured at 28 ℃ for 10 hours under a condition of 150rpm with shaking. To each jar was added 0.8L of grape mash, 0.8mL of sulfurous acid. The yeast is inoculated into the grape mash according to the inoculation amount of 3 percent. Cleaning wide-mouth bottle mouth and bottle cap with gauze dipped with sulfurous acid, placing the bottle cap upside down, and fermenting at 25 deg.C. After the fermentation of the grape mash begins, the temperature, sugar degree and specific gravity are measured and recorded every day. Stirring at proper time according to the fermentation condition. When the specific gravity of the wine is not reduced any more, the supernatant is taken and filled, and the wine is sealed and stored in a refrigerator at 4 ℃.
The contents of reducing sugars, glycerin, ethanol and part of organic acids in wine were measured by High Performance Liquid Chromatography (HPLC), and the measurement results are shown in table 1. And detecting the basic physical and chemical indexes of the wine according to GB/T15038-.
TABLE 1 test of the basic sugar acid content of the wine sample after the Small brewing test of the strains
The results of small-scale wine making tests show that the LH39 has stable fermentation performance, the alcohol content of a wine sample is higher than 11 (V/V%), the total sugar is not higher than 4g/L, and the volatile acid is lower than 1.2g/L, thereby meeting the requirements of GB15038-2006 on related indexes of dry wine. In addition, LH39 ethanol conversion capacity and glycerol production were higher compared to commercial yeast.
Referring to the descriptive examination procedure in GB10220-2012 and the wine scoring rules in GB15038-2006, 10 professional wine tasters were invited to blindly score the test wine samples. The scoring is based on four parts of the wine quality: appearance and color (10 points), aroma (30 points), mouthfeel and flavor (40 points), overall rating (20 points), totaling 100 points. The sensory evaluation table of wine refers to a sensory analysis table (sensory analysis tasting sheet for wire judging recipes) in a wine evaluation game. The results of the sensory evaluation analysis are shown in Table 2.
TABLE 2 sensory evaluation test results of the Small brewing test of the test strains
Sensory evaluation test results show that the wine brewed by the saccharomyces cerevisiae LH39 is superior to commercial saccharomyces cerevisiae in appearance, aroma, taste, overall quality and the total, particularly the fruity aroma is remarkable, and the brewed dry red wine has rich aroma and more layered aftertaste.
Example 3 Pilot-Scale fermentation test of Saccharomyces cerevisiae LH39 and commercial Saccharomyces cerevisiae
Saccharomyces cerevisiae LH39 was used in brewing pilot trials in comparison with commercial yeast F15, produced by LAFFORT, France. Pilot trials were conducted in the source wine house corresponding to LH 39. The fermentation process is recorded as shown in FIGS. 2 and 3, respectively.
LH39 was expanded in the laboratory of Angel Yeast of Hubei to obtain lyophilized yeast powder, and pilot-scale brewing test was performed with Cabernet Sauvignon. As can be known from fermentation records, the saccharomyces cerevisiae LH39 shows stronger fermentation performance, and the fermentation speed of the LH39 test group is obviously higher than that of the control group F15 in the middle and later stages of fermentation. In addition, barrel taste results compared to commercial yeast show that LH39 fermented wine exhibited excellent fruit aroma, balanced with oak aroma, while commercial yeast fermented wine was completely masked by oak aroma.
In conclusion, the preferred wild saccharomyces cerevisiae strain LH39 not only shows excellent fermentation performance in the process of brewing the dry red wine, but also has outstanding fruit aroma and good taste of the brewed wine, is a high-quality saccharomyces cerevisiae strain capable of exerting the characteristics of the native dry red wine, and has the potential of commercial application.
Sequence listing
<110> university of agriculture in China
<120> one strain of fruit-flavor saccharomyces cerevisiae and application thereof in brewing of wine in Beijing area
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 739
<212> DNA
<213> Saccharomyces sp.
<400> 1
atttaataat tttgaaaatg gatttttttt ttttgttttg gcaagagcat gagagctttt 60
actgggcaag aagacaagag atggagagtc cagccgggcc tgcgcttaag tgcgcggtct 120
tgctaggctt gtaagtttct ttcttgctat tccaaacggt gagagatttc tgtgcttttg 180
ttataggaca attaaaaccg tttcaataca acacactgtg gagttttcat atctttgcaa 240
ctttttcttt gggcattcga gcaatcgggg cccagaggta acaaacacaa acaattttat 300
ctattcatta aatttttgtc aaaaacaaga attttcgtaa ctggaaattt taaaatatta 360
aaaactttca acaacggatc tcttggttct cgcatcgata aaaaacgcag cgaaatgcga 420
tacgtaatgt gaattgcaga attccgtgaa tcatcgaatc tttgaacgca cattgcgccc 480
cttggtattc cagggggcat gcctgtttga gcgtcatttc cttctcaaac attctgtttg 540
gaagtgagtg atactctttg gagttaactt gaaattgctg gccttttcat tggatgtttt 600
tttttccaaa aagaggtttc tctgcgtgct tgaggtataa tgcaagtacg gtcgttttag 660
gttttaccaa ctgcggctaa tcttttttta actgagcgta ttggaacgtt atcaaaaaaa 720
aaaaccgcca ggcaaacat 739
Claims (3)
1. A strain of fruit-flavor saccharomyces cerevisiae is characterized in thatThe fruity saccharomyces cerevisiae is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms under the preservation name saccharomyces cerevisiae (Saccharomyces cerevisiae) LH39 with preservation number of CGMCC No. 14592.
2. The use of the fruit-flavored Saccharomyces cerevisiae of claim 1 in the production of Beijing area wine.
3. A method for producing wine by fermenting a grape material with the fruit-flavor Saccharomyces cerevisiae of claim 1.
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