CN1721524A - Acidity reducing yeast of grape wine and its culturing method - Google Patents

Acidity reducing yeast of grape wine and its culturing method Download PDF

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CN1721524A
CN1721524A CN 200510041935 CN200510041935A CN1721524A CN 1721524 A CN1721524 A CN 1721524A CN 200510041935 CN200510041935 CN 200510041935 CN 200510041935 A CN200510041935 A CN 200510041935A CN 1721524 A CN1721524 A CN 1721524A
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yeast
wine
fusant
grape wine
preparation
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李华
刘树文
王�华
何忠宝
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Northwest A&F University
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Abstract

The present invention relates to one kind of acid reducing yeast for grape wine and its culture method. The present invention constitutes acid reducing yeast capable of performing MLF while fermenting alcohol by means of the plasmogamy between the grape wine yeast with excellent wine brewing characteristic and excellent wine coccus SD-2a. The present invention constitutes the acid reducing yeast through plasmogamy with grape wine yeast 1450 and wine coccus SD-2a as parent strains, and the acid reducing yeast for grape wine has cell shape, size and colony characteristic similar to those of grape wine yeast 1450.

Description

A kind of acidity reducing yeast of grape wine and cultural method thereof
One, technical field:
The present invention relates to a kind of microorganism, specifically a kind of acidity reducing yeast of grape wine (F-20-7) and cultural method thereof.
Two, background technology:
Quality vinous depends primarily on the grape quality, too early often cause the quality of grape material not demonstrate fully in the bad time of weather condition (low temperature, high humidity) or owing to gather, cause that the grape berry ripening degree is not enough, acid content is high (mainly being the malic acid content height), so in the Production of Wine process, tend to run into the problem of falling acid.Malo-lactic fermentation (the malolacticfermentation that is undertaken by milk-acid bacteria, MLF), can make L MALIC ACID (diprotic acid) be decomposed into L-lactic acid (monoprotic acid), diprotic acid descends the grape wine total acid to monacid conversion, sour and astringent sense reduces, usually malo-lactic fermentation can make total acid reduce by 1～3g/L, so malo-lactic fermentation is genuine biological acid reduction.MLF can make newborn sour and astringent, harsh feeling disappearance vinous, makes the mouthfeel of wine reach balance, and has increased local flavor vinous and microbially stable; Red wine behind MLF, acidity descends, fruital, sweet-smelling enriching, mouthfeel softness, big and fleshy, according to the ultimate principle of modern wine making, MLF is an indispensable process in the production high-quality dry red winew.Because MLF carries out after zymamsis, belong to Secondary Fermentation, increased operation, and wayward, so people wish to obtain the acid leaven that falls of in the wine making process zymamsis simultaneously and MLF.For addressing this problem, recent two decades comes people oxysuccinic acid-lactalase gene mle of different genera milk-acid bacteria to be carried out the research of clone reorganization, transformed saccharomyces cerevisiae and expression thereof by genetic engineering means, expectation obtains can carry out the wine yeast of MLF in zymamsis, obtained certain progress.Oenococcus Oeni (Oenococcus oeni) is the main startup person and the person of finishing of MLF in the grape wine, and is the lactic acid bacteria culturers that can carry out MLF that MLF finishes unique existence in the grape wine of back.People also attempt the oxysuccinic acid of Oenococcus Oeni-lactalase gene mleA clone reorganization and transformed saccharomyces cerevisiae, but fail to obtain L MALIC ACID is changed into the positive transformant of L-lactic acid.Protoplast Fusion Technique has been used widely in research fields such as agricultural, medicine, food and environmental improvements as a kind of new gene recombination means, and present the advantage that can overcome genetic block, realize distant hybirdization, become one of important method of industrial micro breeding.
Three, summary of the invention:
The present invention is merged by the wine yeast and the protoplastis between good Oenococcus Oeni SD-2a of wine brewing characteristic good, has constructed the acid leaven that falls that can carry out MLF in zymamsis.
For achieving the above object, the technical solution used in the present invention is:
A kind of acidity reducing yeast of grape wine and cultural method thereof, its special character is: it is to be parental plant with wine yeast 1450 and Oenococcus Oeni SD-2a, uses that Protoplast Fusion Technique makes up.
Acidity reducing yeast of grape wine cellular form, size, colony characteristics are all similar to wine yeast 1450.
The acidity reducing yeast of grape wine culture medium prescription is respectively:
YPD substratum: yeast extract paste 1%, peptone 2%, glucose 2%, distilled water preparation, natural pH value, 121 ℃ of sterilization 20min.
ATB substratum: yeast extract paste 0.5%, peptone 1%, glucose 1%, MgSO 47H2O 0.02%, MnSO 44H2O 0.005%, cysteine hydrochloride 0.05%, tomato juice 25% (v/v), distilled water preparation, natural pH value, 115 ℃ of sterilization 20min.Add 1.5% agar during the preparation solid medium, the preparation height adds 17% sucrose when oozing substratum.
Fusant primary dcreening operation substratum: yeast extract paste 0.5%, peptone 1%, glucose 1%, MgSO 47H2O0.02%, MnSO 44H2O 0.005%, cysteine hydrochloride 0.05%, tomato juice 25% (v/v), agar 1.5%, distilled water preparation, natural pH value, 115 ℃ of sterilization 20min.Heating and melting substratum before using, when treating that its temperature is reduced to 55 ℃, in the penbritin of cycloheximide that wherein adds 100ug/mL and 20ug/mL, mixing falls plate rapidly.
The sub-screening culture medium of Target Fusion: yeast extract paste 0.5%, peptone 1%, glucose 10%, MgSO 47H2O0.02%, MnSO 44H2O 0.005%, tomato juice 10% (v/v), and DL-oxysuccinic acid 10g/L, tetrabromo-mcresolsulfonphthalein 0.004%, the distilled water preparation, the pH value transfers to 3.8,115 ℃ of sterilization 20min.
Fusant and wine yeast 1450 zymamsis power---oxysuccinic acid degradability test media: yeast extract paste 0.5%, peptone 1%, glucose 200g/L, tomato juice 20ml/L, L MALIC ACID 3g/L, MnSO44H2O 0.005%, the distilled water preparation, the pH value transfers to 3.0.
Oenococcus Oeni SD-2a oxysuccinic acid degradability test media: yeast extract paste 0.5%, peptone 1%, glucose 2g/L, tomato juice 20ml/L, L MALIC ACID 3g/L, MnSO 44H2O 0.005%, alcoholic strength 11% (v/v), and the distilled water preparation, the pH value transfers to 3.2.
Oenococcus Oeni SD-2a and wine yeast protoplastis prepare process:
For Oenococcus Oeni SD-2a, adopt to be in the logarithmic growth somatic cells in mid-term, with SMM homeo-osmosis liquid, N,O-Diacetylmuramidase enzymolysis concentration is 1mg/mL, in 37 ℃ of water enzyme digestion 30min, the preparation protoplastis.
For wine yeast 1450, adopt to be in the logarithmic growth somatic cells in mid-term, through 0.3% beta-mercaptoethanol-0.1%EDTANa2 in 37 ℃ of pre-treatment 10～15min, be homeo-osmosis liquid again with PBS, helicase concentration is 2%, in 37 ℃ of water enzyme digestion 40min, and the preparation protoplastis.
Protoplastis merges and fusant primary dcreening operation process is:
Adopt several 1/100, the Cacl of PEG-4000 30% (w/v), 30 ℃ of fusion temperatures, time of fusion 30min, yeast count/lactic-acid bacteria cells 2The fusion conditions of concentration 50mmol/L, with the yeast cell through the PEG processing is contrast, with the selective pressure of 100ug/mL cycloheximide+20ug/mL penbritin, after 9 days, obtained the fusant of the similar wine yeasts 1450 of 117 strain colonial morphologies altogether in cultured continuously as the fusant screening.
Compared with prior art, the advantage and the effect that have of the present invention is as follows:
The F-20-7 of the present invention oxysuccinic acid of can in zymamsis, degrading, its zymamsis ability is near wine yeast 1450; The oxysuccinic acid degradation capability is between two parental plants, relatively near Oenococcus Oeni SD-2a.
Four, description of drawings:
Fig. 1 is the leaven line chart of wine yeast 1450, fusant F-20-7;
Fig. 2 is wine yeast 1450, fusant F-20-7, Oenococcus Oeni SD-2a oxysuccinic acid degradability comparison diagram;
Five, embodiment:
The present invention uses Protoplast Fusion Technique, constructs a kind of acidity reducing yeast of grape wine.
The culture medium prescription that the present invention mainly utilizes is respectively
YPD substratum: yeast extract paste 1%, peptone 2%, glucose 2%, distilled water preparation, natural pH value, 121 ℃ of sterilization 20min.
ATB substratum: yeast extract paste 0.5%, peptone 1%, glucose 1%, MgSO 47H2O 0.02%, MnSO 44H2O 0.005%, cysteine hydrochloride 0.05%, tomato juice 25% (v/v), distilled water preparation, natural pH value, 115 ℃ of sterilization 20min.Add 1.5% agar during the preparation solid medium, the preparation height adds 17% sucrose when oozing substratum.
Fusant primary dcreening operation substratum: yeast extract paste 0.5%, peptone 1%, glucose 1%, MgSO 47H2O0.02%, MnSO 44H2O 0.005%, cysteine hydrochloride 0.05%, tomato juice 25% (v/v), agar 1.5%, distilled water preparation, natural pH value, 115 ℃ of sterilization 20min.Heating and melting substratum before using, when treating that its temperature is reduced to 55 ℃, in the penbritin of cycloheximide that wherein adds 100ug/mL and 20ug/mL, mixing falls plate rapidly.
The sub-screening culture medium of Target Fusion: yeast extract paste 0.5%, peptone 1%, glucose 10%, MgSO 47H2O0.02%, MnSO 44H2O 0.005%, tomato juice 10% (v/v), and DL-oxysuccinic acid 10g/L, tetrabromo-mcresolsulfonphthalein 0.004%, the distilled water preparation, the pH value transfers to 3.8,115 ℃ of sterilization 20min.
Fusant and wine yeast 1450 zymamsis power---oxysuccinic acid degradability test media: yeast extract paste 0.5%, peptone 1%, glucose 200g/L, tomato juice 20ml/L, L MALIC ACID 3g/L, MnSO44H2O 0.005%, the distilled water preparation, the pH value transfers to 3.0.
Oenococcus Oeni SD-2a oxysuccinic acid degradability test media: yeast extract paste 0.5%, peptone 1%, glucose 2g/L, tomato juice 20ml/L, L MALIC ACID 3g/L, MnSO 44H2O 0.005%, alcoholic strength 11% (v/v), and the distilled water preparation, the pH value transfers to 3.2.
1, Oenococcus Oeni SD-2a and wine yeast protoplastis preparation
For Oenococcus Oeni SD-2a, adopt to be in the logarithmic growth somatic cells in mid-term, with SMM homeo-osmosis liquid, N,O-Diacetylmuramidase enzymolysis concentration is 1mg/mL, in 37 ℃ of water enzyme digestion 30min, the preparation protoplastis.
For wine yeast 1450, adopt to be in the logarithmic growth somatic cells in mid-term, through 0.3% beta-mercaptoethanol-0.1%EDTANa2 in 37 ℃ of pre-treatment 10～15min, be homeo-osmosis liquid again with PBS, helicase concentration is 2%, in 37 ℃ of water enzyme digestion 40min, and the preparation protoplastis.
2, protoplastis merges and the fusant primary dcreening operation
Adopt several 1/100, the Cacl of PEG-4000 30% (w/v), 30 ℃ of fusion temperatures, time of fusion 30min, yeast count/lactic-acid bacteria cells 2The fusion conditions of concentration 50mmol/L, with the yeast cell through the PEG processing is contrast, with the selective pressure of 100ug/mL cycloheximide+20ug/mL penbritin, after 9 days, obtained the fusant of the similar wine yeasts 1450 of 117 strain colonial morphologies altogether in cultured continuously as the fusant screening.
3, fusant sieves and stability test again
The fusant bacterium colony that will grow on fusant primary dcreening operation culture medium flat plate is numbered, and with the sterilization toothpick it is forwarded on the fusant primary dcreening operation culture medium flat plate.The fusant that still can grow after three times is seeded to after ATB liquid activation culture in the sub-screening culture medium of Target Fusion, in 25 ℃ of standing for fermentation with transferring continuously.After treating that zymamsis finishes, the fermented liquid vibration is shaken up, observe its colour-change situation.The fermented liquid of fusant F-20 is compared with other fermented liquid, and it is blue that color obviously becomes, but preliminary judgement its be Target Fusion.The F-20 fermented liquid has tangible alcohol flavor, and the MLF ply of paper is analysed demonstration, compares with other fermented liquid, and its oxysuccinic acid spot obviously diminishes, and has the lactic acid spot to occur.Therefore decidable F-20 is Target Fusion.After continuous passage 10 times, it still can be grown in fusant primary dcreening operation substratum; Can carry out zymamsis, and its fermented liquid color change is blue, analyses evaluation through ply of paper, it still possesses the MLF ability.So the inherited character of fusant F-20 is stable.
4, the unicellular separation and Culture of fusant F-20
Get the F-20 slant strains is mixed with suitable concentration through YPD bacteria suspension, through micrurgy, bigger unicellular of 20 cell volumes of picking is numbered F-20-1～F-20-20, insert in 20 cuvette cartridge YPD liquid nutrient mediums shaking culture under 25 ℃, 180rpm respectively.Behind the 48h, in 20 test tubes, insert 6 maintenance clarifications of F-20-2, F-20-3, F-20-1, F-20-19, F-20-20, all the other each Guan Junyou muddiness and bacterial sediment in various degree.Keep clarifying test tube to be because the cell that inserted is dead cell or cell not to be inserted and cause in the test tube, the upgrowth situation difference of all the other each pipes may by each cell viability, in the picking process suffered degree of injury and the difference of the aspects such as adaptive faculty of environment caused.Therefrom higher, more mono-clonal bacterial strain F-20-4, F-20-6, F-20-7, F-20-12, F-20-15, the F-20-17 of pipe end bacterial sediment of picking 6 strain opacities behind the mixing that fully vibrates, measures its OD 600, the OD of F-20-7 wherein 600Value is maximum, illustrates that its biomass is big, and cell viability is strong, thus can select for use F-20-7 as measure fusant zymamsis power and oxysuccinic acid degradability for examination mono-clonal bacterial strain.
Mono-clonal bacterial strain F-20-7, its cellular form, size, colony characteristics are all similar to wine yeast 1450.
5, fusant F-20-7 zymamsis power and oxysuccinic acid degradability test
Take the logarithm respectively the growth F-20-7 and the 1450 bacterium liquid in mid-term, by 10% inoculum size, through centrifugal remove supernatant liquor after, with thalline be forwarded to fusant and wine yeast 1450 zymamsis power, the oxysuccinic acid degradability is measured in the substratum, according to CO 2Weightlessness is drawn fermentation diagram, relatively both fermenting speeds; Measure the whole wine degree of both fermented liquids, analyze it and have or not significant difference.
As seen from Figure 1,1450 to finish the zymamsis required time be 5d, and it is 6d that F-20-7 finishes the zymamsis required time, and the fermentation rate of fusant F-20-7 is a little less than wine yeast 1450, illustrates that its zymamsis power is compared with 1450 slightly to descend; The final wine degree of both fermented liquids sees Table 1-1, and through t check, both fermented liquids are on whole wine degree and there was no significant difference.
Can be found out that by Fig. 1 and Fig. 2 acid falls in mono-clonal fusant F-20-7 in zymamsis, it falls mollic acid between wine yeast 1450 and Oenococcus Oeni SD-2a, relatively near SD-2a.1-2 can find out by table, when F-20-7 finishes in zymamsis, and the oxysuccinic acid of degradable 77.6%, its oxysuccinic acid degradability is higher than wine yeast 1450, is lower than Oenococcus Oeni SD-2a.
In sum, the F-20-7 oxysuccinic acid of can in zymamsis, degrading, its zymamsis ability is near wine yeast 1450; The oxysuccinic acid degradation capability is between two parental plants, relatively near Oenococcus Oeni SD-2a.
Wherein: the preparation of SSM damping fluid:
Sucrose 0.5mol/L, MgCl 26H 2O 0.02mol/L, toxilic acid 0.02mol/L, the distilled water preparation, pH transfers to 6.5,121 ℃ of sterilization 20min.
PB and PBS preparation:
Get 0.2mol/L biphosphate sodium water solution 87.7ml, mix 121 ℃ of sterilization 20min, promptly obtain the phosphate buffered saline buffer PB of pH6.0 with the biphosphate sodium water solution 12.3ml of 0.2mol/L.The sorbyl alcohol that adds 1mol/L in PB obtains high phosphatizing phthalate buffer PBS through the degerming of 0.45um mol/L membrane filtration.
Table 1 wine yeast 1450, the mono-clonal fusant F-20-7 whole wine degree that ferments
Bacterial strain Wine yeast 1450 Mono-clonal fusant F-20-7
The whole wine degree % (v/v) of fermented liquid Repeat 1 and repeat 2 11.2 11.4 Repeat 1 and repeat 2 11.1 11.0
Table 2 1450, F-20-7, SD-2a oxysuccinic acid degradability are relatively
Bacterial strain The oxysuccinic acid initial concentration, g/L The oxysuccinic acid final concentration, g/L 1450, F-20-7 zymamsis and SD-2a MLF concluding time, d The oxysuccinic acid degradation rate, % The oxysuccinic acid degradation rate, g/Ld
1450 F-20-7 SD-2a 3.00 3.00 3.00 2.52 0.67 0.40 5 6 4 16.0 77.6 86.6 0.09 0.38 0.65
Above-mentioned wine yeast 1450 and Oenococcus Oeni SD-2a, the public all can obtain from common commercial sources, and the applicant also can answer public's requirement, guarantee to provide to the public in 20 years applyings date.

Claims (5)

1, a kind of acidity reducing yeast of grape wine and cultural method thereof is characterized in that: it is to be parental plant with wine yeast 1450 and Oenococcus Oeni SD-2a, uses that Protoplast Fusion Technique makes up.
2, a kind of acidity reducing yeast of grape wine according to claim 1 and cultural method thereof is characterized in that: acidity reducing yeast of grape wine cellular form, size, colony characteristics are all similar to wine yeast 1450.
3, a kind of acidity reducing yeast of grape wine according to claim 2 and cultural method thereof, it is characterized in that: the acidity reducing yeast of grape wine culture medium prescription is respectively:
YPD substratum: yeast extract paste 1%, peptone 2%, glucose 2%, distilled water preparation, natural pH value, 121 ℃ of sterilization 20min;
ATB substratum: yeast extract paste 0.5%, peptone 1%, glucose 1%, MgSO 47H2O 0.02%, MnSO 44H2O 0.005%, cysteine hydrochloride 0.05%, tomato juice 25% (v/v), distilled water preparation, natural pH value, 115 ℃ of sterilization 20min.Add 1.5% agar during the preparation solid medium, the preparation height adds 17% sucrose when oozing substratum;
Fusant primary dcreening operation substratum: yeast extract paste 0.5%, peptone 1%, glucose 1%, MgSO 47H2O 0.02%, MnSO 44H2O 0.005%, cysteine hydrochloride 0.05%, tomato juice 25% (v/v), agar 1.5%, distilled water preparation, nature pH value, 115 ℃ of sterilization 20min, heating and melting substratum before using is when treating that its temperature is reduced to 55 ℃, in the penbritin of cycloheximide that wherein adds 100ug/mL and 20ug/mL, rapidly mixing falls plate;
The sub-screening culture medium of Target Fusion: yeast extract paste 0.5%, peptone 1%, glucose 10%, MgSO 47H2O0.02%, MnSO 44H2O 0.005%, tomato juice 10% (v/v), and DL-oxysuccinic acid 10g/L, tetrabromo-mcresolsulfonphthalein 0.004%, the distilled water preparation, the pH value transfers to 3.8,115 ℃ of sterilization 20min;
Cream 0.5% fusant and wine yeast 1450 zymamsis power---oxysuccinic acid degradability test media: yeast, peptone 1%, glucose 200g/L, tomato juice 20ml/L, L MALIC ACID 3g/L, MnSO44H2O 0.005%, the distilled water preparation, the pH value transfers to 3.0;
Oenococcus Oeni SD-2a oxysuccinic acid degradability test media: yeast extract paste 0.5%, peptone 1%, glucose 2g/L, tomato juice 20ml/L, L MALIC ACID 3g/L, MnSO 44H2O 0.005%, alcoholic strength 11% (v/v), and the distilled water preparation, the pH value transfers to 3.2.
4, a kind of acidity reducing yeast of grape wine according to claim 3 and cultural method thereof is characterized in that: Oenococcus Oeni SD-2a and wine yeast protoplastis prepare process and are:
For Oenococcus Oeni SD-2a, adopt to be in the logarithmic growth somatic cells in mid-term, with SMM homeo-osmosis liquid, N,O-Diacetylmuramidase enzymolysis concentration is 1mg/mL, in 37 ℃ of water enzyme digestion 30min, the preparation protoplastis;
For wine yeast 1450, adopt to be in the logarithmic growth somatic cells in mid-term, through 0.3% beta-mercaptoethanol-0.1%EDTANa2 in 37 ℃ of pre-treatment 10～15min, be homeo-osmosis liquid again with PBS, helicase concentration is 2%, in 37 ℃ of water enzyme digestion 40min, and the preparation protoplastis.
5, a kind of acidity reducing yeast of grape wine according to claim 3 and cultural method thereof is characterized in that: protoplastis merges and fusant primary dcreening operation process is:
Adopt several 1/100, the Cacl of PEG-400030% (w/v), 30 ℃ of fusion temperatures, time of fusion 30min, yeast count/lactic-acid bacteria cells 2The fusion conditions of concentration 50mmol/L, with the yeast cell through the PEG processing is contrast, with the selective pressure of 100ug/mL cycloheximide+20ug/mL penbritin, after 9 days, obtained the fusant of the similar wine yeasts 1450 of 117 strain colonial morphologies altogether in cultured continuously as the fusant screening.
CN 200510041935 2005-04-11 2005-04-11 Acidity reducing yeast of grape wine and its culturing method Pending CN1721524A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101029300B (en) * 2007-02-06 2010-08-25 韶关学院 Basylous yeast and its use in brewing myrica rubra
CN103215161A (en) * 2013-03-14 2013-07-24 河南科技大学 Method for reducing acidity of wild Kiwi berry wine using Oenococcus oeni
CN103468675A (en) * 2013-08-29 2013-12-25 西北农林科技大学 RAPD marker and SCAR marker of oenococcus antiacid related gene and obtaining method of RAPD marker and SCAR marker

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101029300B (en) * 2007-02-06 2010-08-25 韶关学院 Basylous yeast and its use in brewing myrica rubra
CN103215161A (en) * 2013-03-14 2013-07-24 河南科技大学 Method for reducing acidity of wild Kiwi berry wine using Oenococcus oeni
CN103215161B (en) * 2013-03-14 2014-07-30 河南科技大学 Method for reducing acidity of wild Kiwi berry wine using Oenococcus oeni
CN103468675A (en) * 2013-08-29 2013-12-25 西北农林科技大学 RAPD marker and SCAR marker of oenococcus antiacid related gene and obtaining method of RAPD marker and SCAR marker

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