CN101792719B - Saccharomyces cerevisiae and application thereof in wine brewing - Google Patents

Abstract

The invention discloses saccharomyces cerevisiae and the application thereof in wine brewing. The saccharomyces cerevisiae is saccharomyces cerevisiae NJZCC17 CGMCC No. 3284. The saccharomyces cerevisiae NJZCC17 is used to brew wine. Compared with the widely used reference commercial saccharomyces cerevisiae strain, the sugar content in the wine prepared by the saccharomyces cerevisiae is 1 to 4 percent, the alcohol content thereof is 9 to 14 percent and the glycerol yield thereof is 4 to 11g/L, the yield of hydrogen sulfide, acetaldehyde, acetic acid, urea and other metabolites which are bad for the flavor or the quality of the wine is lower, the malic acid utilization rate is higher, the volatile aroma components are balanced, the total ester yield is higher and the total high-grade alcohol yield is appropriate, the taste is more complicated and the brewing time is shortened; and therefore, the invention displays important roles in stabilizing and improving the quality of the domestic dry red wine, and displaying the brewing potential of cabernet sauvignon and other red wine in Hebei Changli production area.

Description

One Accharomyces cerevisiae and the application in making grape wine thereof

Technical field

The present invention relates to an Accharomyces cerevisiae and the application in wine making thereof, particularly an Accharomyces cerevisiae (Saccharomyces cerevisiae) and the application in brewageing dry red winew thereof.

Background technology

Wine making is the microbiological process of a complexity, and wherein yeast is prevailing microorganism.Brewing grape wine mainly is under the effect of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), adopts spontaneous fermentation or pure-blood ferment, and various potential quality and advantage in the grape material are fully showed in wine.Therefore, the quality of yeast saccharomyces cerevisiae performance is not only very big to mouthfeel vinous and local flavor influence, but also directly has influence on output, quality, economy and the fermentative production management of institute's vintage wine, and is most important to the formation of grape wine characteristic and style.The Wine brewing yeast strain that employing has multiple specific premium properties not only can improve quality vinous, can also shorten fermentation period and reduce production costs, and then improve the competitive edge and the earning capacity of enterprise.These requirements to wine yeast have directly promoted carrying out of good yeast saccharomyces cerevisiae seed selection work.

Utilize the good Wine brewing yeast strain in native country to brewage the high-quality wine with local characteristic, in the time-honored European certain areas of wine brewing, especially the famous brand of wine place of production is widely popular.At present, states such as France, the U.S., Italy, Spain and Australia all attach great importance to the wine brewing characteristic research to the local good yeast saccharomyces cerevisiae of this country, and extensively strain excellent are made active dry yeast and be introduced into the commercialization Production of Wine.Produce shortcut though be one with active dry yeast, because differences such as grape variety, planting environment and processing condition are introduced bacterial classification and can not be shown its better quality fully from these good wine makings of external introducing.There is multiple unartificial yeast to be accompanied in the garden of planting grape for many years, through secular natural selection with evolve and develop, formed the wild yeast saccharomyces cerevisiae of advantage of a collection of adaptation local climate, environment and grape variety gradually with it.Therefore, China must pay attention to the native country Wine brewing yeast strain that seed selection has good brewing characteristic.

Production of Wine enterprise of China and grape wine producing region lack the barms that is fit to own raw material and product, the almost whole dependence on import of the microbial preparation that China's wine industry needs.And external eurytropy grape wine microbial preparation is widely-used, can cause the product homogeneity aggravation, lack place of production characteristic, and a large amount of introducings of external microbial strains, can influence the rich and diversity of native resource, influence the validity that winery carries out the seed selection of nature yeast.The wine yeast that seed selection is possessed of good qualities, significant to the improvement of grape wine color and luster, fragrance, aesthetic quality and style.And China's vinify screening of microbial strains also is in the starting stage.Up to now, there is not China to be applied among wine industry produces as yet from the microorganism strains of row filter and seed selection.

China's wine-growing area is wide, and ground such as Hebei, Shandong, Gansu and Xinjiang all are important viny regions.Wild yeast saccharomyces cerevisiae distributes and has certain regularity in different producing regions and the different varieties grape.Screening has the Wine brewing yeast strain of good brewing characteristic, and this locality (especially country of origin) grape wine that has the high-quality characteristic for exploitation is significant.

Summary of the invention

Purpose of the present invention is intended to solve homemade dry red winew quality owing to the active dry yeast starter that is extensive use of external import is tending towards the problem of " homogeneity ", and an Accharomyces cerevisiae and the application in dry red winew is produced thereof are provided.

Yeast saccharomyces cerevisiae provided by the present invention is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NJZCC17CGMCC № .3284.

Described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NJZCC 17CGMCC № .3284 is circle, oyster white, glossy through the bacterium colony of growing on the 48h solid YEDP substratum, its neat in edge, and the thalline thickness is easily provoked; Colony characteristics is a cream colour band green on the WL nutrient agar, bacterium colony spherical protuberances, smooth surface, opaque, butteriness (as shown in Figure 1); On McClary acetate agar substratum, cultivate 1-4 after week for 28 ℃, have 2-4 thecaspore to form conidial cell wall smooth (as shown in Figure 2).

Described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NJZCC17, be preserved in Chinese microorganism strain preservation board of trustee reason person on September 17th, 2009 and understand common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3284.

Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) NJZCC17 that the present invention obtains from the screening of producing region, China Changli Wine Grape surface, evaluation.The dry red winew advantage of utilizing yeast saccharomyces cerevisiae NJZCC17 of the present invention to produce is: in the dry red winew production process of viny region, Hebei province, compare with contrast commercialization Wine brewing yeast strain, sugared content is 1～4g/L in the grape wine that its fermentation obtains, ethanol content is 9～14%, glycerine output 4～11g/L, hydrogen sulfide, acetaldehyde, acetate and urea etc. are lower to grape wine local flavor or the disadvantageous meta-bolites output of quality, the oxysuccinic acid utilization ratio is higher, volatile aroma becomes balance-dividing, total Ester output is higher and total higher alcohols output is moderate, can improve the content of polyphenol and trans-resveratrol in the grape wine, mouthfeel has more complicacy, brewing time is moderate, and to stabilizing and increasing of native country dry red winew quality, and red grape such as performance producing region, Changli Cabernet Sauvignon is brewageed potentiality and all played a significant role.Yeast saccharomyces cerevisiae NJZCC17 of the present invention especially can bring into play the characteristics of brewageing and the advantage of cabernet sauvignon grape to greatest extent, has the effect of production high-quality producing region Cabernet Sauvignon dry red winew.

Description of drawings

Fig. 1 is the colonial morphology of Wine brewing yeast strain (Saccharomyces cerevisiae) NZJCC17 on the WL nutrient agar medium.

Fig. 2 be Wine brewing yeast strain NJZCC17 on McClary acetate agar substratum 28 ℃ cultivate 1-4 after week, the morphologic observation in microscope.

Embodiment

Method among the following embodiment if no special instructions, is ordinary method.

Simulation Sucus Vitis viniferae culture medium prescription among the following embodiment is as follows:

Glucose, 200g/1000mL; Citric acid, 6g/1000mL; Oxysuccinic acid 6g/1000mL, uridylic 20mg/1000mL; Potassium primary phosphate, 750mg/1000mL; Vitriolate of tartar, 500mg/1000mL; Magnesium sulfate heptahydrate, 250mg/1000mL; Sodium-chlor 200mg/1000mL; Calcium dichloride dihydrate, 155mg/1000mL; Manganese sulfate monohydrate, 4mg/1000mL; Zinc sulfate, 4mg/1000mL; Potassiumiodide, 1mg/1000mL; Cupric sulfate pentahydrate, 1mg/1000mL; Boric acid, 1mg/1000mL; Sodium Molybdate Dihydrate, 1mg/1000mL; CoCL2,0.4mg/1000mL; Inositol, 20mg/1000mL; Nicotinic acid, 2mg/1000mL; Calcium pantothenate 1.5mg/1000mL; Vitamin 0.25mg/1000mL; Pyridoxine hydrochloride (VB6) 0.25mg/1000mL; Vitamin H (biotin), 0.003mg/1000mL; Proline(Pro) (proline), 61.5mg/1000mL; Glutamine (glutamine), 50.7mg/1000mL; Arginine (arginine), 37.5mg/1000mL; Tryptophane (tryptophan), 18mg/1000mL; L-Ala (alanine), 14.7mg/1000mL; L-glutamic acid (glutamic acid), 12mg/1000mL; Serine (serine), 7.8mg/1000mL; Threonine (threonine), 7.8mg/1000mL; Leucine (leucine), 4.8mg/1000mL; Aspartic acid (aspartic acd) 4.5mg/1000mL; Xie Ansuan (valine), 4.5mg/1000mL; Phenylalanine (phenylalanine) 3.9mg/1000mL; Isoleucine (isoleucine), 3.3mg/1000mL; Histidine (histidine), 3.3mg/1000mL; Methionine(Met) (methionine), 3.3mg/1000mL; Tyrosine (tyrosine), 1.8mg/1000mL; Glycine (glycine), 1.8mg/1000mL; Methionin (lysine), 1.8mg/1000mL; Halfcystine (cysteine), 1.2mg/1000mL; Ammonium chloride, 55.8mg/1000mL; Ergosterol, 15mg/1000mL; Sodium oleate, 5mg/1000mL; Tween-80,0.5ml/1000mL.pH＝3.3±0.1。

The YEPD culture medium prescription is as follows among the following embodiment:

Glucose, 20g/1000mL; Peptone, 20g/1000mL; Yeast extract, 10g/1000mL; Nature pH value, 121 ℃, sterilization 15min.Solid medium adds 20g/1000mL agar, and the back sterilization is melted in the microwave oven heating fully.

WL nutrient agar prescription is as follows among the following embodiment:

Yeast extract, 4g/1000mL; Tryptones, 1g/1000mL; Glucose, 50g/1000mL; KH ₂PO ₄, 0.550g/1000mL; KCl, 0.425g/1000mL; CaCl ₂, 0.125g/1000mL; MgSO ₄, 0.125g/1000mL; FeCl ₃, 0.0025g/1000mL; MnSO ₄, 0.0025g/1000mL; Tetrabromo-mcresolsulfonphthalein, 0.022g/1000mL; PH=6.5.

McClary culture medium culturing based formulas is as follows among the following embodiment:

Glucose, 1g/1000mL; KCl, 1.8g/1000mL; Anhydrous sodium acetate, 8.2g/1000mL; Yeast extract paste, 2.5g/1000mL; Agar, 20g/1000mL.

Separation, purifying and the evaluation thereof of embodiment 1, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NJZCC17CGMCC № .3284

The isolating wild yeast of strain more than 200 bacterial strain in different grape varieties surface, viny region, Changli is separated, identifies and screens.Its separation method adopts dull and stereotyped dilution coating method picking list bacterium colony and double purifying.Authentication method adopts traditional form to learn evaluation and Physiology and biochemistry is identified in conjunction with ATB ID 32C yeast automatic identifying system (Biomerieux SA, and be aided with 5.8S-ITS rDNA PCR-RFLP and two kinds of molecular biology methods of 5.8S-ITS rDNA sequential analysis are identified bacterial strain France); Bacterial strain through being accredited as yeast saccharomyces cerevisiae is all with French LAFFORT commercialization of production active dry yeast (the Active Dry Yeast of company, ADY) Zymaflora F15 (following all writing a Chinese character in simplified form made F15) sieve again for control strain carries out test tube fermentation primary dcreening operation, shake flask fermentation, small-sized wine brewing preferred strain screening experiment and excellent sieve bacterial strain pilot scale wine brewing experiment, bacterial strain is screened.Through nearly 4 years systematic study and repeated screening, obtain the Wine brewing yeast strain that the number strain has good brewing characteristic.According to wine making commonly used in the world Wine brewing yeast strain screening index (Efstratios Nikolaou, Evangelos H.Soufleros, ElizabethBouloumpasi, Nikolaos Tzanetakis. Selection of indigenous Saccharomyces cerevisiaestrains according to their oenological characteris

Pass through repeated screening, verify and brewage research, (Saccharomyces cerevisiae) the NJZCC17CGMCC № .3284 (hereinafter to be referred as NZJCC17) that obtains from the cabernet sauvignon grape surface isolation of viny region, Changli County, Hebei province growth has that fermentative activity is vigorous, fermentation starting rapidly, foam yields poorly, the fermentation ends flocculation ability is strong, the fermentation residual sugar is low and alcoholic strength height, glycerine output height, utilize the oxysuccinic acid ability stronger, acetaldehyde, H ₂S and urea etc. have dysgenic material to grape wine quality and yield poorly, to SO ₂, the adverse circumstance life conditions such as high osmotic pressure that caused of alcohol and high pol have good resistance, the grape wine of brewageing through NJZCC17 has good fragrance structure and composition, main secondary volatilization aroma substance (ester class, higher alcohols, carbonyl compound, small molecular organic acid etc.) output is high or approximate than control strain, and the more commercial control strain yeast saccharomyces cerevisiae of kind F15 is many.Think the preferably saccharomyces cerevisiae bacterial strain NJZCC17 fermentation Changli cabernet sauvignon grape that produce in the producing region obtain with separating on the cabernet sauvignon grape of Changli through expert evaluation is consistent, no matter the dry red winew that zythepsary gets all is better than the contrast dry red winew of active dry yeast (France) with the same terms production on color, smell and taste.

To after all qualification result analysis-by-synthesis, identify that NJZCC17 is the new strain of yeast saccharomyces cerevisiae behind the rflp analysis of this bacterial strain process morphological observation, Physiology and biochemistry evaluation, ATB ID 32C yeast rapid evaluation system and 5.8S-ITS rDNA gene amplification and PCR product.Concrete qualification result is as follows:

Morphological observation method and result: cultivate through 48h, the bacterium colony that NJZCC17 grows on the YEDP solid medium is an oyster white, and circle is glossy, neat in edge, thickness is easily provoked, microscopically is viewed as the circular or oval of cell, polygon budding, no pseudohypha; Colony characteristics is a cream colour band green on the WL nutrient agar, the bacterium colony spherical protuberances, smooth surface, opaque, butteriness (as shown in Figure 1) on the McClary nutrient agar 28 ℃ cultivate 1-2 after week, having examined under a microscope 2-4 thecaspore forms, conidial cell wall is smooth, the spore feature (as shown in Figure 2) of NJJCC17.

Physiology and biochemistry authentication method and result: according to the systematic searching table of yeast belong, this research is chosen for yeast saccharomyces cerevisiae and is belonged to assimilative capacity and these four Physiology and biochemistries tests of anti-cycloheximide ability that kind of the more important test event of inner region branch is assimilative capacity, nitrate and the L-Methionin of fermentation capacity, cellobiose and the inositol of glucose, semi-lactosi, sucrose, maltose and lactose.Find through Physiology and biochemistry experiment back, the result of assimilation of the carbon assimilation of NJZCC17, nitrogenous source and cycloheximide growth experiment all with control strain CICC1450 (normal business control strain, available from Chinese industrial microbial strains preservation administrative center, below all write CICC1450) test result is identical, and concrete outcome sees the following form.

The Physiology and biochemistry experimental result of table 1 control strain and representative strain

[notes]: "+" expression thalli growth, the test-results positive; "-" expression thalline is not grown the test-results feminine gender.

Method and result that ID 32C yeast identification systems are identified: utilize full-automatic evaluation of ID 32C yeast identification systems (Mei Liai company, France) and ATB and antibiotics susceptibility test instrument (Mei Liai company, France) that the good yeast of purifying is identified.

ID 32C is suitable for saccharomycetic identification systems by what 32 stdn, little quantized assimilation test and corresponding database were formed.Contain different dehydrated carbohydrates in these 32 apertures.A kind of chemical pure semisolid medium is inoculated in the aperture with bacteria suspension.By saccharomycetic growing state in the ATB reader vision slit, system obtains qualification result according to API LAB ID 32 softwares automatically then behind cultivation 24～48h.

Evaluation likelihood (id%) to (Saccharomyces cerevisiae) NJZCC17 of yeast saccharomyces cerevisiae in this experiment and standard control bacterial strain CICC1450 reaches 99.9, is fabulous qualification result.

5.8S-ITS rDNA PCR-RFLP and 5.8S-ITS rDNA sequential analysis authentication method and result:

(internal transcribed spacer ITS) has the significant interspecific difference opposite sex to the transcribed spacer of zymic rrna 5.8S rDNA and both sides, can be used as the classification foundation of differentiating barms.When yeast strain to be measured being carried out conventional phenotypic evaluation and automatization evaluation, also adopt two kinds of molecular biology methods of 5.8S-ITS rDNA PCR-RFLP and 5.8S-ITSrDNA sequential analysis that bacterial strain is identified, the unity and the reliability of the test-results of several classification authentication methods are verified.Concrete grammar is as follows:

1. the extraction of yeast genomic dna: at first adopt yeast genome DNA extracting reagent kit (tomorrows hundred proud company, Beijing) to extract saccharomycetic genomic dna.

2. the pcr amplification of yeast strain 5.8S-ITS rDNA: the pcr amplification the primer is ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 '), and is synthetic by Shanghai biotechnology Services Co., Ltd; The pcr amplification condition is 95 ℃ * 5min; 94 ℃ * 1min, 55.5 ℃ * 2min, 72 ℃ * 2min, 35 circulations; 72 ℃ * 10min; Pcr amplification system (50 μ L) consists of: template DNA 3-5 μ L, 10 * PCR reaction buffer, 5.0 μ L, 10 μ M primers, 0.5 μ L, 10mM dNTPs1 μ L, Taq archaeal dna polymerase 1U, ddH ₂O replenishes system to 50 μ l, mixing.Subsequently pcr amplification product is detected: get 5 μ L pcr amplification product point samples on 1.4% sepharose, electrophoretic buffer is 1 * TAE, under the 100V voltage, electrophoresis 40min, after ethidium bromide (EB) dyeing, electrophoretogram is taken pictures and analyze by gel imaging system, adopt 100-bp DNA Marker to judge the size of pcr amplification product.

3. the rflp analysis of yeast strain 5.8S-ITS rDNA: the pcr amplification product of 5.8S-ITS rDNA use respectively CfoI, HaeIII and Hinf I (Fermentas, American) 37 ℃ of enzymes of three kinds of restriction enzymes are cut 16h; The enzyme system of cutting of 20 μ L includes 10 μ L PCR products, 2 μ L 10 * Buffer and 10U restriction endonuclease; Get 10 μ L enzymes after enzyme is cut and cut product in 3% sepharose, in 1 * TAE damping fluid, electrophoresis 1h under the 100V voltage, after the EB dyeing, gel is taken a picture, and yeast strain to be measured is identified in last and standard restriction enzyme mapping contrast on the level of planting.

4. the sequential analysis of yeast strain 5.8S-ITS rDNA: 5.8S-ITS rDNA pcr amplification product reclaims test kit (tomorrows hundred proud company through sepharose, Beijing) behind the purifying, Services Co., Ltd checks order by the Shanghai biotechnology, and sequencing primer is with PCR primer I TS1 or ITS4; According to sequencing result, utilize BLAST software from GenBank nucleic acid sequence data storehouse, to carry out homologous sequence search, the relatively similarity degree of strains tested and known yeast strain corresponding sequence.

Use primer I TS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ', sequence 1) and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ', sequence 2) to the 5.8S-ITS rDNA district amplification of isolating representative bacterial strain, the product of gained mostly is between the 450-880bp greatly.Use CfoI, HaeIII and HinfI to carry out enzyme and cut, the gained restriction enzyme mapping has 20 types, and the standard restriction enzyme mapping that people such as restriction enzyme mapping and Guillamon and Esteve-Zarzoao are set up contrasts evaluation.It is as shown in table 2 that the enzyme of the yeast strain that the 5.8S-ITSrDNA-RFLP method of yeast strain NJZCC17 and CICC1450 is identified is cut data.The restriction enzyme mapping of 5.8S-ITS rDNA-RFLP by evidence NJZCC17 and CICC1450 proves that NJZCC17 is the new strain of yeast saccharomyces cerevisiae.

Table 2 yeast strain 5.8S-ITS rDNA pcr amplification product size and restriction endonuclease map

Native country zymic 5.8S-ITS rDNA The sequencing results is as shown in table 3.

The table 3 grape wine yeast strain 5.8S-ITS rDNA sequencing result of being correlated with

The RFLP enzyme is cut type (strain number)	The reference strain gene pool is accepted number	Homology (%)	Qualification result
				CICC1450	AM900396	100	S.cerevisiae
NJZCC17	AM900396	100	S.cerevisiae

According to present yeast molecular systematics research field institute popular viewpoint, the bacterial strain that promptly has same or similar ITS sequence (sequence homology 〉=99%) belongs to same species, the sequence homology of finding reference culture CICC1450 and test strain NJZCC17 and gene pool reference strain 5.8S-ITS rDNA according to sequential analysis is 100%, therefore can confirm qualification result, promptly NJZCC17 is the new strain of yeast saccharomyces cerevisiae.

Adopt four kinds of classification authentication methods NJZCC17 to be identified its qualification result and standard control bacterial strain are identical, so NJZCC17 can be accredited as the new strain of yeast saccharomyces cerevisiae.

Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NZJCC17, be preserved in Chinese microorganism strain preservation board of trustee reason person on September 17th, 2009 and understand common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3284.

The wine brewing effect experiment of embodiment 2, Wine brewing yeast strain (Saccharomyces cerevisiae) NZJCC17

Good grape wine Wine brewing yeast strain NJZCC17 and the control strain CICC1450 brewing characteristic in the simulation fermenting must compares, and concrete grammar is as described below:

The yeast saccharomyces cerevisiae NJZCC17 and the yeast saccharomyces cerevisiae CICC1450 that are stored in-80 ℃ are gone down to posterity twice respectively on YEPD solid and liquid nutrient medium, by 10 ⁶CFU/mL is inoculated in the 1.8L simulation Sucus Vitis viniferae nutrient solution, 25 ℃ of cultivations.

The yeast saccharomyces cerevisiae NJZCC17 of picking overnight incubation or yeast saccharomyces cerevisiae CICC1450 control strain insert and contain in the test tube of 10mL liquid YEPD substratum, 28 ℃ leave standstill to cultivate and draw 1mL respectively behind the 12h and transfer in the triangular flask that contains 100mL YEPD liquid nutrient medium, and 28 ℃ leave standstill and cultivate 12h.Then according to 10 ⁶The inoculum size of CFU/mL is inoculated in the simulation Sucus Vitis viniferae, and 25 ℃ are cultured to residual sugar amount＜4g/L, brewage and obtain simulating grape wine.

Detect brewing characteristic in the simulation fermenting must according to following method:

CO ₂Weightless: weighing method (M.S.P é rez-Coello1, A.I.Briones P é rez, J.F.Ubeda Iranzo andP.J.Martin Alvarez.Characteristics of wines fermented with different Saccharomycescerevisiae strains isolated from the LaMancha region.Food Microbio.1999,16 (6): 563-573);

Residual sugar content: film reagent method (GB/T 15038-2006);

Ethanol content: pycnometric method (GB/T 15038-2006);

Total acidity: volumetry (GB/T 15038-2006);

Acetaldehyde output: the test kit method, acetaldehyde is measured test kit (Acetaldehyde Assay Kit, Megazyme company, Ireland);

Glycerine output: the test kit method, glycerine is measured test kit (Glycerol Assay Kit, Megazyme company, Ireland);

Urea production: the test kit method, urea is measured test kit (Urea Assay Kit, Megazyme company, Ireland);

Malic acid content: liquid phase chromatography (GB/T 15038-2006);

Volatile aroma substances content such as ester class, higher alcohols, organic acid: detect various volatile aroma substance classes and content in the making grape wine of above-mentioned acquisition with Agilent 6890 gas-chromatographies (GC) and Agilent 5975 mass spectrums (MS) combined instrument (Agilent, the U.S.).Actual conditions is: capillary column HP-INNOWAX PolyethyleneGlycol 60m * 0.25mm * 0.25 μ m (J﹠amp; W scientific, U.S.) carrier gas is high-purity helium, flow velocity 1mL/min; The headspace solid-phase microextraction hand sampling adopts not shunt mode, inserts the injection port of gas-chromatography, 250 ℃ of injector temperatures, and 25min is analysed in pyrolysis.The heating schedule of column oven is: 40 ℃ keep 5min, and the speed with 3 ℃/min is warming up to 200 ℃ then, keep 2min.Mass spectrum excuse temperature is 280 ℃, ion source temperature is 230 ℃, ionization mode EI, ion energy 70ev, mass scanning scope 20-450amu (Zhang Mingxia. the research of grape wine fragrance Changing Pattern---focus on of the influence [D] of crucial making method to grape wine fragrance. the doctorate paper .2007 of China Agricultural University);

Maximum anti-ethanol content: press document (J.A.Regod ó n, F.Per é z, M.E.Vald é s, C.De Miguel andM.Ram í rez.A simple and effective procedure for selection of wine yeast strains.FoodMicrobio.1997,14 (3): 247-254) method of Miao Shuing is carried out;

Maximum anti-SO ₂Content: press document (J.A.Regod ó n, F.Per é z, M.E.Vald é s, C.De Miguel andM.Ram í rez.A simple and effective procedure for selection of wine yeast strains.Food Microbio.1997,14 (3): 247-254) method of Miao Shuing is carried out;

Produce H ₂S ability: each test strain dibbling on the BIGGY agar of commercially producing, is cultivated 4～7d for 30 ℃, continue to observe colony colour and change, investigate each test strain according to shade and produce the hydrogen sulfide ability.1=white (white), 2=cream (cream color), 3=light brown (light brown), 4=brown (brown), 5=dark brown (dark-brown), 6=black (black).The high more then H of numerical value ₂S produces the strong more (Giudici of ability, P., Kunkee, R.E.The effect of nitrogen deficiency and sulfur-containing amino acids on the reduction ofsulfate to hydrogen sulfide by wines yeasts.Am.J.Enol.Vitic.1994,45:107-112);

2,3-butyleneglycol: utilize magnetic resonance detection (Hong-Seok Son, Geum-Sook Hwang, Ki MyongKim, Eun-Young Kim, Frans van den Berg, Won-Mok Park, Cherl-Ho Lee, andYoung-Shick Hong. ¹H NMR-Based Metabolomic Approach for Understanding theFermentation Behaviors of Wine Yeast Strains.Anal Chem.2009,81:1137-1145).

Yeast saccharomyces cerevisiae NJZCC17 and CICC1450 brewing characteristic comparative result in the simulation fermenting must sees Table 4.

Table 4 yeast saccharomyces cerevisiae NJZCC17 and CICC1450 brewing characteristic in the simulation fermenting must compares

Bacterial strain	CO 2Weightless (g/100mL)	Residual sugar content (g/L)	Ethanol content (%, v/v)	Total acidity (g/L)	Acetaldehyde output (mg/L)
						NZJCC17	11.3±0.1	1.06±0.02	10.58±0.02	11.2±0.017	0.818±0.023
CICC1450	10.5±0.1	1.19±0.03	10.29±0.02	10.6±0.051	2.142±0.091

Table 4 (continuous 1) yeast saccharomyces cerevisiae NJZCC17 and CICC1450 brewing characteristic in the simulation fermenting must compares

Bacterial strain	Glycerine output (g/L)	Urea production (mg/L)	Oxysuccinic acid residual quantity (g/L)	Acetic acid content (mg/L)	Phenylethyl alcohol (mg/L)
						NZJCC17	4.33±0.012	0.57±0.21	4.51±0.067	1.161±0.012	15.80±2.42
CICC1450	4.16±0.035	3.62±0.10	5.43±0.043	1.365±0.016	12.01±0.64

Table 4 (continuous 2) yeast saccharomyces cerevisiae NJZCC17 and CICC1450 brewing characteristic in the simulation fermenting must compares

Bacterial strain	Ethyl acetate (mg/L)	Ethyl hexanoate (mg/L)	Hexyl acetate (mg/L)	Caproic acid phenethyl ester (mg/L)	Total ester (mg/L)
						NZJCC17	6.76±0.54	6.32±1.12	0.13±0.02	0.094±0.016	60.31±1.34
CICC1450	6.37±0.54	4.29±0.26	0.096±0.034	0	51.88±0.98

Table 4 (continuous 3) yeast saccharomyces cerevisiae NJZCC17 and CICC1450 brewing characteristic in the simulation fermenting must compares

Bacterial strain	The volatile aroma substance classes	Maximum anti-ethanol content (%, v/v)	Maximum anti-SO 2Content (mg/L)	Produce H 2The S ability	2,3-butyleneglycol (mg/L)
						NZJCC17	34	17％	600	Low (light brown)	0.456±0.021

CICC1450

28

16％

600

High (dark-brown)

0.346±0.017

In the simulation Sucus Vitis viniferae, Wine brewing yeast strain carried out brewing characteristic research can with the closely similar trophic component condition of natural grape juice under get rid of of the influence of wild assorted yeast fully to fermentation character.By the experimental result of table 4 as can be seen, CICC1450 compares with the commercial criterion control strain, and the fermenting power of NJZCC17 is stronger, its CO ₂Higher with alcohol output, and the residual sugar amount is lower; The product that grape wine quality and local flavor are had disadvantageous effect is as acetaldehyde, acetate, H ₂S and urea production, the output of NJZCC17 is all lower; And grape wine local flavor and mouthfeel are had the material of positive influence, and as glycerine, ethyl acetate, hexyl acetate, ethyl hexanoate, phenylethyl alcohol, 2,3-butyleneglycol and total ester, the output of NJZCC17 all is higher than control strain; On volatile aroma composition formation, NJZCC17 can produce 34 kinds of volatile aroma compositions, and CICC1450 only can produce 30 kinds.Producing on the Ester ability, NJZCC17 is better than control strain F15, belongs to good brewing characteristic.And, NJZCC17 can produce the ester class Phenylethyl ethanoate that CICC 1450 can't produce (0.094 ± 0.016mg/L), caproic acid-3-methyl butyl ester (0.17 ± 0.021mg/L), 3-octylenic acid ethyl ester (0.1 ± 0.001mg/L) and 2-methyl-propionic acid (0.054 ± 0.002mg/L); From the comparative studies to stress resistance, the anti-alcohol ability of NJZCC17 is a little more than CICC1450, and to SO ₂Resistance is identical.The low time that can shorten the milk-acid bacteria malic-lactic acid fermentation of malic acid content shortens brewing period in the grape wine.Though Wine brewing yeast strain does not possess the film system that transports oxysuccinic acid outside film, different strains is different to the utilization ratio of oxysuccinic acid, compares the oxysuccinic acid utilization ratio of NJZCC17 higher (NJZCC17 is 24.83%, and CICC1450 is 9.5%) with CICC1450

Embodiment 3, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NZJCC17 and the small-sized wine brewing comparative experiments of commercial strain F15 in cabernet sauvignon grape juice

With Wine brewing yeast strain NJZCC17 of the present invention and control strain active dry yeast ADY F15, for brewageing system, compare brewing characteristic with 4L cabernet sauvignon grape juice.Followingly produce cabernet sauvignon grape destemming, removal of impurities with Changli in 2007, broken then, the Sucus Vitis viniferae that obtains of squeezing the juice is a raw material, brewages experiment.Following 2007 Changli is produced the main physical and chemical index of cabernet sauvignon grape and is: reducing sugar (with glucose meter) content, 210.99 ± 1.20g/L; Total acid content (in tartrate), 5.06 ± 0.46g/L; PH=3.83 ± 0.04.

The yeast saccharomyces cerevisiae NZJCC17 that is stored in-80 ℃ is gone down to posterity twice on YEPD solid and liquid nutrient medium, commercial strain active dry yeast F15 (control strain with 7 ℃, commercialization active dry yeast Zymaflora F15 is available from French LAFFORT company) by producer's suggesting method water activation.Get good yeast saccharomyces cerevisiae NJZCC17 of activation or control strain F15 respectively and be inoculated into respectively and be equipped with in the 5L Glass Containers that produces cabernet sauvignon grape juice in Changli, 4L Hebei province in 2007, add sulfurous acid, reach 60mg/L to free sulphur content, inoculum size is 10 ⁶CFU/mL, room temperature is fermented, and monitors the temperature and the proportion of fermented liquid every day, is considered as fermentation ends when proportion no longer descends, and obtains dry red winew.

Measure the CO of the above-mentioned dry red winew that obtains according to embodiment 2 described methods ₂Weightlessness, residual sugar amount, ethanol content, glycerine output, volatile acid, higher alcohols, ester class content and product urea ability.

Measure the grape wine product foam ability of Wine brewing yeast strain in cabernet sauvignon grape juice of gained, this experiment adds the 10mL filtration sterilization in 16mm * 160mm test tube cabernet sauvignon grape juice carries out, with 10 ⁶CFU/mL inoculation is cultivated 5d for 25 ℃, and experimental session is observed the foamy height change every day.Foam height h＜2mm represents that test strain product foam ability is low, 2mm≤h≤4mm represents that test strain product foam ability is medium, h＞4mm represents strong (the J.A.Regod ó n of test strain product foam ability, F.Per é z, M.E.Vald é s, C.De Miguel and M.Ram í rez.A simple and effective procedure for selection of wine yeast strains.FoodMicrobio.1997,14 (3): 247-254).

Concrete outcome sees Table 5.After utilizing the cabernet sauvignon grape zymamsis to finish as can be seen from Table 5, the CO of the dry red winew that the NJZCC17 fermentation obtains ₂Weightlessness is higher than commercial control strain, and its residual sugar content is less than control strain, and alcohol output is higher than control strain, and this illustrates that its fermenting power is better than control strain, but under equal conditions its utilization fermentable carbohydrates ability and ethanol to produce ability stronger.Other several main brewing characteristic indexs in addition, as glycerine output, volatile acid output, ethyl acetate output, foam output, urea production, NJZCC17 all is better than commercial control strain F15.On total ester output, NJZCC17 belongs to good brewing characteristic apparently higher than F15; And on total higher alcohols output, F15 is higher than NJZCC17.Think that in the world the higher alcohols content in the grape wine should not be higher than 300mg/L, otherwise to the grape wine local flavor can have a negative impact (

D., Tominac, V.P.,

V.On higher alcohols in wine.Periodicum Biologorum.2007,109 (2): 205-217).

The physical and chemical index of test strain fermented liquid in the table 5 cabernet sauvignon grape lab scale wine brewing experiment

Bacterial strain	CO 2Weightless (g/100mL)	Residual sugar content (g/L)	Ethanol content (%, v/v)	Glycerine output (g/L)	Volatile acid (g/L)
						NJZCC17	13.7±0.1	3.10±0.06	10.01±0.09	10.83	0.15±0.01
F15 **	12.8±0.1	3.88±0.03	9.89±0.14	9.46	0.22±0.03

The physical and chemical index of test strain fermented liquid in table 5 (continuing) the cabernet sauvignon grape lab scale wine brewing experiment

Bacterial strain	Ethyl acetate (mg/L)	Produce foam ability (mm)	Urea production (mg/L)	Total higher alcohols (mg/L)	Total ester (mg/L)
						NJZCC17	16.15	h*＜2	4.23±0.08	275.68±5.89	76.13±12.7

F15 **

12.78

2＜h＜4

9.78±0.05

309.94±8.67

68.91±8.83

h ^*: the expression foam height.F15 ^*: expression commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

Embodiment 4, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NZJCC17 and commercial strain F15 are in uncommon small-sized wine brewing comparative experiments of drawing in the Sucus Vitis viniferae

With Wine brewing yeast strain NJZCC17 of the present invention and control strain active dry yeast F15, for brewageing system, compare brewing characteristic with the uncommon Sucus Vitis viniferae that draws of 4L.Following wishing with Changli product in 2007 drawn grape destemming, removal of impurities, and broken then, the Sucus Vitis viniferae that obtains of squeezing the juice is a raw material, brewages experiment.Following 2007 Changli is produced to wish and is drawn the main physical and chemical index of grape to be: reducing sugar (with glucose meter) content, 188.81 ± 1.27g/L; Total acid content (in tartrate), 7.20 ± 0.40g/L; PH=3.56 ± 0.02.

The yeast saccharomyces cerevisiae NJZCC17 that is stored in-80 ℃ is gone down to posterity twice on YEPD solid and liquid nutrient medium, commercial strain active dry yeast F15 (control strain with 7 ℃, commercialization active dry yeast S.cerevisiaeZymaflora F15, available from French LAFFORT company) by producer's suggesting method water activation, getting good NJZCC17 of activation and commercial control strain F15 respectively is inoculated into and 4L Changli in 2007 is housed produces uncommon drawing in the 5L Glass Containers of Sucus Vitis viniferae, add sulfurous acid, reach 60mg/L to free sulphur content, inoculum size is 10 ⁶CFU/mL, room temperature is fermented, and monitors the temperature and the proportion of fermented liquid every day, is considered as fermentation ends when proportion no longer descends, and obtains dry red winew.

Measure the CO of the dry red winew of above-mentioned acquisition according to embodiment 2 described methods ₂Weightlessness, residual sugar amount, ethanol content, glycerine output, volatile acid, higher alcohols, ester class content and product urea ability.

Measure bacterial strain to be measured in uncommon product foam ability of drawing in the Sucus Vitis viniferae according to the method shown in the embodiment 3.

Concrete outcome sees Table 6.As can be seen from Table 6, after utilization is wished and is drawn grape alcohol fermentation ends, the CO of the dry red winew that the NJZCC17 fermentation obtains ₂Weightlessness is higher than commercial control strain, and its residual sugar content is less than control strain, and alcohol output is higher than control strain, and this illustrates that its fermenting power is better than control strain, but under equal conditions its utilization fermentable carbohydrates ability and ethanol to produce ability stronger.Other several main brewing characteristic indexs in addition are as glycerine output, volatile acid output, ethyl acetate output, foam output, H ₂S output, NJZCC17 all is better than commercial control strain F15.On total ester output, NJZCC17 belongs to good brewing characteristic apparently higher than F15; And on total higher alcohols output, F15 is higher than NJZCC17.Think that in the world the higher alcohols content in the grape wine should not be higher than 300mg/L, otherwise to the grape wine local flavor can have a negative impact (

D., Tominac, V.P., V.On higher alcohols in wine.Periodicum Biologorum.2007,109 (2): 205-217).

The uncommon physical and chemical index that draws test strain fermented liquid in the grape lab scale wine brewing experiment of table 6

Bacterial strain	CO 2Weightless (g/100mL)	Residual sugar content (g/L)	Ethanol content (%, v/v)	Glycerine output (g/L)	Volatile acid (g/L)
						NJZCC17	12.9±0.1	2.26±0.08	9.55±0.36	9.08	0.21±0.02
F15 *	12.1±0.1	2.67±0.04	9.35±0.35	8.31	0.37±0.04

The uncommon physical and chemical index that draws test strain fermented liquid in the grape lab scale wine brewing experiment of table 6 (continuing)

Bacterial strain	Ethyl acetate (mg/L)	Produce foam ability (mm)	Urea production (mg/L)	Total higher alcohols (mg/L)	Total ester (mg/L)
						NJZCC17	17.89	h *＜2	4.06±0.13	296.23±7.89	96.13±9.94

F15 **

14.42

2＜h＜4

10.11±0.04

382.88±10.12

78.91±2.88

h ^*: the expression foam height.F15 ^*: expression commercialization active dry yeast S.cerevisiae ZymafloraF15, available from French LAFFORT company.

Embodiment 5, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NZJCC17 and the small-sized wine brewing comparative experiments of commercial strain F15 in the happy Sucus Vitis viniferae of U.S.

With Wine brewing yeast strain NJZCC17 of the present invention and control strain active dry yeast F15, for brewageing system, compare brewing characteristic with the U.S. happy Sucus Vitis viniferae of 4L.Followingly produce U.S. happy grape destemming, removal of impurities with Changli in 2007, broken then, the Sucus Vitis viniferae that obtains of squeezing the juice is a raw material, brewages experiment.Following 2007 Changli is produced the main physical and chemical index of U.S. happy grape and is: reducing sugar (with glucose meter) content, 199.14 ± 1.05g/L; Total acid content (in tartrate), 4.87 ± 0.20g/L; PH=3.99 ± 0.01.

The NJZCC17 that is stored in-80 ℃ is gone down to posterity twice on YEPD solid and liquid nutrient medium, commercial strain active dry yeast F15 (control strain with 7 ℃, commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company) by producer's suggesting method water activation, getting good NJZCC17 of activation and control strain F15 respectively is inoculated into and 4L Changli in 2007 is housed produces in the 5L Glass Containers of U.S. happy Sucus Vitis viniferae, add sulfurous acid, reach 60mg/L to free sulphur content, inoculum size is 10 ⁶CFU/mL, room temperature is fermented, and monitors the temperature and the proportion of fermented liquid every day, is considered as fermentation ends when proportion no longer descends, and obtains dry red winew.

According to measuring CO vinous after the embodiment 2 described method fermentation ends ₂Weightlessness, residual sugar amount, ethanol content, glycerine output, volatile acid, higher alcohols, ester class content and urea production.

Measure the product foam ability of bacterial strain to be measured in the happy Sucus Vitis viniferae of U.S. according to the method shown in the embodiment 3.

Concrete outcome sees Table 7.As can be seen from Table 7, utilize U.S. happy grape alcohol fermentation ends after, the CO of the dry red winew that NJZCC17 fermentation obtains ₂Weightlessness is higher than commercial control strain, and its residual sugar content is less than control strain, and alcohol output is higher than control strain, and this illustrates that its fermenting power is better than control strain, but under equal conditions its utilization fermentable carbohydrates ability and ethanol to produce ability stronger.Other several main brewing characteristic indexs in addition, as glycerine output, volatile acid output, ethyl acetate output, foam output, urea production, NJZCC17 all is better than commercial control strain F15.On total ester output, NJZCC17 belongs to good brewing characteristic apparently higher than F15; And on total higher alcohols output, F15 is higher than NJZCC17.Think that in the world the higher alcohols content in the grape wine should not be higher than 300mg/L, otherwise to the grape wine local flavor can have a negative impact ( D., Tominac, V.P.,

V.On higher alcohols in wine.Periodicum Biologorum.2007,109 (2): 205-217).

The physical and chemical index of test strain fermented liquid in the U.S. happy grape lab scale wine brewing experiment of table 7

Bacterial strain	CO 2Weightless (g/100mL)	Residual sugar content (g/L)	Ethanol content (%, v/v)	Glycerine output (g/L)	Volatile acid (g/L)
						NJZCC17	13.1±0.1	3.14±0.08	10.04±0.08	8.11	0.36±0.01

F15 **

12.8±0.1

3.25±0.04

10.01±0.15

7.31

0.39±0.08

The physical and chemical index of test strain fermented liquid in the U.S. happy grape lab scale wine brewing experiment of table 7 (continuing)

Bacterial strain	Ethyl acetate (mg/L)	Produce foam ability (cm)	Urea production (mg/L)	Total higher alcohols (mg/L)	Total ester (mg/L)
						NJZCC17	11.34	h *＜2	5.21±0.02	249±8.01	79.43±7.86
F15 **	9.52	2＜h＜4	12.62±0.03	324.11±3.16	63.65±4.03

h ^*: the expression foam height.F15 ^*: expression commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

Embodiment 6, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NZJCC17 and the commercial strain F15 pilot scale wine brewing comparative experiments in 400L cabernet sauvignon grape juice

Following 2008 Changli is produced the main physical and chemical index of fresh cabernet sauvignon grape and is: reducing sugar (with glucose meter) content, 229.12 ± 1.02g/L; Total acid content (in tartrate), 6.14 ± 0.42g/L; PH=3.39 ± 0.03.

The NJZCC17 that is stored in-80 ℃ is gone down to posterity twice on YEPD solid and liquid nutrient medium, with 7 ℃ commercial strain active dry yeast F15 water activation.

Fresh Cabernet Sauvignon Wine Grape destemming, removal of impurities are produced in 2 tons of Changli in 2008, broken then, squeezing the juice obtains cabernet sauvignon grape juice, amount branch with every jar of 400L is filled in the stainless steel jar, divide 3 times by amount interpolation sulfurous acid, reach 60mg/L to free sulphur content, and add the 20mg/L polygalacturonase, beat the circulation mixing after, total acid, total reducing sugar amount, free sulphur, proportion and the product temperature of mensuration Sucus Vitis viniferae.Every strain bacterium is fermented two jars as parallel check experiment.The experiment place is pilot scale base, China Agricultural University grape wine center.

Meanwhile, the above-mentioned cabernet sauvignon grape juice that adopts fresh accent sulphur to free sulphur content to reach 60mg/L carries out the bacterial strain activation, and concrete grammar is NJZCC17 strain passage to be measured to be cultivated the back join in the above-mentioned cabernet sauvignon grape juice of 12L with 3% inoculum size.After leaving standstill 24h, the NJZCC17 bacterial strain after obtaining activating after 22 ℃ of rising again.

The bacterial strain NJZCC17 to be measured that activation is good joins in the above-mentioned fermentor tank with 3% inoculum size, and each bacterial strain to be measured is established 2 parallel laboratory test jars, and control canisters is added the good F15 of activation with the 20mg/L inoculum size.Beating circulation 3min with pump makes stock liquid and yeast thorough mixing even.Divide every day and play circulation early, middle and late three times, and the temperature and the proportion of the monitor fermentation liquid of after circulation, taking a sample.Respectively at after the zymamsis, skin juice separate before and take a sample behind the malic-lactic acid fermentation, measure the physical and chemical index of wine sample according to detecting needs, index mainly comprises volatile acid, residual sugar, alcoholic strength, total phenol etc., and utilize SPME-GC-MS to measure main aroma component and formation, organize jury of experts that grape wine is carried out subjective appreciation.And each detected result of NJZCC17 and control strain F15 be analyzed, concrete outcome is as described below:

(1) comparison of strain fermentation curve and physical and chemical index

Yeast NJZCC17 plays the ferment time slightly early than F15, reaches the climax behind postvaccinal 24-30h, and contrast commercial strain F15 30-42h after inoculation reaches the climax, and contrast F15 finishes fermentation at 4.5d; The NJZCC17 fermentation time is slightly long, finishes at 5d.It is fast that yeast plays ferment speed, can suppress the growth and breeding of other assorted bacterium, guarantees composition vinous and quality to greatest extent.Simultaneously fermentation period is wanted suitably, too shortly can not fully finish fermentation, brings into play the local flavor expressive force in the Sucus Vitis viniferae fully; The long chance that then may increase pollution produces the by product of not expecting.

The physical and chemical index of grape wine sample is shown in Table 8 after the zymamsis.

In the table 8, the detection method of ethanol content is a pycnometric method, and concrete steps are pressed the procedure operation of GB/T 15038-2006 defined;

The detection method of residual sugar is a film reagent method, and concrete steps are pressed the procedure operation of GB/T 15038-2006 defined;

The detection method of glycerol content is test kit method (glycerine is measured test kit, Glycerol Assay Kit, Megazyme company, Ireland), and concrete steps are by company's recommended program operation;

The volatile acid content detection method is a volumetry, and concrete steps are pressed the procedure operation of GB/T 15038-2006 defined;

The wine degree of preferably saccharomyces cerevisiae NJZCC17 fermented wine sample is higher than commercial control strain F15, shows that the product alcohol ability of excellent sieve bacterial strain is better than F15.But the F15 fermentation is more complete, and the residual sugar amount in the fermented liquid is lower, but in allowed band (≤4.0g/L).By detection to all wine sample volatile acids, find volatile acid all less than 0.6g/L, show the no abnormal situation of fermentation, vinosity is good, detects data and can be used for analyzing.Comprehensive evaluation thus, the fermentative activity of bacterial strain NJZCC17 is higher.NJZCC17 is a little more than control strain for glycerine output.

The physical and chemical index of grape wine sample after the zymamsis of table 8 test strain

Bacterial strain

Ethanol (%, V/V)

Residual sugar is (with glucose meter, g/L)

Glycerine (g/L)

Volatile acid is (with acetometer, g/L)

NJZCC17	13.57±0.01	2.23±0.05	5.766	0.15±0.01
					F15 *	13.32±0.02	2.77±0.03	5.124	0.18±0.01

F15 ^*: commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

(2) comparison of bacterial strain main volatile aroma component kind and content

The aroma substance that produces in the Fermentation of Grape Wine is also referred to as " secondary fragrance ", mainly comprises higher alcohols, short-chain aliphatic ester class, short chain fatty acid and aromatic acid and carbonyl complex.

Table 9 shown utilize SPME-GC-MS (Zhang Mingxia. grape wine fragrance Changing Pattern research---focus on of the influence [D] of crucial making method to grape wine fragrance. the doctorate paper .2007 of China Agricultural University) technology (mainly is ester, pure and mild sour three class materials to preferred strain NJZCC17 and the industrial bacterial strain F15 of contrast at malic-lactic acid fermentation secondary fermentation aroma substance, other class materials except that this three classes material, as aldehyde, ketone etc., because kind is less, content is low and unlisted) kind.The kind of ester and alcohol accounts for the overwhelming majority of fermentation aroma component as seen from Table 9.Ester is respectively 13 kinds and 12 kinds in NJZCC17 and the F15 fermented wine sample; Alcohols material is respectively 21 kinds and 17 kinds; The total kind of volatile aroma material is respectively 44 kinds and 35 kinds.Compare with F15, aspect the complicacy of fermentation fragrance, no matter preferred strain NJZCC17 is the total kind of aroma substance, or Ester and alcohols material kind are all more than F15.Complicated aroma component will be given the mouthfeel and the sense of taste of grape wine complexity, thereby gives more individual character of grape wine and speciality.The mean yield of the total Ester of NJZCC17 is 109.76 ± 6.78mg/L, apparently higher than 91.57 ± 7.08mg/L of F15; The mean yield of NJZCC 17 total senior alcohols materials is 169.34 ± 6.99mg/L, and F15 is 211.37 ± 5.71mg/L.

By above analysis as can be seen, the NJZCC17 preferred strain all can produce the aroma component of more complicated, has given full play to the potentiality of brewageing of producing region, Changli cabernet sauvignon grape.

Table 9 bacterial strain NJZCC17 and the industrial bacterial strain F15 of contrast detect at malic-lactic acid fermentation secondary fermentation aroma substance

F15 ^*: commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

(3) comparison of polyphenol substance content after the bacterial strain zymamsis

Grape wine is the main alcoholic beverage that contains polyphenols.Polyphenols is present in Pericarpium Vitis viniferae, grape berry, Semen Vitis viniferae and the carpopodium, is extracted in the grape wine by brewing process, and they have constituted the main body of grape wine color, gives the gorgeous color of grape wine, strong fragrance and mellow structure.Grape wine also comes from these polyphenols to the distinctive benefit of human body, and they have tangible vasodilation, strengthen the effect of vascular permeability.Therefore say that kind, structure and the content of aldehydes matter and color vinous, mouthfeel, fragrance and nutritive value etc. are closely related, they are one of determinatives of wine quality and typicalness.

In recent years result of study shows that the difference of aldehydes matter content has much relations with different yeast brewing characteristics in the grape wine.Utilize the LC-UV-MS/MS technology (Sun Jianping. aldehydes matter LC-UV-MS/MS spectrum storehouse makes up and uses [D] in grape and the grape wine. the doctorate paper .2006 of China Agricultural University) polyphenol result of study in preferred strain NJZCC17 and the commercial control strain F15 fermented wine sample is shown: anthocyanin class material and flavan-3-alcohol class material are apparently higher than the wine sample of active dry yeast fermentation in the grape wine of preferably saccharomyces cerevisiae strain fermentation; Diphenylethylene and flavonol content also are higher than contrast wine sample; In addition, hydroxy-benzoic acid in the preferred strain fermented wine sample and hydroxycinnamic acid class and contrast wine sample do not have significant difference.

The polyphenols content of table 10 test strain fermentating wine sample

Polyphenols (mg/L)	NJZCC17	F15 *
			Anthocyanin class	340.1±2.02	319.8±1.49
Diphenylethylene	0.81±0.04	0.66±0.02
			Hydroxy-benzoic acid	45.17±0.71	45.93±0.57
Flavonol	15.63±0.22	15.21±0.13
			The flavan-3-alcohol class	3357.83±12.26	2994.64±6.73
The hydroxycinnamic acid class	16.43±1.02	22.41±0.44

F15 ^*: commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

This shows, adopt the nutritive value of the grape wine sample of preferred native country yeast strain NJZCC17 fermentation will significantly be better than the grape wine of import active dry yeast fermentation, four class polyphenol substance growing amounts are more than contrast, in addition two class material growing amounts and the no significant difference of contrast.

(4) expert's subjective appreciation that pilot scale is made grape wine

Seminar's tissue is to expert's sensory evaluation of pilot scale fermented wine sample, ten experts have the senior engineer officer (two) from enterprise, are engaged in professor, associate professor's (four are taught two) of grape wine research for many years from colleges and universities, grape wine specialty postgraduate (four, two of doctors).The experts' evaluation group is taken into account enterprise, teaching and scientific research personnel, has higher authority and reasonableness.The appraisal result that evaluation expert's group provides is shown in Table 8.

Table 11 fermentation pilot scale grape wine subjective appreciation result (expert is blind to be commented)

F15 ^*: commercialization active dry yeast S.cerevisiae ZymafloraF15, available from French LAFFORT company

By expert's subjective appreciation result as seen, most experts (8) to the evaluation of yeast strain NJZCC17 fermented wine sample than higher, with contrast F15 fermented wine sample organoleptic quality more excellent (7) or quite (1).Yeast strain NJZCC17 comes from Changli in 2006 and produces in the natural fermentation broth of Cabernet Sauvignon, and the Sucus Vitis viniferae that pilot scale is this time adopted is the squeezing juice of Changli product Cabernet Sauvignon in 2008, this shows, the local grape wine that derives from the wild yeast strain fermentation production in same producing region has better quality, and its brewing characteristic has obtained more perfectly representing and bringing into play.

Embodiment 7, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NZJCC17 and the commercial strain F15 pilot scale wine brewing comparative experiments in 1000L cabernet sauvignon grape juice

Following 2009 Changli is produced the main physical and chemical index of fresh cabernet sauvignon grape and is: reducing sugar (with glucose meter) content, 240.06 ± 2.00g/L; Total acid content (in tartrate), 6.94 ± 0.62g/L; PH=3.49 ± 0.01.

The NJZCC17 that is stored in-80 ℃ is being gone down to posterity twice on YEPD solid and the liquid nutrient medium respectively, with 7 ℃ commercial strain active dry yeast F15 water activation.

Fresh Cabernet Sauvignon Wine Grape destemming, removal of impurities are produced in 2.5 tons of Changli in 2009, broken then, squeezing the juice obtains Sucus Vitis viniferae, amount branch with every jar of 1000L is filled in the stainless cylinder of steel of 1500L, divide 3 times by amount interpolation sulfurous acid, reach 60mg/L to free sulphur content, and add the 20mg/L polygalacturonase, beat the circulation mixing after, total acid, total reducing sugar amount, free sulphur, proportion and the product temperature of mensuration Sucus Vitis viniferae.The experiment place is a COFCO Huaxia Great Wall Wine Co., Ltd.

Meanwhile, the above-mentioned cabernet sauvignon grape juice that adopts fresh accent sulphur to free sulphur content to reach 60mg/L carries out the bacterial strain activation, and concrete grammar is NJZCC17 strain passage to be measured to be cultivated the back join in the above-mentioned cabernet sauvignon grape juice of 30L with 3% inoculum size.After leaving standstill 24h, the NJZCC17 bacterial strain after obtaining activating after 22 ℃ of rising again.

The bacterial strain NJZCC17 to be measured that activation is good joins in the above-mentioned fermentor tank with 3% inoculum size, and control canisters is added the good F15 of activation with the 20mg/L inoculum size, and concrete mode recommends rules to carry out according to manufacturer.Beating circulation 3min with pump makes stock liquid and yeast thorough mixing even.Divide every day and play circulation early, middle and late three times, and the temperature and the proportion of the monitor fermentation liquid of after circulation, taking a sample.Take a sample after zymamsis, measure the physical and chemical index of wine sample according to detecting needs, index mainly comprises volatile acid, residual sugar, alcoholic strength, total phenol etc., and utilizes SPME-GC-MS to measure main aroma component and formation, and LC-MS measures anthocyanogen and polyphenol.Behind malic-lactic acid fermentation, organize jury of experts that grape wine is carried out subjective appreciation.Each detected result of NJZCC17 and control strain F15 are analyzed, and concrete outcome is as described below:

(1) comparison of strain fermentation curve and physical and chemical index

Yeast NJZCC17 plays the ferment time slightly early than F15, reaches the climax behind postvaccinal 24-30h, and contrast commercial strain F15 30-42h after inoculation reaches the climax, and contrast F15 finishes fermentation at 5d; The NJZCC17 fermentation time is slightly long, finishes at 5.5d.It is fast that yeast plays ferment speed, can suppress the growth and breeding of other assorted bacterium, guarantees composition vinous and quality to greatest extent.Simultaneously fermentation period is wanted suitably, too shortly can not fully finish fermentation, brings into play the local flavor expressive force in the Sucus Vitis viniferae fully; The long chance that then may increase pollution produces the by product of not expecting.

The physical and chemical index of grape wine sample is shown in Table 12 after the zymamsis.

In the table 12, the detection method of ethanol content is a pycnometric method, and concrete steps are pressed the procedure operation of GB/T 15038-2006 defined;

The detection method of residual sugar amount is a film reagent method, and concrete steps are for pressing the procedure operation of GB/T 15038-2006 defined;

The detection method of glycerol content is test kit method (glycerine is measured test kit, Glycerol Assay Kit, Megazyme company, Ireland), and concrete steps are by company's recommended program operation;

The detection method of volatile acid amount is a volumetry, and concrete steps are pressed the procedure operation of GB/T 15038-2006 defined;

The pH pH-value determination pH is the pH meter method, and concrete steps are for pressing the procedure operation of GB/T 15038-2006 defined;

The detection of total acid content is a potentiometric titration, and concrete steps are for pressing the procedure operation of GB/T 15038-2006 defined;

Free SO ₂The detection of amount is direct iodimetry,iodometry, and concrete steps are for pressing the procedure operation of GB/T 15038-2006 defined;

The detection method of acetaldehyde output is test kit method (acetaldehyde is measured test kit, Aldehyde Assay Kit, Megazyme company, Ireland), and concrete steps are by company's recommended program operation;

The detection method of urea production is test kit method (urea is measured test kit, Urea Assay Kit, Megazyme company, Ireland), and concrete steps are by company's recommended program operation;

The detection of oxysuccinic acid is the HPLC method, and concrete steps are for pressing the procedure operation of GB/T 15038-2006 defined;

The detection of trans-resveratrol is the HPLC method, and concrete steps are for pressing the procedure operation of GB/T 15038-2006 defined;

The detection of 2,3 butyleneglycols is nuclear magnetic resonance method (Hong-Seok Son, Geum-Sook Hwang, Ki MyongKim, Eun-Young Kim, Frans van den Berg, Won-Mok Park, Cherl-Ho Lee, andYoung-Shick Hong. ¹H NMR-Based Metabolomic Approach for Understanding theFermentation Behaviors of Wine Yeast Strains.Anal Chem.2009,81:1137-1145).

The wine degree of preferably saccharomyces cerevisiae NJZCC17 fermented wine sample shows that a little more than commercial control strain F15 the product alcohol ability of excellent sieve bacterial strain is better than F15.But F15 fermentation is more complete, and the residual sugar amount in the fermented liquid is lower, but the two residual sugar content all in allowed band (≤4.0g/L).By detection to wine sample volatile acid, find the two volatile acid all less than 0.3g/L (NJZCC17 volatile acid amount is lower), show the no abnormal situation of fermentation.Comprehensive evaluation thus, the fermentation starting of bacterial strain NJZCC17 is very fast, vigor is higher, and leavening property is more steady.Glycerine, trans-resveratrol, 2, the material output that 3-butyleneglycol etc. have a positive effect to grape wine quality, NJZCC17 all is higher than control strain, and wherein the content of trans-resveratrol is more than 2 times of control strain.Trans-resveratrol has good biological activity, it is a kind of natural antioxidant, but blood viscosity lowering, suppressing thrombocyte condenses and vasorelaxation, keep unobstructed blood, but the generation of preventing cancer and development has atherosclerosis and coronary heart disease, ischemic heart disease, the preventive and therapeutic effect of hyperlipidemia.It also has the tumour of inhibition and the estrogenic effect of class, can be used for treating diseases such as mammary cancer.The free SO of NJZCC17 ₂, acetaldehyde, urea etc. is all low than control strain F15 to the material output that grape wine quality has potential for adverse effects.

The physical and chemical index of grape wine sample after the zymamsis of table 12 test strain

Bacterial strain	Ethanol (%, V/V)	Residual sugar is (with glucose meter, g/L)	Glycerine (g/L)	Volatile acid is (with acetometer, g/L)
					NJZCC17	14.02±0.01	3.56±0.08	6.198	0.193±0.06
ADY *	13.91±0.02	3.52±0.24	5.679	0.225±0.31

The physical and chemical index of grape wine sample after table 12 (continuous 1) the test strain zymamsis

Bacterial strain	pH	Total acid is (in tartrate, g/L)	Free SO 2 (mg/L)	Acetaldehyde (mg/L)
					NJZCC17	3.40±0.01	8.12±0.07	11.03±0.21	0.498±0.13
ADY *	3.46±0.01	7.34±0.11	13.35±0.17	0.519±0.22

The physical and chemical index of grape wine sample after table 12 (continuous 2) the test strain zymamsis

Bacterial strain	Urea (mg/L)	Oxysuccinic acid (g/L)	Trans-resveratrol (mg/L)	2,3-butyleneglycol (mg/L)
					NJZCC17	1.89±0.44	3.04±0.10	4.987±0.058	1.225±0.64
ADY *	6.92±0.18	3.52±0.33	2.174±0.067	1.293±0.31

^*ADY: commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

It can also be seen that from table 12 through after the zymamsis, the pH value in the grape wine is reduced for NZJCC17 and F15 two strain bacterium but total acid raises, this proves that two strain bacterium have generated other acidic substance during the fermentation.And wherein the variation of NJZCC17 institute vintage wine is particularly evident.The pathways metabolism that this explanation NJZCC17 produces in the cabernet sauvignon grape in Changli is more complicated.Yet malic acid content is lower in the vintage wine of NJZCC17 institute after zymamsis finishes, and the time that this can reduce the later stage malic-lactic acid fermentation, has the effect of shortening fermentation period.

(2) comparison of bacterial strain main volatile aroma component kind and content

The aroma substance that produces in the Fermentation of Grape Wine is also referred to as " secondary fragrance ", mainly comprises higher alcohols, short-chain aliphatic ester class, short chain fatty acid and aromatic acid and carbonyl complex.

Table 13 shown utilize SPME-GC-MS (Zhang Mingxia. grape wine fragrance Changing Pattern research---focus on of the influence [D] of crucial making method to grape wine fragrance. the doctorate paper .2007 of China Agricultural University) preferred strain NJZCC17 and the industrial bacterial strain F15 of contrast are finished the secondary fermentation aroma substance in zymamsis (mainly is ester, pure and mild sour three class materials to technology, other class materials except that this three classes material, as aldehyde, ketone etc., because kind is less, content is low and unlisted) kind.The kind of ester and alcohol accounts for the overwhelming majority of fermentation aroma component as seen from Table 13.Ester is respectively 20 kinds and 18 kinds in NJZCC17 and the F15 fermented wine sample.Total Ester content NJZCC17 is far above F15, wherein ethyl acetate, ethyl hexanoate, hexyl acetate, ethyl octylate, ethyl decylate, sad-3-methyl butyl ester, diethyl succinate, Laurate ethyl, ten tetra-carbonic ethyl esters and ethyl palmitate etc. are to the Ester content of vinous flavor moral character frame outbalance, NJZCC17 all is higher than F15, and NJZCC17 can produce oil of cognac and caproic acid phenethyl ester that F15 can't produce.Higher alcohols also is assorted oleyl alcohol or potato spirit, is the big class volatile aroma material that yeast produces.But the metabolism burden that too much higher alcohols material can increase liver is drunk in studies show that in recent years, may make hepatic tissue produce the pathology similar to the chronic alcoholism hepatitis; Higher alcohols also can produce in various degree toxic action to neural system, and the poisoning of its generation and anesthetic action are all strong than ethanol, makes drinking person produce headache, so-called drinking " the higher authorities " phenomenon such as dizzy.The toxic action of potato spirit aggravates (Dirk W.Lachenmeier along with the increase of molecular weight, Simone Haupt, Katja Schulz.Defining maximum levels ofhigher alcohols in alcoholic beverages and surrogate alcohol products.RegulatoryToxicology and Pharmacology.2008,50:313-321).And because the higher alcohols of too high amount can produce negative effect to local flavor vinous and mouthfeel on the contrary, make grape wine produce so-called " fusel taste " and " paint-like flavour ", so it is generally acknowledged higher alcohols in the grape wine should not be higher than 300mg/L ( D., Tominac, V.P.,

V.On higher alcohols in wine.Periodicum Biologorum.2007,109 (2): 205-217).Therefore, when international grape wine circle screens Wine brewing yeast strain a kind of like this trend is arranged at present, exactly under the prerequisite that does not influence grape wine quality and flavor complexity, screening ester class output is higher, and the low Wine brewing yeast strain of higher alcohols output.Though the higher alcohols ultimate production of NJZCC17 is lower than F15, its kind that produces higher alcohols is more than F15 (the higher alcohols material that NJZCC17 and F15 produce is respectively 21 kinds and 17 kinds).This shows that NJZCC17 has more complicated metabolic rule compared to F15.NJZCC17 and F15 produce VFA class material and are 5 kinds, and kind is identical, but the volatile acid total amount that NJZCC17 produces is lower than F15, and wherein the output of NJZCC17 acetate also is lower than F15.These all are good brewing characters that grape wine needs with yeast saccharomyces cerevisiae.The total kind of volatile aroma material is respectively 49 kinds and 43 kinds.Compare with F15, aspect the complicacy of fermentation fragrance, no matter preferred strain NJZCC17 is the total kind of aroma substance, or Ester and alcohols material kind are all more than F15.Complicated aroma component will be given the mouthfeel and the sense of taste of grape wine complexity, thereby gives more individual character of grape wine and speciality.

By above analysis as can be seen, the NJZCC17 preferred strain all can produce the aroma component of more complicated, and Ester output is higher, and the content of higher alcohols and VFA is lower, has given full play to the potentiality of brewageing of producing region, Changli cabernet sauvignon grape.

Table 13 bacterial strain NJZCC17 and the industrial bacterial strain F15 of contrast detect at malic-lactic acid fermentation secondary fermentation aroma substance

(3) comparison of polyphenol substance content after the bacterial strain zymamsis

Utilize the LC-UV-MS/MS technology (Sun Jianping. aldehydes matter LC-UV-MS/MS spectrum storehouse makes up and uses [D] in grape and the grape wine. the doctorate paper .2006 of China Agricultural University) polyphenol result of study in preferred strain NJZCC17 and the commercial control strain F15 fermented wine sample is shown: anthocyanin class material and flavan-3-alcohol class material are apparently higher than the wine sample of active dry yeast F15 fermentation in the grape wine of preferably saccharomyces cerevisiae strain fermentation; Diphenylethylene and flavonol content also are higher than contrast wine sample; In addition, hydroxy-benzoic acid in the preferred strain NJZCC17 fermented wine sample and hydroxycinnamic acid class and contrast wine sample do not have significant difference.

The polyphenols content of table 14 test strain fermentating wine sample

Polyphenols (mg/L)	NJZCC17	F15 *
			Anthocyanin class	436.5±1.38	389.2±2.29
Diphenylethylene	1.07±0.06	0.84±0.03
			Hydroxy-benzoic acid	39.65±0.41	37.97±0.39
Flavonol	21.03±0.42	23.11±0.18
			The flavan-3-alcohol class	3561.41±17.08	3197.34±10.03
The hydroxycinnamic acid class	12.19±0.82	14.49±0.39

F15 ^*: commercialization active dry yeast S.cerevisiae Zymaflora F15, available from French LAFFORT company.

This shows, adopt the nutritive value of the grape wine sample of preferred native country yeast strain NJZCC17 fermentation will significantly be better than the grape wine of import active dry yeast fermentation, four class polyphenol substance growing amounts are more than contrast, in addition two class material growing amounts and the no significant difference of contrast.

(4) expert's subjective appreciation that pilot scale is made grape wine

Seminar's tissue is to expert's sensory evaluation of pilot scale fermented wine sample, 14 experts have the senior engineer officer () from enterprise, assistant engineer (one), national grape wine teacher of the sampling wine (), the professor, the associate professor's (four are taught two) that are engaged in grape wine research from domestic colleges and universities for many years, foreign nationality's yeast saccharomyces cerevisiae researcher one people (professor, Holland Wageningen University), grape wine specialty postgraduate (six, four of doctors).The experts' evaluation group is taken into account enterprise, teaching, specialty is judged and the scientific research personnel, has higher authority and reasonableness.The appraisal result that evaluation expert's group provides is shown in Table 15.

By expert's subjective appreciation result as seen, most experts (12) than higher, think that comparison is according to F15 fermented wine sample organoleptic quality more excellent (11) or quite (1) to the evaluation of yeast strain NJZCC17 fermented wine sample.Yeast strain NJZCC17 comes from Changli in 2006 and produces in the natural fermentation broth of Cabernet Sauvignon, and the Sucus Vitis viniferae that pilot scale is this time adopted is the squeezing juice of Changli product Cabernet Sauvignon in 2009, this shows, the local grape wine that derives from the wild yeast strain fermentation production of the same kind in same producing region has better quality, and its brewing characteristic has obtained more fully representing and bringing into play.

Table 15 fermentation pilot scale grape wine subjective appreciation result (expert is blind to be commented)

F15 ^*: commercialization active dry yeast S.cerevisiae ZymafloraF15, available from French LAFFORT company.

Sequence table

<160>2

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

tccgtaggtg aacctgcgg 19

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

tcctccgctt attgatatgc 20

Claims (6)

1. yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NJZCC17CGMCC № .3284.

2. the application of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) NJZCC17CGMCC № .3284 in making grape wine.

3. application according to claim 2 is characterized in that: described grape wine is dry red winew.

4. according to claim 2 or 3 described application, it is characterized in that: the kind of the described grape material that makes grape wine is cabernet sauvignon grape, uncommon grape or the U.S. happy grape of drawing.

5. according to claim 2 or 3 described application, it is characterized in that: the described grape material that makes grape wine is cabernet sauvignon grape, uncommon grape or the U.S. happy grape of drawing that produce in producing region, Changli, Hebei.

6. application according to claim 4 is characterized in that: the described grape material that makes grape wine is cabernet sauvignon grape, uncommon grape or the U.S. happy grape of drawing that produce in producing region, Changli, Hebei.

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