CN116515654B - Saccharomyces cerevisiae and application thereof in longan wine brewing - Google Patents
Saccharomyces cerevisiae and application thereof in longan wine brewing Download PDFInfo
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- CN116515654B CN116515654B CN202310357273.0A CN202310357273A CN116515654B CN 116515654 B CN116515654 B CN 116515654B CN 202310357273 A CN202310357273 A CN 202310357273A CN 116515654 B CN116515654 B CN 116515654B
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- wine
- saccharomyces cerevisiae
- brewing
- grape
- fermentation
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention relates to the technical field of microorganisms and brewing, and discloses a saccharomyces cerevisiae and application thereof in brewing longan wine. The saccharomyces cerevisiae provided by the invention has good stress resistance, is high in fermentation speed, can complete wine fermentation at a wider fermentation temperature, and in addition, the wine brewed by using the saccharomyces cerevisiae also contains rich aroma substances, so that the taste of the wine is more complicated. The saccharomyces cerevisiae is particularly suitable for brewing wine by taking the grapes in the producing area of the Huai as a raw material, and the obtained wine has the characteristics of stronger and rich aroma substances, high quality, good flavor, prominent style in the producing area and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms and brewing, in particular to a saccharomyces cerevisiae and application thereof in brewing longan wine.
Background
Saccharomyces cerevisiae is used as a main microbial fermentation strain, has great influence on aspects of wine quality, yield, fermentation production management and the like, and the selection of the Saccharomyces cerevisiae strain is also very important for the formation of characteristics and styles of wine products. Saccharomyces cerevisiae is involved in the process of wine brewing and can metabolize to produce a variety of wine flavours, mainly including alcohols, esters, aldehydes, ketones, acids, terpenes and some other minor ingredients. The aroma of wine affects the style and characteristic differences of wine products and is an important sensory index for evaluating the quality of wine.
At present, commercial active dry yeast is generally adopted for fermentation in grape winery in China, the breeding and application of native yeast strains are still in a starting stage, and the yeast strains which are bred in the native country and can be applied to the industrial production of grape winery are seriously lacking. The excessive dependence on imported active saccharomyces cerevisiae increases brewing cost, limits industrial independent development space, and also leads to serious homogenization of wine quality, thereby restricting the development of wine industry in China. Although the strain screening work for wine brewing has been developed gradually along with the development of economy in recent years, the strain screening work is mainly focused on the aspects of fermentation speed, aroma substance yield and the like, and the improvement of aroma quality of wine and the brewing of special high-quality wine are less involved.
Disclosure of Invention
The invention aims to solve the problems of the prior art that the aroma quality of the wine is lack of improvement, the excellent brewing strain of the special high-quality wine is brewed, and the like, and provides a saccharomyces cerevisiae and application thereof in wine brewing. The saccharomyces cerevisiae provided by the invention has good stress resistance, is high in fermentation speed, can complete wine fermentation at a wider fermentation temperature, and in addition, the wine brewed by using the saccharomyces cerevisiae also contains rich aroma substances, so that the taste of the wine is more complicated.
In order to achieve the aim, the first aspect of the invention provides a saccharomyces cerevisiaeSaccharomyces cerevisiae) The preservation number of the saccharomyces cerevisiae is CGMCC No.25847.
In a second aspect, the invention provides the use of a Saccharomyces cerevisiae according to the first aspect in wine brewing.
In a third aspect the present invention provides a method of brewing wine, the method comprising inoculating the Saccharomyces cerevisiae of the first aspect into grape juice and/or grape fruit pulp, fermenting under brewing conditions to obtain wine.
According to a fourth aspect of the present invention there is provided wine prepared according to the method of the third aspect.
Through the technical scheme, the invention at least has the following beneficial effects:
(1) The saccharomyces cerevisiae provided by the invention has higher stress resistance, and can normally grow and ferment under the conditions of 18 volume percent alcohol, 500g/L glucose, pH1.5 and 350mg/L sulfur dioxide. Moreover, the saccharomyces cerevisiae can also start fermentation at a lower temperature, hydrogen sulfide is not generated in the fermentation process, and fermentation production of wine can be realized at a wider temperature range, so that the requirements on control and management of the brewing condition of the wine are reduced.
(2) The wine brewed by the saccharomyces cerevisiae has rich types of aroma substances, has higher contents of acetate, fatty acid ethyl ester, C6 alcohol and the like, also contains terpene substances with rich flower fragrance, has moderate content of higher alcohols, increases the complexity of the wine, and is far lower than the concentration which has negative influence on the aroma of the wine, so that the wine has higher flavor quality. And compared with the existing commercial strains, the wine brewed by using the saccharomyces cerevisiae provided by the invention has more complexity.
(3) The saccharomyces cerevisiae provided by the invention is particularly suitable for brewing basin earthworm-eye grape. The longan dry white wine brewed by the saccharomyces cerevisiae has stronger flower fragrance and fruit fragrance, and has the characteristics of high quality, prominent style in a production area and good taste.
Drawings
FIG. 1 is a colony morphology of Saccharomyces cerevisiae CGMCC No.25847 cultured on YPD plates in example 1.
FIG. 2 is a colony morphology of Saccharomyces cerevisiae CGMCC No.25847 cultured on a WL nutrient agar culture plate in example 1.
Fig. 3A to 3E are respectively an ethanol inhibition rate curve, a glucose inhibition rate curve, a sulfur dioxide inhibition rate curve, a pH inhibition rate curve and a temperature inhibition rate curve of saccharomyces cerevisiae CGMCC No.25847 drawn in example 2.
FIG. 4 is a graph showing the comparison of growth curves of Saccharomyces cerevisiae CGMCC No.25847 and commercial yeast VL2 of example 3.
FIG. 5 is a fragrance radar chart of longan dry white wine obtained by fermenting Saccharomyces cerevisiae CGMCC No.25847 and commercial yeast VL2 in example 4.
Preservation of organisms
The Saccharomyces cerevisiae provided by the invention is classified and named asSaccharomyces cerevisiaeHas been preserved in China at 2022, 09 and 30 daysThe general microbiological culture collection center of the microbiological culture collection center is provided with an address of 1 # 3 of North Chen West Lu of the Chaoyang area of Beijing city and a collection number of 25847 of CGMCC.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the present invention, unless specifically stated otherwise, "(provided by the present invention) s.cerevisiae" means s.cerevisiae CGMCC No.25847, s.cerevisiae BC 60771 (abbreviated as 60771) the inventors have numbered this strain during the course of the study, both of which are the same strain, and the preservation number and the number (during the course of the study) are used interchangeably hereinafter.
The basin is positioned near 40 degrees north latitude, and is one of the most ideal grape raw material bases in China. Due to the unique geological and geomorphic characteristics, the unique climate characteristics in the basin are caused, and excellent survival conditions are provided for the growth of the grapes. The heat in the basin is rich, the day and night temperature difference is large, the solar radiation is strong, the frost free period is long, the annual rainfall is low, and the like, the perfect soil permeability, drainage and natural soil fertility are all favorable for grape growth, the altitude is high, the ultraviolet rays and blue-violet light are rich, and the grape is very powerful in photosynthesis by combining with good ventilation conditions, especially favorable for grape anthocyanin synthesis, so that the grape fruits cultivated in the basin of the bosom are bright in color and luster, and rich in aroma, and are one of the excellent areas of wine grapes in China.
Longan grape is an ancient grape cultivar in China, is widely distributed in northwest, north China and other places, has purple red fruits, higher sugar content and acid content, is sour, sweet and tasty, and is a good product in fresh grape and a main raw material for brewing high-grade white wine. Because of long-term cultivation and domestication, the Hebei basin has become one of the most suitable cultivation areas for longan grapes.
In the research process, the inventor of the invention separates and obtains a strain of saccharomyces cerevisiae from wine grape mash naturally fermented in a sand city production area of a basin Saccharomyces cerevisiae) The Saccharomyces cerevisiae has good fermentation performance, outstanding aroma-producing capability, and can highlight the style of the producing area. The popularization and application of the saccharomyces cerevisiae not only can enrich the market of the wine brewing strain and improve the independent development process of the wine industry in China, but also can avoid the problem of the homogenization of the wine in different areas of production in China, improve the quality of the wine and highlight the characteristics of the wine in basin areas. Through further research, the inventor also discovers that when the saccharomyces cerevisiae is adopted for brewing the longan dry white wine, the saccharomyces cerevisiae has the characteristics of being capable of starting fermentation at a lower temperature and high in fermentation speed. Moreover, the longan dry white wine obtained by brewing has good taste and rich aroma substances, so that the longan dry white wine has higher aroma complexity, greatly highlights characteristics of a production area and improves the quality of the wine.
Based on the above findings, the first aspect of the invention provides a saccharomyces cerevisiae with a preservation number of CGMCC No.25847.
In a second aspect, the invention provides the use of a Saccharomyces cerevisiae according to the first aspect in wine brewing. In particular to the application in the brewing of longan (dry white) wine in (Huai Zhuo) producing area.
In a third aspect the present invention provides a method of brewing wine, the method comprising inoculating the Saccharomyces cerevisiae of the first aspect into grape juice and/or grape fruit pulp, fermenting under brewing conditions to obtain wine.
In the method provided by the invention, the source of the raw material (wine grape) for brewing wine is not particularly limited, and any fruit juice or grape pulp made of grape which can be used for brewing wine can be suitable for the method provided by the invention. Through a great deal of research, the inventor finds that the saccharomyces cerevisiae CGMCC No.25847 provided by the invention is particularly suitable for brewing wine by taking the saccharomyces cerevisiae in a basin production area as a raw material. According to the characteristics of grapes in the producing area, the method is particularly suitable for brewing wine by taking the wine-making grapes with high sugar and acid content as raw materials.
According to a preferred embodiment of the invention, wherein the reducing sugar content in the grape juice and/or grape pulp is 150-500 g/L and the total acid content is 1-10 g/L.
Preferably, the content of reducing sugar in the grape juice and/or grape pulp is 150-350g/L, and the total acid content is 3-10g/L.
More preferably, the reducing sugar content in the grape juice and/or grape pulp is 160-200 g/L and the total acid content is 3-8g/L.
The method provided by the invention can be used for brewing wine by taking any variety of wine-making grapes with the characteristics as raw materials. According to a preferred embodiment of the invention, wherein the grape variety used for preparing the grape juice and/or grape fruit pulp is selected from at least one of longan, ma Selan, cabernet Sauvignon, merlot, pink, sila, nibino, danifett, hairyveromyces, gui-ren and Chardonnay.
According to a particularly preferred embodiment of the invention, the grapes used for the preparation of the grape juice and/or grape puree are longan grapes produced from a basin of origin.
The saccharomyces cerevisiae provided by the invention is particularly suitable for brewing dry white wine, so that peeled grape juice and/or peeled grape pulp are preferably adopted in the method provided by the invention.
The grape juice or grape pulp used in the method provided by the invention can be prepared and obtained by any method existing in the field, and can also be directly purchased from a commercial way, so long as the raw materials thereof meet the characteristics.
In the method provided by the invention, the inoculation amount of the saccharomyces cerevisiae is not particularly limited, and can be adjusted according to actual conditions (such as raw material sources, fermentation conditions, product requirements and the like). According to a preferred embodiment of the present invention, wherein the inoculum size of Saccharomyces cerevisiae is 10 5 -10 10 CFU/mL. Said 10 5 -10 10 CFU/mL means that the content of Saccharomyces cerevisiae in the post-inoculation fermentation system is 10 5 -10 10 On the order of CFU/mL, e.g., 10 5 The level of CFU/mL is 1X 10 or more 5 CFU/mL to less than 1X 10 6 The CFU/mL range, i.e., as 1X 10 5 CFU/mL、5×10 5 CFU/mL、9.9×10 5 CFU/mL etc. all belong to 10 5 On the order of CFU/mL. Therefore, in the method provided by the invention, the inoculation amount of the saccharomyces cerevisiae is more than or equal to 1 multiplied by 10 5 CFU/mL to less than 1X 10 11 CFU/mL.
Preferably, the inoculation amount of the saccharomyces cerevisiae is 10 6 -10 8 CFU/mL. More preferably 10 6 -10 7 CFU/mL。
Any brewing conditions known in the art to be suitable for wine brewing may be suitable for the method provided by the present invention. In order to further adapt to the characteristics of wine grapes with high sugar content and low acidity, the quality of the wine is improved, according to a preferred embodiment of the present invention, wherein the brewing conditions comprise: the temperature is 10-40deg.C (preferably 12-38deg.C, more preferably 13-32deg.C) for 5-25 days (preferably 5-20 days, more preferably 10-20 days).
In the method provided by the invention, auxiliary materials such as auxiliary materials for helping yeast fermentation, auxiliary materials for further improving the fragrance and flavor of the wine and the like can be added into the raw materials (namely grape juice and/or grape pulp) for brewing the wine for the purpose of improving the quality of the wine. According to a preferred embodiment of the present invention, wherein at least one of pectinase, tannin, a nitrogen source, zymosan and oak products is further comprised in the grape juice and/or grape pulp. The method provided by the invention is not particularly limited in the amount of the auxiliary materials, and can be adjusted according to actual conditions (such as raw material characteristics, auxiliary material characteristics, product requirements and the like). The nitrogen source may be a nitrogen source common in the art, such as peptone, yeast extract, and the like. Oak products are used to further improve the aroma and flavor of wine.
In the process of the invention, the strains may also be activated prior to fermentation by conventional methods. Preferably, the conditions of the activation may include: the temperature is 25-35℃and the time may be 10-30 hours (preferably 15-25 hours).
According to a fourth aspect of the present invention there is provided wine prepared according to the method of the third aspect.
Any wine prepared by the method provided by the invention belongs to the content of the invention, and specific components in the wine are not particularly limited.
Because the components of the wine are related to brewing materials, brewing conditions and the like, the components in the wine obtained by adopting different sources and varieties of brewing grapes or adopting different brewing conditions are different.
According to a particularly preferred embodiment of the invention, the wine is obtained by brewing longan must (peeled) with a content of reducing sugars of 150-300g/L (preferably 150-200 g/L) and a total acid content of 5-8g/L as starting material, using brewing conditions as described above. Preferably, in the wine, the content of acetate is not less than 50mg/L, the content of higher alcohol is not more than 250mg/L, the content of fatty acid ethyl ester is not less than 12mg/L, and the content of terpene substances is not less than 0.001mg/L. Acetate is formed by ethanol or higher alcohol and acetyl CoA under the catalysis of alcohol acyl transferase, and can provide flower fragrance and fruit fragrance for wine. "higher alcohols" generally refer to alcohols having 3 or more C atoms, which are one of the important aroma substances in wine, but too high a concentration (generally a total concentration of more than 400 mg/L) can negatively affect the aroma of wine, and at concentrations of not more than 300mg/L can increase the aroma complexity of wine. Fatty acid ethyl ester is another important ester compound in wine, and is produced by alcoholysis of acyl-CoA produced in the metabolic process of yeast fatty acid, and different fatty acid ethyl esters have different flavors, for example, ethyl caproate can add apple aroma and strawberry aroma to wine body, and ethyl lactate can provide cream aroma.
Preferably, the wine comprises: 5-20% by volume of ethanol, 50-70mg/L of acetic ester, 150-250mg/L of higher alcohol, 12-25mg/L of fatty acid ethyl ester, 1.8-2mg/L of C6 alcohol, 1-10mg/L of fatty acid and 0.001-0.01mg/L of terpene substances. C6 alcohols generally impart green and grassy flavors to wine. Fatty acids generally exhibit cheese and fatty notes in wine, and terpenes generally have a rich floral note.
More preferably, the wine comprises: 5-15 vol% of ethanol, 60-70mg/L of acetic ester, 180-220mg/L of higher alcohol, 15-20mg/L of fatty acid ethyl ester, 1.8-2mg/L of C6 alcohol, 4-7mg/L of fatty acid and 0.005-0.01mg/L of terpene substances.
The invention provides a wine, which comprises the following components: the ethanol content may be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% by volume, or may be any intermediate value between any two of the above values.
The acetate content may be 60mg/L, 61mg/L, 62mg/L, 63mg/L, 64mg/L, 65mg/L, 66mg/L, 67mg/L, 68mg/L, 69mg/L, 70mg/L, or any intermediate value between any two of the above values.
The higher alcohol content may be 180mg/L, 185mg/L, 190mg/L, 195mg/L, 200mg/L, 205mg/L, 210mg/L, 215mg/L, 220mg/L, or may be any intermediate value between any two of the above values.
The content of the fatty acid ethyl ester may be 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, or may be any intermediate value between any two of the above values.
The C6 alcohol content may be 1.8mg/L, 1.85mg/L, 1.9mg/L, 1.95mg/L, 2mg/L, or any intermediate value between any two of the above values.
The fatty acid content may be 4mg/L, 4.5mg/L, 5mg/L, 5.5mg/L, 6mg/L, 6.5mg/L, 7mg/L, or any intermediate value between any two of the above values.
The terpene may be present in an amount of 0.005mg/L, 0.006mg/L, 0.007mg/L, 0.008mg/L, 0.009mg/L, 0.01mg/L, or any intermediate value between any two of the above.
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only and are not intended to limit the invention.
Commercial strain VL2 used as a control strain in the following examples was purchased from laffort corporation, france. Unless otherwise indicated, reagents and materials were used as purchased from standard chemical or biological suppliers, and the reagents were all analytically pure in purity.
The preparation method of the culture medium used in the following examples is as follows:
YPD medium:
yeast extract 10 g/1000mL, glucose 20g/1000mL, peptone 20g/1000 mL. And weighing the reagent according to the dosage, and dissolving the reagent in deionized water without adjusting pH to obtain the liquid YPD medium. Agar was added to the liquid YPD medium at a rate of 1.5g/1000mL to obtain a solid YPD medium. The resulting culture medium was autoclaved at 121℃for 15min and cooled for further use.
Cooling the sterilized solid YPD medium to a temperature not to scald hands (about 45-50 ℃), pouring the solid YPD medium into a plate culture dish, and cooling and solidifying to obtain a YPD culture plate.
WL nutrient agar medium:
yeast extract 4. 4 g/1000mL, ferric chloride 0.0025 g/1000mL, glucose 50 g/1000mL, manganese sulfate 0.0025 g/1000mL, potassium chloride 0.425 g/1000mL, bromocresol green 0.022 g/1000mL, calcium chloride 0.125 g/1000mL, magnesium sulfate 0.125 g/1000mL, acid hydrolyzed casein 5g/1000mL, and potassium dihydrogen phosphate 0.55 g/1000 mL. And weighing the reagent according to the dosage, dissolving the reagent in deionized water, and regulating the pH to 5.5+/-0.2 to obtain the liquid WL culture medium. Adding agar into the liquid WL culture medium according to the proportion of 20g/1000mL to obtain the solid WL culture medium. The resulting culture medium was autoclaved at 121℃for 15min and cooled for further use.
Cooling sterilized solid WL culture medium to no scalding hands (about 45-50deg.C), pouring into a plate culture dish, cooling and solidifying to obtain WL culture plate.
BIGGY medium:
bismuth ammonium citrate 5g/1000mL, sodium sulfite 3 g/1000mL, glucose 10 g/1000mL, glycine 10 g/1000mL, yeast extract 1 g/1000 mL. Weighing the reagent according to the dosage, dissolving in deionized water, regulating the pH to 6.8+/-0.2, and boiling for not more than 1min to obtain the BIGGY liquid culture medium. Adding agar according to the ratio of 16 g/1000mL before boiling to obtain solid BIGGY culture medium.
Cooling the boiled solid BIGGY culture medium to a temperature not to scald hands (about 45-50 ℃), pouring the solid BIGGY culture medium into a plate culture dish, and cooling and solidifying to obtain the BIGGY culture plate.
Grape juice simulation medium:
glucose 100 g/1000mL, fructose 100 g/1000mL, tartaric acid 3 g/1000mL, citric acid 0.3 g/1000mL, L-malic acid 0.3 g/1000mL, ammonium sulfate 0.3 g/1000mL, asparagine 0.6 g/1000mL, manganese sulfate monohydrate 4 mg/1000 mL, zinc sulfate monohydrate 4 mg/1000 mL, potassium dihydrogen phosphate 2 g/1000mL, copper sulfate pentahydrate 1 mg/1000 mL, potassium iodide 1 mg/1000 mL, boric acid 1 mg/1000 mL, (NH) 4 )6MO 7 O 24 ·4H 2 O 1 mg/1000 mL，COCl 2 ·6H 2 O0.4 mg/1000 mL, inositol 0.3 g/1000mL, biotin 0.04 mg/1000 mL, vitamin B 1 1 mg/1000 mL, vitamin B 6 1 mg/1000 mL, nicotinic acid 1 mg/1000 mL, pantothenic acid 1 mg/1000 mL, p-aminobenzoic acid 1 mg/1000 mL. Weighing the reagent according to the dosage, dissolving in deionized water, regulating the pH to 5.8, and filtering and sterilizing to obtain the grape juice simulated culture medium.
Example 1
The embodiment is used for explaining the acquisition and preservation of the saccharomyces cerevisiae CGMCC No.25847.
Screening and identification of strains
Performing laboratory test fermentation with longan grape juice in the production area of basin, adding non-sterilized grape juice into fermentation container, and fermenting at 18deg.C. The reducing sugar content is detected during the fermentation process, and sampling is carried out at different stages of fermentation according to the consumption of the reducing sugar. The sample is coated on a WL culture medium plate after gradient dilution, after the wine yeast grows out, the strain with better growth vigor and obvious characteristics is selected from the strain, colony morphology and molecular biology identification (5.8S-rDNA ITS method) are carried out, and then a high-throughput platform is used for carrying out inhibition rate screening experiments on the identified wine yeast, and the method comprises the following steps ofAfter the yield of ethanol, glucose, sulfur dioxide, temperature, pH and hydrogen sulfide is evaluated, 5 strains of saccharomyces cerevisiae with high inhibition rate are screened. The brewing characteristics of 5 strains of Saccharomyces cerevisiae were studied and each of them was 1X 10 6 CFU/mL inoculum size was inoculated into a simulated grape juice medium, a fermentation primary screening experiment was performed in 500 mL conical flasks, fermented at 18deg.C for 14 d, and the growth curve (OD 600 nm ) And judging the progress of fermentation by a carbon dioxide weight loss curve, judging the strength of the fermentation capacity and the speed of fermentation according to the slope, and comprehensively screening out a strain of saccharomyces cerevisiae with stronger fermentation capacity, wherein the number of the strain is BC 60771 (called 60771 for short).
The strain is subjected to morphological observation and 5.8S-ITS rDNA gene amplification sequencing, and all identification results are comprehensively analyzed. The specific identification results are as follows:
(1) Morphological observation method and results: after 48h of culture, bacterial strain 60771 grows on YPD culture plates, and the bacterial colony is round, milky white and glossy and has neat edges (refer to FIG. 1 for details); the colony features on the WL nutrient agar plate are cream-colored, round, smooth, opaque, and creamy (see fig. 2 for details).
(2) ITS identification: the total DNA of the strain was taken and amplified using fungal ITS rDNA universal primers according to the PCR system and amplification procedure of Table 1.
TABLE 1
The PCR product was sent to the engineering (Shanghai) limited to complete sequencing. Comparison of ITS rDNA sequencing results with data in NCBI, strain 60771 andSaccharomyces cerevisiaehas 100 percent of homology and is comprehensively identified as saccharomyces cerevisiae by combining physiological and biochemical identification results.
(II) preservation of strains
The Saccharomyces cerevisiae obtained by the screening is treated by the method in 2022, 09 and 30 daysSaccharomyces cerevisiae) BC 60771 is preserved in China Committee for culture Collection of microorganismsThe common microorganism center has an address of Hospital No. 3 of North Chen West Lu 1 in the Chaoyang area of Beijing city and a preservation number of CGMCC No.25847.
Example 2
The embodiment is used for explaining the stress resistance of the saccharomyces cerevisiae CGMCC No.25847.
(1) Evaluation of bacterial Strain inoculation
A single colony of strain 60771 was inoculated into a 96-well plate of a sterile YPD liquid medium, and culture activation was performed at 30℃and 200 rpm overnight. The stress resistance of the strain was then examined as follows.
Ethanol tolerance: the activated strain in 96-well plate was transferred to YPD medium containing 6%, 8%, 10%, 12%, 14%, 16%, 18% ethanol concentration respectively at 3% inoculum size for ethanol concentration inhibition ratio experiment, and after overnight culture at 30℃and 200 rpm, OD was measured by enzyme-labeled instrument 600 nm Values, plotting ethanol concentration inhibition rate curve of Saccharomyces cerevisiae.
Glucose tolerance ability: the activated strain in 96-well plate was transferred to YPD medium containing 20%, 30%, 40%, 50% and 60% glucose concentration respectively at 3% inoculum size for glucose concentration inhibition ratio experiment, and after overnight culture at 30℃and 200 rpm, OD was measured by enzyme-labeled instrument 600 nm Values, glucose concentration inhibition curves for Saccharomyces cerevisiae were plotted.
Sulfur dioxide tolerance: the activated strain in the 96-well plate is transferred to YPD culture medium containing 150 mg/L, 200mg/L, 250mg/L, 300mg/L and 350mg/L sulfur dioxide concentration respectively at 3% inoculation amount for sulfur dioxide concentration inhibition ratio experiment, after 30 ℃ and 200 rpm overnight culture, OD is measured by enzyme-labeled analyzer 600 nm And (3) value, drawing a sulfur dioxide concentration inhibition rate curve of the saccharomyces cerevisiae.
Evaluation of pH inhibition: the activated strain in the 96-well plate was transferred to YPD medium containing pH1.5, pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, pH 4.5 and pH 5.0 respectively at an inoculum size of 3% for pH inhibition experiments, and after overnight culture at 30℃and 200 rpm, OD was measured by a microplate reader 600 nm And (3) values, drawing a pH inhibition rate curve of the saccharomyces cerevisiae.
Temperature inhibition evaluation: activated strains in 96 well platesTransferring 3% of the inoculum size into YPD medium for temperature inhibition ratio experiment, culturing at 10deg.C, 13deg.C, 18deg.C, 32deg.C, 40 deg.C and 200 rpm overnight, and measuring OD by enzyme-labeled instrument 600 nm And (3) values, drawing a temperature inhibition rate curve of the saccharomyces cerevisiae.
Hydrogen sulfide production test of the strain: activated strains in 96-well plates were spotted onto BIGGY medium plates and colony growth was observed. The high hydrogen sulfide producing strain can reduce bismuth sulfite in the culture medium, thereby making the colony brown to black. And evaluating the hydrogen sulfide production condition of the saccharomyces cerevisiae according to the color of the bacterial colony.
(2) Evaluation results
The ethanol inhibition rate curve, glucose inhibition rate curve, sulfur dioxide inhibition rate curve, pH inhibition rate curve, and temperature inhibition rate curve of strain 60771 are shown in fig. 3A-3E, respectively.
As can be seen from the figure, 60771 can be normally grown and fermented under conditions of 18 vol.% ethanol, 500g/L glucose, pH1.5, 350mg/L sulfur dioxide. The strain can also start fermentation under the condition of lower temperature (for example, 10 ℃) so as to ensure the normal brewing production of the wine.
In addition, the test of hydrogen sulfide production shows that the strain 60771 does not produce hydrogen sulfide, does not bring peculiar smell to the wine, and can effectively improve the flavor quality of the wine.
Example 3
The embodiment is used for explaining the fermentation effect and aroma producing performance of the saccharomyces cerevisiae CGMCC No.25847.
The saccharomyces cerevisiae 60771 obtained by screening in the embodiment 1 is subjected to a small-scale fermentation experiment by adopting a grape juice simulation medium, and the fermentation effect and the aroma-producing performance of the saccharomyces cerevisiae are verified, and the specific method is as follows:
saccharomyces cerevisiae 60771 was passaged on YPD plates, and single colonies were picked from the YPD plates and individually inoculated into triangular flasks containing 30 mL sterilized liquid YPD liquid medium, and cultured in a shaker (180 rpm,30 ℃) for 18 h as seed liquid.
According to 1X 10 6 CFU/mL inoculum size, seed solution was inoculated into 300 mL grape juice simulation medium (500 mL Erlenmeyer flask), and the primary sugar concentration was achievedAt a temperature of 200. 200g/L, the fermentation is stopped by sealing with a fermentation plug liquid and standing at 18 ℃ for 10-14 days (until the final sugar content is less than 4 g/L).
The brewing characteristics of wine were measured as follows:
ethanol, fructose, glucose, glycerol, acetic acid, citric acid, malic acid, succinic acid: liquid chromatography (GB/T15038-2006);
volatile aroma content of esters, higher alcohols, organic acids, etc.: the various volatile aroma species and contents in the brewed wine obtained above were detected by an Agilent 6890 Gas Chromatograph (GC) and Agilent 5975 Mass Spectrometer (MS) in combination (Agilent, usa). The specific conditions include: the capillary column HP-INNOWAX Polyethylene Glycol 60m ×0.25 mm ×0.25 μm (J & W scientific, USA) carrier gas is high purity helium gas with a flow rate of 1 mL/min; the headspace solid-phase microextraction is manually injected, a non-split flow mode is adopted, the sample is inserted into a sample injection port of gas chromatography, the temperature of the sample injection port is 250 ℃, and thermal desorption is carried out for 25 min. The temperature rise program of the column temperature box is as follows: the temperature was kept at 40℃for 5min, and then heated to 200℃at a rate of 3℃per min for 2 min. The temperature of the mass spectrum interface is 280 ℃, the temperature of the ion source is 230 ℃, the ionization mode EI, the ion energy is 70 and ev, and the mass scanning range is 20-450 amu.
The commercial strain VL2 was used for cultivation under the above-mentioned pilot fermentation experimental conditions, and the growth characteristics of Saccharomyces cerevisiae 60771 and commercial strain VL2 during fermentation were compared, and the results are shown in FIG. 4. From the graph, the growth trends of the two strains are similar, the growth rates of the two strains are relatively close in the early stage of fermentation, and the growth rate of 60771 is obviously higher after the 9 th day of fermentation.
Table 2 and table 3 show the physical and chemical index detection results and the volatile aroma substance detection results of the wine samples after the brewing of 60771 using the grape juice simulation medium, respectively.
TABLE 2 physical and chemical indicators of wine samples after fermentation of the test strains
The results of the small test of the simulated grape juice culture medium shaking bottle by the saccharomyces cerevisiae 60771 can show that the saccharomyces cerevisiae can complete fermentation and the residual sugar is less than 4 g/L, meets the index requirements of dry type grape wine, has good fermentation performance and has the potential of being popularized and applied as a new special commercial strain of grape wine.
TABLE 3 test results of volatile aroma substances in wine samples after fermentation of strains
The detection result shows that the saccharomyces cerevisiae 60771 can generate more higher alcohols (the content of the higher alcohols does not exceed the highest value of the bad flavor of 400 mg/L) and acid and ester aroma substances, and particularly the higher alcohols and the acid substances are more, which indicates that the strain has stronger aroma-producing capability. Therefore, the invention provides a new high-quality special wine brewing strain, and enriches the choice of the wine strain.
In addition, it is noted that fatty acids are generally expressed as cheese flavor and fatty flavor in wine, and 4 kinds of fatty acids are detected in total in the wine body after fermentation of Saccharomyces cerevisiae 60771, and the total content is high. Meanwhile, the fatty acid ethyl ester is another important ester compound and is generated by alcoholysis of ester acyl-CoA generated in the metabolic process of the yeast fatty acid. A total of 4 fatty acid ethyl esters were detected in this experiment, including: ethyl butyrate, ethyl caproate, ethyl lactate and ethyl caprylate, wherein the ethyl caproate can add apple aroma and strawberry aroma to the wine body, and the ethyl lactate can add cream aroma to the wine body, thus providing a foundation for brewing high-quality wine with special aroma.
Example 4
The embodiment is used for explaining the fermentation effect and aroma-producing performance of the wine of the saccharomyces cerevisiae CGMCC No.25847.
The fermentation effect and aroma-producing performance of strain 60771 and commercial yeast VL2 during fermentation of longan grape juice are compared by using a 5t fermentation tank. The specific method comprises the following steps: seed liquid preparation was carried out as in example 3, followed by 10 6 CFU/mInoculating seed solution into 80L longan grape juice (prepared from basin, squeezing, peeling, filtering, sterilizing, wherein the content of reducing sugar is about 170+ -5 g/L, the content of total acid is about 7+ -1 g/L), standing, activating and culturing for 24 h, adding activated 60771 and VL2 culture solution into a 5t fermentation tank for fermentation when the strain grows and the temperature returns to less than 10deg.C, and the fermentation temperature is set to 10deg.C. The fermentation process was monitored and the fermentation was completed (reducing sugar concentration<4 g/L), and sampling and detecting physicochemical index and aroma substances of the obtained wine sample by liquid chromatography (GB/T15038-2006). Wherein, 60771 ends fermentation for about 20 days and VL2 ends fermentation for about 15 days. The specific results are shown in tables 4 and 5.
TABLE 4 physical and chemical index detection results of longan grape juice fermentation wine samples
From the results in table 4, it can be seen that: the residual sugar content after 60771 fermentation is 2.36 g/L, the residual sugar content after VL2 fermentation is 2.2 g/L, and both yeasts can completely complete the alcohol fermentation stage. The alcohol body after fermentation of the two strains of yeast is basically consistent in the aspects of alcohol degree, volatile acid, total acid content and the like, and has no obvious difference. 60771 has the potential to be used as a new commercial yeast for wine scale brewing popularization.
TABLE 5 fragrance content of wine-like fermented longan grape juice
Note that: n D it is not detected
As can be seen from the contents of Table 5, the longan wine obtained by fermentation of strain 60771 has higher acetate, fatty acid ethyl ester, other esters and C6 alcohol contents than the wine obtained by fermentation with commercial strain VL 2. In addition, 5 kinds of higher alcohols are detected in the longan wine obtained by fermenting the strain 60771, the total concentration is less than 400mg/L, the complexity of the wine is increased, and the fragrance of the wine is not negatively influenced. In addition, terpene substances with rich flower fragrance are detected in longan wine obtained by fermenting with strain 60771, so that the fragrance quality of the wine is further improved.
Based on OAV aroma classification, the longan wine aroma classification obtained by fermenting strains 60771 and VL2 is compared, and the result of comparing the aroma radar maps of the two longan wines is shown in fig. 5. From the graph, the longan dry white wine obtained by fermenting the indigenous yeast 60771 has stronger flower fragrance and fruit fragrance than the wine body obtained by fermenting the commercial yeast VL2, can more fully show the quality and the characteristics of the basin earthworm eye dry white wine, highlights the style of a producing area, improves the taste of the wine and shows good brewing quality.
In addition, after the expertise, the longan dry white wine brewed by the prior commercial strain has lighter fragrance and slightly insufficient complexity. The longan dry white wine brewed by adopting the indigenous yeast 60771 has fresh taste, has the characteristics of prominent flower and fruit fragrance and high fragrance complexity, greatly highlights the characteristics of the longan wine in the production area , and has important significance for developing regional characteristic wine, enriching the types of wine products in China and improving the yield of high-quality wine.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. Saccharomyces cerevisiaeSaccharomyces cerevisiae) The method is characterized in that the preservation number of the saccharomyces cerevisiae is CGMCC No.25847.
2. Use of the saccharomyces cerevisiae of claim 1 in wine brewing.
3. A method of brewing wine, comprising inoculating the saccharomyces cerevisiae of claim 1 into grape juice and/or grape fruit pulp, and fermenting under brewing conditions to obtain wine.
4. A method according to claim 3, wherein the reducing sugar content in the grape juice and/or grape puree is 150-500 g/L and the total acid content is 1-10 g/L.
5. The method of claim 3 or 4, wherein the grape variety used to prepare the grape juice and/or grape fruit pulp is selected from at least one of longan, ma Selan, cabernet Sauvignon, merlot, pink, sila, heibuo, danfite, hairyveromyces, gui-ren and Chardonnay.
6. A method according to claim 3, wherein the saccharomyces cerevisiae is inoculated in an amount of 10 5 -10 10 CFU/mL。
7. A method according to claim 3, wherein the brewing conditions comprise: the temperature is 10-40 ℃ and the time is 5-25 days.
8. A method according to claim 3, wherein at least one of pectinase, tannin, a nitrogen source, zymosan and oak products is also included in the grape juice and/or grape pulp.
9. Wine prepared by the method according to any one of claims 3-8.
10. The wine according to claim 9, wherein the content of acetate is not less than 50mg/L, the content of higher alcohol is not more than 250mg/L, the content of fatty acid ethyl ester is not less than 13mg/L, and the content of terpenes is not less than 0.001mg/L.
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| CN115651852A (en) * | 2022-11-18 | 2023-01-31 | 中粮营养健康研究院有限公司 | Space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof |
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| EP2837698A1 (en) * | 2012-04-13 | 2015-02-18 | Yeastwine - Wine Solutions, Lda. | Saccharomyces cerevisiae strain |
| CN113717870A (en) * | 2021-11-01 | 2021-11-30 | 中粮营养健康研究院有限公司 | Saccharomyces cerevisiae, leavening agent and application of saccharomyces cerevisiae and leavening agent in wine brewing |
| CN115651852A (en) * | 2022-11-18 | 2023-01-31 | 中粮营养健康研究院有限公司 | Space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof |
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