CN111363686B - Yeast for brewing aromatized wine and application and method thereof - Google Patents

Yeast for brewing aromatized wine and application and method thereof Download PDF

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CN111363686B
CN111363686B CN202010147613.3A CN202010147613A CN111363686B CN 111363686 B CN111363686 B CN 111363686B CN 202010147613 A CN202010147613 A CN 202010147613A CN 111363686 B CN111363686 B CN 111363686B
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CN111363686A (en
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林元山
刘其酉
田云
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Hunan Agricultural University
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Abstract

The invention discloses a yeast strain for brewing aromatized wine, and application and a method thereof. The yeast strain is Saccharomyces cerevisiae lys225(Clavispora lusitaniae lys225), and the preservation number is CGMCC No. 18933. Can be used for preparing fruit wine starter and brewing wine.

Description

Yeast for brewing aromatized wine and application and method thereof
Technical Field
The invention belongs to the technical field of food biology for brewing wine, and particularly relates to a saccharomyces cerevisiae strain for brewing aromatized wine, and application and a method thereof.
Background
According to the regulation of the national wine standard GB15037-2006, the wine is an alcohol beverage which is prepared by taking fresh grapes or grape juice as a raw material and fermenting all or part of the fresh grapes or the grape juice, and the alcohol content is not lower than 7.0%; the content of methanol in wine per liter must be below 400mg, and if the methanol content in wine is too high, the methanol content can cause damage to nerves and eyes, and blindness can be caused.
The wine is classified according to color, and is generally divided into three types, namely red wine, white wine and pink wine; dividing into dry wine according to sugar content, wherein the sugar content is lower than 4 g/L; semi-dry wine with sugar content of 4-12 g/L; the semi-sweet wine has a sugar content of 12-45 g/L; the sugar content of the sweet wine is more than 45 g/L; based on whether carbon dioxide is contained in the still wine or the sparkling wine, the method does not contain self-fermentation or artificial addition of CO2The grape wine is called static wine, namely static grape wine; sparkling wine and sparkling wine contain a certain amount of CO2Gaseous wine containing CO2Is sparkling wine produced by adding sugar into wine and fermenting, and artificially adding CO2The product can be added into wine as sparkling wine due to CO2The effect makes the wine have fresh, pleasant and pleasant taste.
Vitis davidii (vitas davidii)
Figure GDA0002501241080000011
) The woody vine is perennial deciduous leaf of Vitaceae, is originally produced in China, is mainly distributed in Hunan, Jiangxi, Fujian, Zhejiang, Anhui, Yunnan, Guangdong and other provinces, and is a wild germplasm resource widely distributed in east Asia population; because of high temperature resistance, high humidity, low illumination and high disease resistance, the cultivation history in the south has been many years; at present, the planting area of the Aleuropaea Hunanensis is 6800hm2, the resources are very rich, and the Aleuropaea Hunanensis is widely distributed in the snow mountain; the vitis davidii has high yield, the total sugar content in the fruits is 15.53 percent, the total acid content is 0.33 percent, the sugar-acid ratio is high, the tannin content is low, the fruit grains are small, the seeds are more, the fruit peel is thick, the meat sacs and the seeds are difficult to separate, the edible rate is low, and the market price of fresh food is low.
The research result of Dengjihong shows that the skin of Vitis davidii has abundant anthocyanin content, and the color value is 2.43 times of that of Cabernet Sauvignon grape skin, 2.87 times of that of red grape skin and 21.75 times of that of Kyoho grape skin; the content of the anthocyanin reaches 2.52mg/gFW (calculated by 3' delphinidin-3-glucoside).
Jianghui research shows that the content of procyanidine in the spine grape juice is 3147 mu g/mL and is far higher than that of procyanidine in other grape juice, the content of procyanidine in Welch's purple grape juice is 2476 mu g/mL and is also obviously higher than that of the other grape juice, but the content of Welch's white grape juice is extremely low and cannot be detected. Therefore, the Vitis davidii Murr is a raw material suitable for brewing red wine.
According to the research result of Zhengzhou fruit tree research on Sunyilai of Chinese academy of agricultural sciences, the total amount of anthocyanin in the peel of wild grapes in China is obviously higher than that of European-Asia grape varieties, the European-Asia grape varieties mainly contain monosaccharide anthocyanin and have little content of disaccharide anthocyanin, the European-Asia grape varieties mainly contain dimethyl delphinidin (Mv) anthocyanin and have little content of malvidin (Pg) anthocyanin; the content of disaccharide anthocyanin in most wild grapes in China is high, and Pg anthocyanin with high content is detected; in the Vitis cuneata, Vitis davidii and Vitis davidii, the content of Pg anthocyanin is highest, accounting for 81%, 95% and 87% of total anthocyanin, respectively, and the anthocyanin component in the 3 strains is mainly disaccharide anthocyanin.
Disclosure of Invention
The invention aims to provide a yeast strain for brewing aromatized wine, application and a method thereof according to the production current situation of the spine grape in Hunan province and by using aromatized yeast screened from rotten fruits of strawberry with wine aroma, thereby promoting the practical production application.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the invention provides a microbial strain capable of fermenting grape wine and forming special flavor, the strain is obtained by screening from strawberry rotten fruit with wine aroma, the yeast strain is a saccharomyces cerevisiae lys225(Clavispora lusitaniae lys225), and is preserved in the China grape microbial center of China Committee for culture Collection of microorganisms (CGMCC No. 18933) 11.11.2019; can be used for preparing fruit wine starter.
The physiological and biochemical characteristics of the saccharomyces cerevisiae lys225(Clavispora lusitaniae lys225) are as follows:
(1) the bacterial strain is on a wort culture medium, the colony form is milky white, the surface is smooth and moist, no bulge exists, the edge is neat, the bacterial strain is in an oval or elliptic rod shape under an optical microscope, the bacterial strain is propagated in teething, the size is 3-6 microns multiplied by 5-15 microns, and the bacterial strain presents pleasant fragrance in a culture dish;
(2) the strain grows well on a grape bacterium culture medium, a mildew culture medium and a yeast culture medium; can grow well under the condition of 15-38 ℃, and the most suitable temperature is 30-35 ℃; can grow at pH 3.8-7.0, and has optimum pH of 4.5-6.2; in a yeast extract glucose liquid culture medium (YPD), the propagation speed is high, the strain enters a logarithmic growth phase within 8-12 hours, and the viable count reaches 1.2-1.8 multiplied by 109 CFU/mL; the strain can ferment and generate gas by utilizing various monosaccharides and disaccharides such as glucose, sucrose, lactose, fructose, arabinose, xylose, maltose, sorbose and the like, and can not directly utilize starch;
(3) the strain can be used for producing fruit wine by fermenting fruit juice or jam of fruits such as grapes, strawberries, kiwifruits, blueberries, oranges, mulberries, apples and waxberries, and is very suitable for fermenting the fruit wine by the berries;
(4) the strain can tolerate the alcoholic strength of 11.0-14.5, and the strain cannot survive in fruit wine mash with the alcoholic strength of more than 14.5 degrees;
(5) the strain is not sensitive to penicillin and streptomycin.
The invention provides a preparation method of a fruit wine leaven, which is characterized in that the yeast strain is prepared into a Corynebacteria cinerea active yeast paste or a Corynebacteria cinerea active yeast powder, namely the fruit wine leaven.
Specifically, the preparation method of the corynebacterium viticola active yeast paste comprises the following steps:
(1) preparing a shake flask seed culture medium: the mass percent content of the nitrogen source, the mass percent content of the carbon source and the mass percent content of the sodium chloride in the shake flask seed culture medium are respectively 0.5-1.5%, 2.0-4.0% and 0.3-0.6%; sterilizing at 121 deg.C for 20-30min, cooling, and inoculating fresh slant strain of yeast strain; shake culturing for 12-18h at 28-34 deg.C and 150-; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(2) preparing liquid seeds: preparing a fermentation tank seed culture medium, wherein the mass percent content of a nitrogen source in the fermentation tank seed culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 25-30min, cooling, inoculating liquid fermentation shake flask seeds, wherein the volume ratio of the liquid fermentation shake flask seeds to a seed culture medium in a fermentation tank is 1 (10-20) during inoculation, mechanically stirring and culturing for 12-18h in the fermentation tank at 28-32 ℃ and the rotation speed of 150-; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(3) fermentation production of the corynespora viticola: preparing a production tank culture medium, wherein the mass percent content of a nitrogen source in the production tank culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 30-40min, cooling, inoculating the fermentation tank seeds obtained in the step (2), wherein the volume ratio of the fermentation tank seeds to the culture medium of the production tank is 1 (10-20) during inoculation, mechanically stirring and culturing for 18-24h in a fermentation tank at 28-32 ℃ and the rotating speed of 150-; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(4) and (3) concentrating and producing the fermentation liquor of the corynespora viticola: when the volume of the fermentation liquor is less than or equal to 10L, directly centrifuging by using a high-speed centrifuge to obtain yeast paste; when the volume of the fermentation liquor is more than 10L and less than or equal to 100L, directly centrifuging by using a tubular high-speed centrifuge to obtain yeast paste; when the volume of the fermentation liquor is more than 500L, directly centrifuging by adopting a disc type high-speed centrifuge to obtain yeast paste; the viable count of the yeast paste is more than 5.0 × 1010 CFU/g. The prepared yeast paste leaven has extremely high activity, can be immediately put into fruit wine for fermentation, and has the quality guarantee period of 7-10 days.
The preparation method of the corynespora viticola active yeast powder comprises the following steps:
(1) preparing a shake flask seed culture medium: the mass percent content of the nitrogen source, the mass percent content of the carbon source and the mass percent content of the sodium chloride in the shake flask seed culture medium are respectively 0.5-1.5%, 2.0-4.0% and 0.3-0.6%; sterilizing at 121 deg.C for 20-30min, cooling, and inoculating fresh slant strain of yeast strain; shake culturing for 12-18h at 28-34 deg.C and 150-; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(2) preparing liquid seeds: preparing a fermentation tank seed culture medium, wherein the mass percent content of a nitrogen source in the fermentation tank seed culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 25-30min, cooling, inoculating liquid fermentation shake flask seeds, wherein the volume ratio of the liquid fermentation shake flask seeds to a seed culture medium in a fermentation tank is 1 (10-20) during inoculation, mechanically stirring and culturing for 12-18h in the fermentation tank at 28-32 ℃ and the rotation speed of 150-; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(3) preparing a secondary seed of the saccharomyces cerevisiae: preparing a secondary seeding tank culture medium, wherein the mass percent content of a nitrogen source in the secondary seeding tank culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 30-40min, cooling, inoculating the primary seeds of the fermentation tank, wherein the volume ratio of the primary seeds of the fermentation tank to the culture medium of the secondary seed tank is 1 (10-20) during inoculation, mechanically stirring and culturing for 12-18h in the fermentation tank at 28-32 ℃ and the rotating speed of 150-;
(4) fermentation production of the corynespora viticola: preparing a production tank culture medium, wherein the mass percent content of a nitrogen source in the production tank culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 30-40min, cooling, inoculating the seeds of the liquid fermentation tank obtained in the step (3), wherein the volume ratio of the seeds of the liquid fermentation tank to the culture medium of the production tank is 1 (10-20) during inoculation, mechanically stirring and culturing for 18-24h in the fermentation tank at 28-32 ℃ and the rotating speed of 150-;
(5) and (3) concentrating and producing the fermentation liquor of the corynespora viticola: when the volume of the fermentation liquor is more than 1000L, the yeast is concentrated by adopting ceramic membrane microfiltration, the concentration ratio is (40-50):1, and high-density yeast slurry with the viable count of more than or equal to 5.0 multiplied by 1010CFU/mL is obtained;
(6) mixing the high-density yeast slurry with dextrin according to the weight ratio of 1 (1.2-1.5), stirring to prepare yeast paste, and controlling the water content to be 40-45%; then putting the yeast mud into a yeast mud silk pressing forming machine, and controlling the diameter of the yeast silk to be 1.0-1.5 mm; drying the yeast filaments in a fluidized bed dryer, controlling the air inlet temperature to be less than or equal to 65 ℃, the air outlet temperature to be less than or equal to 34 ℃, controlling the water content of the yeast powder to be 5-6%, and controlling the viable count to be more than or equal to 2.0 multiplied by 1010CFU/g, thus obtaining the dentate clavaria active yeast powder. Can be packaged according to different specifications of 100g, 150g, 500g, 1000g and the like.
In the fruit wine fermentation process, the starter is added to quickly start fermentation, so that the special aroma of the fruit wine is increased, the acidity of the fruit wine is obviously improved, the mixed bacteria pollution in the fermentation process is reduced, the success rate of sulfur-free fermentation is improved, and the generation of methanol in the wine is obviously inhibited. Therefore, the invention also provides a preparation method of wine, which is to prepare the wine by fermenting the yeast strain. The grape is Vitis davidii.
Specifically, the preparation method of the wine comprises the following steps:
the method A comprises the following steps: brewing dry type natural spine grape wine: removing stalks of mature spine grapes, crushing the spine grapes, and adding the grapes into the yeast paste prepared by the method, or adding the grapes into the yeast powder prepared by the method; the mass percentage content of the yeast paste in the grapes is 0.5-1.0 per mill, and the mass percentage content of the yeast powder in the grapes is 0.2-0.4 per mill; no other additives (no sulfur dioxide and no sugar are added), stirring, mixing, sealing with a respirator to ensure that gas only can not enter, naturally fermenting at 15-25 deg.C for 30-40d, continuously pressing grape skin into wine mash during natural fermentation, separating skin and residue, clarifying, and filtering to obtain dry red grape wine; the brewed dry natural spine grape wine has purplish red color, comfortable, fragrant and harmonious aroma, higher acidity but refreshing and mellow; the dry spine grape wine has the accuracy of 10.0-11.5 degrees, the sugar content is less than or equal to 3.0g/L, the methanol content is 60.0-150mg/L which is far lower than the national standard of 400 mg/L;
and B, method: brewing of semi-dry type, semi-sweet type and sweet type spine grape wine: removing stalks of mature spine grapes, crushing the spine grapes, and adding the grapes into the yeast paste prepared by the method, or adding the grapes into yeast powder prepared by the method; the mass percentage content of the yeast paste in the grapes is 0.5-1.0 per mill, and the mass percentage content of the yeast powder in the grapes is 0.2-0.4 per mill; sealing the fermentation tank with a respirator after stirring and mixing uniformly to ensure that gas only flows out and cannot enter, naturally fermenting for 30-40d at the temperature of 15-25 ℃, continuously pressing grape skin into wine mash during natural fermentation, separating skin and residue, clarifying and filtering to obtain dry red grape wine; the brewed semi-dry type, semi-sweet type and sweet type spine grape wine has purplish red color, pleasant aroma, strong fragrance, purity and harmony, moderate acidity, but good and fresh taste, mellow, smooth and smooth mouthfeel, but low astringency, and obviously increased alcoholic strength; the dry spine grape wine has the accuracy of 12.5-16.5 degrees, the sugar content is more than or equal to 6.0g/L, the methanol content is 50.0-100mg/L, and is far lower than the national standard of 400 mg/L.
Preferably, in the method B, sucrose is also added during fermentation, and the mass percentage of the sucrose in the grapes is 5% -15%.
Compared with a control without the yeast starter, in the fruit wine fermented by the yeast strain, isoamyl alcohol is increased by 3.5-5.0 times, ethyl acetate is increased by 6.5-8.0 times, acetic acid is increased by 2.0-3.5 times, and the fruit wine is mild, pleasant and sweet in the sense.
More specifically, the process of brewing wine is as follows:
(1) selecting the vitis davidii: removing mildewed fruits, rotten fruits and immature fruits from mature Vitis davidii by manpower, and removing impurities (no washing fruits) such as fruit leaves, grape twigs, dregs and the like;
(2) removing stalks and crushing: putting the artificial spine grape into a crushing and stem removing machine, removing stem and breaking grape skin (only fruit pulp is squeezed out of peel), feeding the processed grape into a fermentation tank by a screw pump, and adding fresh active yeast paste (or active dry yeast powder of 0.2-0.4 per mill) according to the proportion of 0.5-1.0 per mill of the grape;
(3) adding sugar: measuring and calculating sugar content of fresh grape according to grape wine type (dry type, semi-sweet type, sweet type) and final alcohol content requirement, adding 0-15% sucrose, stirring to dissolve sucrose, and sealing fermentation tank equipped with respirator;
(4) pre-fermentation: and (3) observing the fermentation state and temperature of the grape wine every day when fermentation is started, continuously pressing grape skin into wine mash after gas production, wherein the temperature of the tank body is preferably 15-25 ℃, otherwise, the thorn grape wine is kept for natural fermentation for 30-40 days at the temperature of 15-25 ℃ through jacket heating or cooling control, and a large amount of wine aroma and fruit aroma are continuously released from a fermentation tank until skin dregs sink.
(5) Squeezing and filtering: after grape skin sinks, filtering with a screen to obtain self-retained wine, and preparing pressed wine from the residual skin residue with a screw squeezer, or collecting wine filtrate without separating self-retained wine and pressed wine.
(6) Filtering and deslagging by diatom, and sterilizing: adding food grade diatomite into the squeezed and filtered wine according to the ratio of 200-.
(7) And (3) after-fermentation: filtering wine with diatomaceous earth meeting quality requirements, delivering to the fermentation tank again, adding 0.1-0.2% lactobacillus or 0.5-0.8% oak chip as required, anaerobic fermenting at 20-25 deg.C for 2-4 days, and naturally standing for 10-20 days.
(8) Coarse filtration and fine filtration of diatomite for sterilization: adding food-grade diatomite according to the ratio of 150-.
(9) Aging and stabilizing wine body: and (3) conveying the finely filtered wine to a wine storage tank or an oak barrel to ensure that the wine body is stable and full for more than or equal to 30 days.
(10) Microfiltration and sterilization: performing online cleaning and sterilization on the wine storage tank, the stainless steel conveying pipeline, the wine bottle and the filling machine by using a CIP online sterilization system at the temperature of 121 ℃ for 30 min; the microfiltration filter is provided with two-stage or three-stage series microfiltration membranes with the diameters of 0.45 mu m, 0.22 mu m and 0.1 mu m, the microfiltration filter is filter-pressed by a sanitary 304 stainless steel centrifugal pump, the lift is more than or equal to 30m, and the subsequent microfiltration pipelines of the centrifugal pump are sterilized by the CIP sterilization system; the wine after microfiltration is conveyed into an aseptic wine storage tank, the wine storage tank is provided with a foldable aseptic filtering respirator, pressure balance is kept, and external mixed bacteria pollution is isolated, at the moment, the spine wine is purplish red, the aroma is strong and harmonious, and the taste is refreshing and mellow; sterilizing cork at 80-100 deg.C for 60-90 min.
(11) Packaging and warehousing: according to the requirements, the wine is filled and corked in an aseptic room by adopting an aseptic packaging machine, then is sent to the subsequent thermal shrinkage wine sealing and labeling processes to complete packaging, and the wine bottles are horizontally placed and put in storage.
Compared with the natural fermentation of wild yeast on the surface of the existing Vitis davidii, the invention has the following remarkable advantages and progresses:
(1) the stable yeast starter is added, so that the fermentation of the wine is started quickly, the quality of the wine is easy to control, the growth of mixed bacteria is effectively inhibited, the content of methanol is effectively controlled, the brewing power of the wine is obviously improved, and the wine is safer;
(2) the fermentation technology of sulfur-free addition is adopted, the growth of mixed bacteria is controlled through acidity, the pollution rate and the failure rate are reduced, and the wine is safer;
(3) by adding high-quality saccharomycetes, the special aroma is increased, the quality of the wine is improved, and the unique wine style and characteristics are formed;
(4) the strain preparation link adopts a ceramic membrane concentration technology, the process is simple, safe, rapid and efficient, CIP online cleaning and sterilization operations can be added, and the technology is advanced; the process is environment-friendly and safe, the filtering link adopts diatomite filtration deslagging and sterilization, CIP online cleaning and online sterilization and 2-3 grade membrane filtration thorough sterilization, avoids destroying anthocyanin in the wine at high temperature, is safe and sanitary, and is beneficial to prolonging the quality guarantee period of the wine.
Detailed Description
Example 1 acquisition of flavored yeast strains:
(1) primary screening by a flat plate: sampling fruits such as grapes, apples, pears, bananas, mangos, pineapples, strawberries, oranges, phoenix pears and the like which are slightly rotten and have bouquet, diluting and coating malt juice agar plates according to a ten-fold dilution method, culturing for 2-4 days in an incubator at 30 ℃, selecting milky single colonies similar to yeasts, inoculating the milky single colonies to a slant culture medium, preserving the strains, and obtaining the single colony slant 508 strain.
(2) Re-screening the shaking bottle: inoculating the primary-screened strains into 150mL secondary-screened culture medium (4.0% of cane sugar and 0.5% of yeast extract), sealing the primary-screened strains one by one, culturing the primary-screened strains for 36h at 30 ℃ by a shaking table at 130 rpm, then performing static culture in a 30 ℃ incubator for 48h, then performing sensory screening on 27 strains with good aroma by an aroma smelling method, further determining the conversion rate of cane sugar and alcohol by a distillation method, selecting 6 strains with the conversion rate higher than 40%, and finally performing a wine brewing experiment on the Chinese strains to obtain 2 strains of the good strains with good aroma, wherein 1 strain is identified as the saccharomyces cerevisiae (Clavispora luxitaae lys225) by molecular biology and morphology, and the strain is preserved in the China microorganism culture collection and management committee grape microbial center at 11 months 11 days in 2019, and has the preservation number of CGMCC No. 18933;
example 2 morphological characteristics of Saccharomyces cerevisiae (Clavispora lusitaniae lys 225):
culturing strain Corynebacterium viticola (Clavispora lusitaniae lys225) in wort culture medium for 3-4 days, wherein the strain is in milky colony form, has smooth and moist surface, no protrusion, regular edge, elliptic or elliptic rod shape under optical microscope, and can propagate when going out of teeth, and the size is 3-6 μm × 5-15 μm, and the strain presents pleasant fragrance in culture dish.
Example 3 physiological and biochemical characteristics of Saccharomyces cerevisiae (Clavispora lusitaniae lys 225):
culturing strain Corynebacterium viticola (Clavispora lusitaniae lys225) in different culture media, observing, and detecting, wherein the strain can grow under 15-38 deg.C, and the optimum temperature is 30-35 deg.C; can grow at pH 3.8-7.0, and has optimum pH of 4.5-6.2; in a yeast extract peptone glucose liquid culture medium (YPD), the propagation speed is high, the strain enters a logarithmic growth phase within 8-12 hours, and the viable count reaches 1.2-1.8 multiplied by 109 CFU/mL; the strain can ferment and generate gas by utilizing various monosaccharides and disaccharides such as glucose, sucrose, lactose, fructose, arabinose, xylose, maltose, sorbose and the like, and can not directly utilize starch; the strain can be used for producing fruit wine by fermenting fruit juice or jam of fruits such as grapes, strawberries, kiwifruits, blueberries, oranges, mulberries, apples and waxberries, and is very suitable for fermenting the fruit wine by the berries; the strain can tolerate the alcoholic strength of 11.0-14.5, and the strain cannot survive in fruit wine mash with the alcoholic strength of more than 14.5 degrees; the strain is not sensitive to penicillin and streptomycin.
Example 4 flavouring effect of inoculation of fresh Vitis vinifera with Saccharomyces cerevisiae lys 225:
preparing 100mL of culture medium according to 0.5 percent of yeast extract and 2.0 percent of cane sugar in the formula, sterilizing at 121 ℃ for 30min, and inoculating a fresh slant strain of the saccharomyces cerevisiae CGMCC No.18933 after cooling; shake culturing for 12-18h in a shaking table with the temperature of 30 ℃ and the rotating speed of 150 r/min, the viable count is 3.0 multiplied by 108CFU/mL, 4.5g of yeast paste is obtained by centrifuging with an aseptic centrifugal bottle, the obtained yeast paste is added into a 5L glass jar of 3.5kg of Vitis vinifera to assist anaerobic fermentation, skin and slag are continuously pressed into a wine body after gas production, the wine is filtered after 35 days, and relevant indexes are tasted and detected, and the brewed dry type natural Vitis vinifera wine has purplish red color and luster, is comfortable, fragrant and harmonious in aroma, and presents unique mild and full sweet aroma; the acidity is high, but the taste is refreshing and fresh, the alcohol content is 10.8 degrees, the sugar content is 2.9g/L, the methanol content is 65.0mg/L, and the quality meets the requirements.
Example 5 brewing and flavour enhancement effects of semi-sweet wine:
preparing 100mL of culture medium according to 0.5 percent of yeast extract and 5.0 percent of cane sugar in the formula, sterilizing at 121 ℃ for 30min, and inoculating a fresh slant strain of the saccharomyces cerevisiae CGMCC No.18933 after cooling; shake culturing for 12-18h in a shaking table with the temperature of 30 ℃ and the rotating speed of 150 r/min, the viable count is 3.5 multiplied by 108CFU/mL, 4.8g of yeast paste is obtained by centrifuging with an aseptic centrifugal bottle, the obtained yeast paste is added into a 5L glass jar containing 3.5kg of Vitis davidii and 200g of cane sugar to assist anaerobic fermentation, skin and slag are continuously pressed into wine after gas production, the wine is filtered after 40 days, sensory taste and related indexes are detected, and the semi-sweet natural Vitis davidii wine is purplish red in color and luster, strong and lasting in fragrance and presents unique mild and full sweet fragrance; the acidity is always fresh and cool, the alcohol content is 14.7 degrees, the sugar content is 36.5g/L, the methanol content is 71.0mg/L, and the quality meets the requirements.
Example 6 preparation of a fermentation agent of Vitis davidii active yeast paste:
(1) preparing 400mL of a shake flask seed culture medium according to 0.5% of formula source yeast extract, 4.0% of sucrose and 0.3% of sodium chloride, sterilizing at 121 ℃ for 30min, cooling, and inoculating a fresh slant strain of the saccharomyces cerevisiae CGMCC No. 18933; shake culturing in shaking table at 30 deg.C and rotation speed of 250 r/min for 18 h;
(2) preparing 8000mL of fermentation medium according to 0.5% of formula source yeast extract, 4.0% of sucrose and 0.3% of sodium chloride, sterilizing for 30min at 121 ℃, cooling, inoculating 400mL of liquid shake flask seeds, and mechanically stirring and culturing for 18h in a fermentation tank at 32 ℃ and the rotating speed of 200 r/min to obtain 7.5L of log-phase yeast with the viable count of 4.0 multiplied by 108 CFU/mL;
(3) concentrating the fermentation liquor of the corynespora viticola, and directly centrifuging by a high-speed centrifuge to obtain yeast paste; the viable count is 6.0 × 1010CFU/g (wet thallus), the prepared yeast paste leavening agent has extremely high activity, can be immediately put into fruit wine for fermentation, and the shelf life of the yeast paste is 7-10 days.
Example 7 preparation of an active yeast powder of a fermentation agent for Vitis davidii:
(1) preparing shake flask seeds: preparing 500mL of a shake flask seed culture medium according to 0.5% of formula source yeast extract, 4.0% of sucrose and 0.3% of sodium chloride, sterilizing for 30min at 121 ℃, and inoculating a fresh slant strain of the saccharomyces cerevisiae CGMCC No.18933 after cooling; shake culturing in shaking table at 30 deg.C and rotation speed of 250 r/min for 18 h;
(2) first-stage fermentation tank seeds: preparing 8.0L of fermentation medium according to 0.5 percent of formula source yeast extract, 4.0 percent of sucrose and 0.3 percent of sodium chloride, sterilizing in a 10L fermentation tank at 121 ℃ for 30min, cooling, inoculating 500mL of liquid shake flask seeds, and mechanically stirring and culturing in a fermentation tank at 32 ℃ and the rotating speed of 250 r/min for 18h to obtain 8.0L of first-stage yeast seeds;
(3) second-stage fermentation tank seeds: preparing 80.0L of fermentation medium according to 0.5% of formula source yeast extract, 4.0% of sucrose and 0.3% of sodium chloride, sterilizing in a 100L fermentation tank at 121 ℃ for 30min, cooling, pressing in the 8.0L of second-stage seeds by adopting sterile air, and mechanically stirring and culturing in the fermentation tank at 32 ℃ and 150 r/min for 12h to obtain 80.0L of log-phase yeast;
(3) preparing 1600.0L of fermentation medium according to 0.5 percent of formula source yeast extract, 50 percent of cane sugar and 0.4 percent of sodium chloride, sterilizing for 30min at 121 ℃ in a 1000L fermentation tank, pressing the 80.0L of second-stage seeds by adopting sterile air after cooling, and mechanically stirring and culturing for 14h in the fermentation tank at 32 ℃ and the rotating speed of 100 r/min to obtain 1600.0L of logarithmic phase yeast, wherein the viable count reaches 3.0 multiplied by 109 CFU/mL;
(4) and (3) thallus concentration: circularly concentrating yeast thallus by adopting ceramic membrane microfiltration equipment to obtain 50.0L of high-density yeast slurry with thallus density of 8.5 multiplied by 1010CFU/mL for further preparing yeast powder;
(5) adsorption and shred pressing of yeast strain slurry: adding 60.0kg of dextrin into the 50.0L of high-density yeast strain slurry for adsorption, stirring to prepare yeast strain mud with the water content of 42.5%, and then putting the yeast strain mud into a yeast mud silk pressing forming machine, wherein the diameter of the yeast silk is 1.5 mm;
(6) and (3) drying and packaging: further drying the yeast filaments in a fluidized bed dryer, wherein the inlet air temperature is less than or equal to 65 ℃, the outlet air temperature is less than or equal to 34 ℃, obtaining 68kg of active dry yeast powder, the water content of the yeast powder is 5.5%, the viable count is 5.2 multiplied by 1010CFU/g, and packaging the active dry yeast powder into the corynespora viticola yeast active dry yeast powder according to the specification of 500 g.
Example 8 brewing of semi-dry type spine wine:
(1) selecting the vitis davidii: removing mildewed fruits, rotten fruits and immature fruits from 2600kg of ripe spine grapes manually, and removing impurities such as fruit leaves, grape branches and dregs;
(2) removing stalks and crushing: putting the artificial spine grape into a crushing and stemming machine, feeding the processed grape into a 3000L fermentation tank by using a screw pump, adding 1kg of the active strain powder of the corynespora viticola, adding 130kg of cane sugar, dissolving the cane sugar by using grape juice, stirring and uniformly mixing, and then sealing the fermentation tank with a respirator;
(3) pre-fermentation: and (3) observing the fermentation state and temperature of the grape wine every day when fermentation is started, wherein the temperature of the tank body is 15-25 ℃, continuously pressing grape skin into wine mash after gas generation, controlling the temperature through a jacket, and fermenting for 40 days, wherein a large amount of wine aroma and fruit aroma are continuously released from a fermentation tank until skin dregs sink.
(4) Squeezing and filtering: after grape skin sinks, filtering by a screen to obtain self-remaining wine, preparing pressed wine by using the residual skin dregs through a screw squeezer, and collecting 1600L of wine filtrate; adding 6.5kg of food-grade diatomite into the filtrate, stirring and pre-coating, and carrying out primary filtration by using a diatomite filter; the filtered wine is tasted organoleptically, the alcoholic strength is 13.5 degrees, the total sugar is 9.1g/L, the methanol content is less than or equal to 71m g/L, and the wine is safe.
(5) And (3) after-fermentation: filtering the wine with diatomite meeting the quality requirement, conveying to the fermentation tank again, adding 200g of lactobacillus powder and 500g of oak chips according to the requirement, performing anaerobic fermentation for 4 days at the temperature of 20-25 ℃ and naturally standing for 20 days.
(6) Coarse filtration and fine filtration of diatomite for sterilization: adding 8kg of food-grade diatomite into the post-fermented wine, stirring, pre-coating, performing secondary coarse filtration and third fine filtration by using a diatomite filter, removing particles, thalli and residues in the wine body, performing sensory taste on the finely filtered wine, measuring the alcoholic strength, the total acidity, the total sugar content and the methanol content, and ensuring that all indexes of the wine reach the specification of GB 15037-2006.
(7) Aging and stabilizing wine body: and (4) conveying the finely filtered wine to a wine storage tank or an oak barrel for storage for 30 days to ensure that the wine body is stable and full.
(8) Microfiltration and sterilization: performing online cleaning and sterilization on the wine storage tank, the stainless steel conveying pipeline, the wine bottle and the filling machine by using a CIP online sterilization system at the temperature of 121 ℃ for 30 min; carrying out three-stage serial microfiltration by adopting microfiltration machines with the diameters of 0.45 mu m, 0.22 mu m and 0.1 mu m, carrying out filter pressing by a sanitary 304 stainless steel centrifugal pump with the head of 30m, and sterilizing the subsequent microfiltration pipelines of the centrifugal pump by using the CIP sterilization system; the wine after microfiltration is conveyed into an aseptic wine storage tank, the wine storage tank is provided with a foldable aseptic filtering respirator, pressure balance is kept, and external mixed bacteria pollution is isolated, at the moment, the spine wine is purplish red, the aroma is strong and harmonious, and the taste is refreshing and mellow; the cork is sterilized at 80 deg.C for 90 min.
(9) Packaging and warehousing: and (3) filling 750mL of Bordeaux red wine bottles with the wine in an aseptic room by using an aseptic packaging machine, pressing and plugging, then sending the wine into the subsequent thermal shrinkage wine sealing and labeling processes, completing packaging, and horizontally putting the wine bottles in storage.

Claims (10)

1. The yeast strain for brewing the aromatized wine is saccharomyces cerevisiae (Clavispora lusitaniae) lys225 with the preservation number of CGMCC No. 18933.
2. The use of the yeast strain of claim 1 in the preparation of a fruit wine starter.
3. The use according to claim 2, wherein the yeast strain produces a wine-outcome starter in the form of a puree of, or a powder of, portuguese active yeast.
4. A method for preparing a fruit wine starter, which is characterized in that the yeast strain of claim 1 is prepared into a Corynebacteria cinerea active yeast paste or a Corynebacteria cinerea active yeast powder, namely the fruit wine starter.
5. The method of claim 4, wherein the preparation of the Bacteroides ivorangii active yeast paste is as follows:
(1) preparing a shake flask seed culture medium: the mass percent content of the nitrogen source, the mass percent content of the carbon source and the mass percent content of the sodium chloride in the shake flask seed culture medium are respectively 0.5-1.5%, 2.0-4.0% and 0.3-0.6%; sterilizing at 121 deg.C for 20-30min, cooling, and inoculating fresh slant strain of yeast strain; shake culturing at 28-34 deg.C and rotation speed of 150-8CFU/mL as liquid fermentation shake flask seed; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(2) preparing liquid seeds: preparing a fermentation tank seed culture medium, wherein the mass percent content of a nitrogen source in the fermentation tank seed culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 25-30min, cooling, inoculating liquid fermentation shake flask seeds, wherein the volume ratio of the liquid fermentation shake flask seeds to a fermentation tank seed culture medium is 1: 10-1: 20 during inoculation, mechanically stirring and culturing for 12-18h in a fermentation tank at 28-32 ℃ and a rotation speed of 150-8CFU/mL as the next seed in the liquid fermentation tank; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(3) fermentation production of the corynespora viticola: preparing a production tank culture medium, wherein the mass percent content of the nitrogen source in the production tank culture medium is 0.5-1.5%, and the mass percent content of the carbon source in the production tank culture medium is 2.0-4.0%The mass percentage content of the sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 30-40min, cooling, inoculating the fermentation tank seeds obtained in the step (2), wherein the volume ratio of the fermentation tank seeds to the culture medium in the production tank is 1: 10-1: 20 during inoculation, mechanically stirring and culturing for 18-24h in a fermentation tank at 28-32 ℃ and the rotating speed of 150-9Obtaining fermentation liquor for preparing the yeast paste when CFU/mL; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(4) and (3) concentrating and producing the fermentation liquor of the corynespora viticola: when the volume of the fermentation liquor is less than or equal to 10L, directly centrifuging by using a high-speed centrifuge to obtain yeast paste; when the volume of the fermentation liquor is more than 10L and less than or equal to 100L, directly centrifuging by using a tubular high-speed centrifuge to obtain yeast paste; when the volume of the fermentation liquor is more than 500L, directly centrifuging by adopting a disc type high-speed centrifuge to obtain yeast paste; the viable count of yeast paste is greater than 5.0 × 1010CFU/g。
6. The method of claim 4, wherein the powdered active yeast of Cladosporium vitis is prepared by the following method:
(1) preparing a shake flask seed culture medium: the mass percent content of the nitrogen source, the mass percent content of the carbon source and the mass percent content of the sodium chloride in the shake flask seed culture medium are respectively 0.5-1.5%, 2.0-4.0% and 0.3-0.6%; sterilizing at 121 deg.C for 20-30min, cooling, and inoculating fresh slant strain of yeast strain; shake culturing at 28-34 deg.C and rotation speed of 150-8CFU/mL as liquid fermentation shake flask seed; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(2) preparing liquid seeds: preparing a fermentation tank seed culture medium, wherein the mass percent content of a nitrogen source in the fermentation tank seed culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 deg.C for 25-30min, cooling, inoculating liquid, and fermentingShaking the flask seed, the volume ratio of the liquid fermentation shaking flask seed to the seed culture medium in the fermentation tank is 1: 10-1: 20 during inoculation, the liquid fermentation shaking flask seed and the seed culture medium in the fermentation tank are mechanically stirred and cultured for 12-18h at 28-32 ℃ and the rotation speed of 150-8CFU/mL as the primary seed of the liquid fermentation tank; wherein the nitrogen source is at least one of yeast extract, protein and beef extract, and the carbon source is at least one of glucose, sucrose, lactose and fructose;
(3) preparing a secondary seed of the saccharomyces cerevisiae: preparing a secondary seeding tank culture medium, wherein the mass percent content of a nitrogen source in the secondary seeding tank culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 deg.C for 30-40min, cooling, inoculating the first-stage seed in the fermentation tank at volume ratio of 1: 10-1: 20, mechanically stirring and culturing at 28-32 deg.C and rotation speed of 150-8CFU/mL as the next seed in the liquid fermentation tank;
(4) fermentation production of the corynespora viticola: preparing a production tank culture medium, wherein the mass percent content of a nitrogen source in the production tank culture medium is 0.5-1.5%, the mass percent content of a carbon source is 2.0-4.0%, and the mass percent content of sodium chloride is 0.3-0.6%; sterilizing at 121 ℃ for 30-40min, cooling, inoculating the seeds obtained in the step (3), wherein the volume ratio of the seeds in the liquid fermentation tank to the culture medium in the production tank is 1: 10-1: 20 during inoculation, mechanically stirring and culturing for 18-24h in a fermentation tank at 28-32 ℃ and the rotating speed of 150-9Obtaining fermentation liquor for preparing yeast powder when CFU/mL;
(5) and (3) concentrating and producing the fermentation liquor of the corynespora viticola: when the volume of the fermentation liquor is more than 1000L, the yeast is concentrated by ceramic membrane microfiltration, the concentration ratio is 40:1 to 50:1, and the obtained viable count is more than or equal to 5.0 multiplied by 1010CFU/mL high density yeast slurry;
(6) mixing the high-density yeast slurry with dextrin according to the weight ratio of 1:1.2 to 1:1.5, stirring to prepare yeast paste, and controlling the water content to be 40-45%; however, the device is not suitable for use in a kitchenThen putting the yeast mud into a yeast mud silk pressing forming machine, and controlling the diameter of the yeast silk to be 1.0-1.5 mm; drying the yeast filaments in a fluidized bed dryer, controlling the air inlet temperature to be less than or equal to 65 ℃, the air outlet temperature to be less than or equal to 34 ℃, controlling the water content of the yeast powder to be 5-6%, and controlling the viable count to be more than or equal to 2.0 multiplied by 1010CFU/g, and obtaining the dentate clavispora active yeast powder.
7. A method for producing wine by fermentation using the yeast strain of claim 1.
8. The method of claim 7, wherein the grape is a Vitis vinifera.
9. The method of claim 7, wherein the method comprises:
the method A comprises the following steps: brewing dry type natural spine grape wine: destemming and crushing mature Vitis davidii, adding the Vitis davidii to the yeast paste prepared by the method of claim 5, or adding the Vitis davidii to the yeast powder prepared by the method of claim 6; the mass percentage content of the yeast paste in the grapes is 0.5-1.0 per mill, and the mass percentage content of the yeast powder in the grapes is 0.2-0.4 per mill; mixing without any other additives, sealing with respirator to ensure that gas only can not enter, naturally fermenting at 15-25 deg.C for 30-40d, continuously pressing grape skin into wine mash during natural fermentation, separating skin and residue, clarifying, and filtering to obtain dry natural spine grape wine; the dry natural spine grape wine has the accuracy of 10.0-11.5 degrees, the sugar content is less than or equal to 3.0g/L, and the methanol content is 60.0-150 mg/L;
and B, method: brewing of semi-dry type, semi-sweet type and sweet type spine grape wine: destemming and crushing mature Vitis davidii, adding the Vitis davidii to the yeast paste prepared by the method of claim 5, or adding the Vitis davidii to the yeast powder prepared by the method of claim 6; the mass percentage content of the yeast paste in the grapes is 0.5-1.0 per mill, and the mass percentage content of the yeast powder in the grapes is 0.2-0.4 per mill; sealing the fermentation tank with a respirator after uniformly mixing, naturally fermenting at 15-25 deg.C for 30-40d, continuously pressing grape skin into wine mash during natural fermentation, separating skin and residue, clarifying, and filtering to obtain semi-dry type, semi-sweet type, and sweet type spine grape wine; the semi-dry type, semi-sweet type and sweet type spine grape wine has the accuracy of 12.5-16.5 degrees, the sugar content is more than or equal to 6.0g/L, and the methanol content is 50.0-100 mg/L.
10. The method of claim 9, wherein sucrose is further added during fermentation in the method B, and the mass percentage of the sucrose in the grapes is 5% -15%.
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