CN115651852B

CN115651852B - Space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof

Info

Publication number: CN115651852B
Application number: CN202211443627.5A
Authority: CN
Inventors: 郑晓卫; 刘沛通; 李泽福; 丁子元; 罗飞; 商华; 董宣; 都振江; 何少华; 孙建平; 范佳硕; 孙玉婷; 陈晓园; 庞建
Original assignee: Cofco Great Wall Wine Ningxia Co ltd; Cofco Huaxia Great Wall Wine Co ltd; Cofco Nutrition and Health Research Institute Co Ltd
Current assignee: Cofco Great Wall Wine Ningxia Co ltd; Cofco Huaxia Great Wall Wine Co ltd; Cofco Nutrition and Health Research Institute Co Ltd
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2023-04-11
Anticipated expiration: 2042-11-18
Also published as: CN115651852A

Abstract

The invention relates to the technical field of fermentation engineering, and discloses a space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof. The saccharomyces cerevisiae used by the invention is obtained by space mutation breeding, has the advantages of high osmotic stress tolerance, high ethanol tolerance, outstanding aroma-producing characteristics and the like, and is particularly suitable for brewing high-quality wine from grapes with higher sugar content produced in western production areas in China. The saccharomyces cerevisiae is used for replacing the existing commercial strains, not only provides more strain selections for wine brewing, but also can effectively improve the homogenization problem of wine products, is beneficial to the independent research and development of core strains for wine brewing in China, perfects the industrial chain and improves the enterprise benefits.

Description

Space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof

Technical Field

The invention relates to the technical field of fermentation engineering, in particular to a space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof.

Background

Saccharomyces cerevisiae (C.,)Saccharomyces cerevisiae) Is a key microorganism for brewing the wine, converts sugar in the grape fruits into ethanol, metabolizes the ethanol to generate a plurality of fermentation aroma substances, and directly determines the style of the wine. At present, the wine industry in China seriously depends on imported active dry yeast, the safety of yeast germplasm is threatened, the homogenization of wine products is gradually serious, and the industry urgently needs to obtain excellent indigenous yeast by various breeding means.

The production area in the western part of China has large day and night temperature difference and long sunshine time, and is one of excellent wine production areas. However, in hot climates in summer, strong illumination causes high sugar content in grape fruits, which brings high osmotic stress in the early fermentation stage and high ethanol stress in the middle and late fermentation stages for saccharomyces cerevisiae, so that the risks of incomplete fermentation and poor biological stability of the wine exist, and adverse effects are caused on the quality of the wine. With the development of the domestic wine industry, the development of a wine brewing process suitable for grape fruits with high sugar content is urgently needed to improve the quality of the wine.

In order to further improve the autonomous rate of core strains and select the saccharomyces cerevisiae with more excellent performance, the industrial chain is perfected, and the production cost is reduced. However, the productivity of yeast obtained by traditional mutation breeding techniques has reached the bottleneck of "mutation screening". More and more research has found that the bottleneck of "mutagenesis" can be broken by means of cosmic radiation and microgravity. Microgravity effect, extreme temperature difference, high vacuum, high-energy particle radiation, weak magnetic field and the like in the space environment can generate mutagenesis effect on space microorganisms, and gene mutation frequency is obviously improved, so that biological properties (including colony characteristics, physiological and biochemical characteristics, individual morphology and the like) and fermentation production performance (including enzyme activity, aroma-producing capability and the like) of the microorganisms are changed, and the space breeding becomes a new technological means for strain mutagenesis breeding which is concerned in recent years. Through aviation breeding, yeast with more excellent fertility can be selected, and the productivity and competitiveness of wine enterprises in China are improved.

Disclosure of Invention

The invention aims to solve the problem that the quality of high-alcohol-stress wine caused by high sugar content cannot be guaranteed by the existing commercial active dry yeast in the prior art, and provides a space breeding saccharomyces cerevisiae with excellent stress resistance and application thereof. The saccharomyces cerevisiae provided by the invention is obtained by space mutation breeding, has the advantages of high fermentation speed, high osmotic stress tolerance, high ethanol tolerance, outstanding aroma-producing characteristics and the like, and is particularly suitable for brewing high-quality wine from grapes with high sugar content.

In order to realize the aim, the invention provides a strain of saccharomyces cerevisiae, wherein the saccharomyces cerevisiae has a preservation number of CGMCC No.24017.

In a second aspect, the present invention provides the use of the saccharomyces cerevisiae of the first aspect in saccharomyces cerevisiae.

In a third aspect, the invention provides a method for brewing wine, which comprises inoculating the saccharomyces cerevisiae of the first aspect into grape juice, and fermenting under brewing conditions to obtain the wine.

In a fourth aspect of the invention there is provided a wine prepared according to the method of the third aspect.

Through the technical scheme, the invention can obtain the following beneficial effects:

(1) The saccharomyces cerevisiae provided by the invention has higher stress resistance, and particularly has higher ethanol and osmotic pressure stress tolerance (caused by high-concentration sugar in fermentation raw materials);

(2) The saccharomyces cerevisiae provided by the invention has good aroma production performance, particularly has higher yield of ester substances, and can endow wine with richer fruit aroma and flower aroma. In addition, the yield of metabolites which are unfavorable for the flavor of the wine is low in the fermentation process, the method plays an important role in improving and stabilizing the quality of the wine, and the method is suitable for brewing high-quality wine;

(3) The saccharomyces cerevisiae provided by the invention has high acid yield, and is particularly suitable for brewing grapes with low acidity and high sugar degree (such as grapes in western production areas in China);

(4) The saccharomyces cerevisiae provided by the invention can replace the existing strains for use, provides more strain selections for wine brewing, improves the homogenization problem of wine products, is beneficial to breaking the situation that the core strains adopted by the wine industry in China are limited by monopoly abroad, perfects the industry chain and reduces the production cost.

Biological preservation

The saccharomyces cerevisiae provided by the invention is named as saccharomyces cerevisiae in classificationSaccharomyces cerevisiaeIt has been preserved in China general microbiological culture Collection center (CGMCC) No. 1 at 12 months and 02 days in 2021, with the preservation number of CGMCC No.24017, and the address of No. 3, west Lu No. 1, north Cheng, the south China area of Beijing.

Drawings

FIG. 1 is a characteristic diagram of colonies obtained by culturing HTZL03-5 in YPD medium in example 1;

FIG. 2 is a characteristic diagram of a colony obtained when HTZL03-5 was cultured in WL medium in example 1;

FIG. 3 is a glucose tolerance plot of HTZL03-5, plotted in example 2;

FIG. 4 is a graph comparing the sensory evaluation results of Western production Matherland dry red wine brewed using HTZL03-5 and XR in example 4.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In the invention, under the condition that no special description is provided, the 'saccharomyces cerevisiae (provided by the invention)' and 'saccharomyces cerevisiae HTZL 03-5' both refer to the saccharomyces cerevisiae CGMCC No.24017 provided by the invention.

In the research process, the inventor of the invention carries out space mutagenesis on a saccharomyces cerevisiae strain obtained by separating and screening natural fermented grape mash, and then screens the saccharomyces cerevisiae strain to obtain a saccharomyces cerevisiae strainSaccharomyces cerevisiae) The high-alcohol-content fermented grape wine has the characteristics of good fermentation performance, high osmotic stress tolerance, high ethanol tolerance, outstanding aroma production characteristic and the like, and is particularly suitable for brewing high-quality grape wine by adopting the wine grapes with low acidity and high sugar degree.

In the first aspect of the invention, the saccharomyces cerevisiae (Saccharomyces cerevisiae) The saccharomyces cerevisiae is the saccharomyces cerevisiae with the preservation number of CGMCC No.24017.

In a second aspect the present invention provides the use of a s.cerevisiae as described in the first aspect for the brewing of wine.

In a third aspect, the present invention provides a method for brewing wine, said method comprising inoculating the saccharomyces cerevisiae of the first aspect into grape juice and/or grape pulp, and fermenting under brewing conditions to obtain wine.

In the method of the present invention, the source of the raw material (wine grape) used for wine brewing is not particularly limited, and any grape juice or grape pulp made of a grape that can be used for wine brewing can be applied to the method of the present invention. The saccharomyces cerevisiae CGMCC No.24017 provided by the invention has high acid yield in the wine brewing process, so the saccharomyces cerevisiae is particularly suitable for brewing wine (such as grapes produced in western production areas of China) by taking the wine grapes with higher sugar-acid ratio (namely the content ratio of reducing sugar to total acid) as raw materials.

In view of the above-mentioned characteristics of the Saccharomyces cerevisiae provided by the present invention, according to some preferred embodiments of the present invention, the ratio between the content of reducing sugars and the total acid content used in the preparation of said grape juice and/or grape pulp is not lower than 50, more preferably between 50 and 100. Wherein, the reducing sugar content and the total acid content (calculated by tartaric acid) are mass contents, the unit is usually g/L, and the reducing sugar content and the total acid content can be respectively measured by a Fehling reagent method and a titration method.

The method provided by the invention can be used for brewing wine by adopting any variety of wine-brewing 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 pulp is selected from at least one of mashela, sauvignon, merlot, pindol, cilar, nobilex, danrpht, acacia longissima, guirenxiang and chardonnay. The grape variety used for preparing the grape juice and/or grape pulp is, for example, grapes produced in western production.

Any grape juice or grape pulp made of wine grapes characterized as described above can be applied to the method provided by the present invention, and there is no particular limitation on the components thereof. According to a preferred embodiment of the invention, the grape juice and/or grape must has a reducing sugar content of 200 to 500g/L and a total acid content of 1 to 10 g/L.

Preferably, the content of reducing sugar in the grape juice and/or the grape pulp is 200-400g/L, and the total acid content is 3-8g/L.

More preferably, the content of reducing sugar in the grape juice and/or grape pulp is 250-350g/L, and the total acid content is 4-7 g/L.

The grape juice or grape pulp used in the method provided by the invention can be prepared by any method existing in the field, and can also be directly purchased from commercial sources, as long as the raw materials meet the characteristics.

In the method provided by the present invention, the inoculation amount of the saccharomyces cerevisiae is not particularly limited, and may be adjusted according to actual conditions (for example, source of raw material, fermentation conditions, product requirements, etc.). According to a preferred embodiment of the present invention, wherein the inoculation amount of Saccharomyces cerevisiae is 10 ⁵ -10 ¹⁰ CFU/mL. The above-mentioned 10 ⁵ -10 ¹⁰ CFU/mL means that the content of the saccharomyces cerevisiae in a fermentation system after inoculation is 10 ⁵ -10 ¹⁰ Of the order of CFU/mL, e.g. 10 ⁵ Levels on the order of CFU/mL represent 1X 10 or greater ⁵ CFU/mL to less than 10 ⁶ CFU/mL range, i.e., such as 1X 10 ⁵ CFU/mL、5×10 ⁵ CFU/mL、9.9×10 ⁵ CFU/mL, etc. all belong to 10 ⁵ An order of magnitude level 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 × 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。

Any brewing conditions existing in the art and suitable for brewing wine can be applied to the method provided by the invention. In order to further adapt to the characteristics of wine grapes with high sugar content and low acidity and improve the quality of wine, according to a preferred embodiment of the invention, the wine brewing conditions comprise: the temperature is 10-30 deg.C (preferably 12-28 deg.C, more preferably 25-28 deg.C), and the time is 5-20 days (preferably 5-15 days).

In the method provided by the invention, auxiliary materials, such as auxiliary materials for assisting yeast fermentation, auxiliary materials for further improving the aroma and flavor of the wine and the like can be added into the raw materials for brewing the wine (namely the grape juice and/or the grape pulp) for improving the quality of the wine. According to a preferred embodiment of the invention, the grape juice and/or grape pulp further comprises at least one of pectinase, tannin, a nitrogen source, zymosan and oak product. The amount of the auxiliary materials used in the method provided by the invention is not particularly limited, 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 commonly used 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 method of the present invention, the strain may be activated by a conventional method before fermentation. The conditions for activation may include: the temperature is 25-30 deg.C, and the time can be 20-30h.

In a fourth aspect of the invention there is provided wine prepared according to the method of the third aspect.

Any wine prepared by the above method provided by the present invention is within the scope of the present invention, and the specific components thereof are not particularly limited.

Since the components of the wine are related to wine brewing raw materials, wine brewing conditions and the like, the components of the wine obtained by adopting wine brewing grapes of different sources and varieties or wine brewing conditions are different.

According to a particularly preferred embodiment of the invention, the wine is brewed from masher grape juice with a reducing sugar content of 280-320g/L and a total acid content of 4.5-6.5g/L using the brewing conditions as described above, wherein the wine comprises: 10-20% of ethanol, 250-320 mg/L of higher alcohol, 1-5 mg/L of ester substance and 0.1-1.5mg/L of acid aroma substance. The "acid aroma" refers to acids in volatile aroma in wine, for example, caproic acid, caprylic acid, capric acid, etc.

Preferably, the wine contains: 13-17% of ethanol, 270-310 mg/L of higher alcohol, 2-4 mg/L of ester substances and 0.5-1 mg/L of acid aroma substances.

The present invention will be described in detail below by way of examples. It should be understood that the following examples are only intended to further illustrate and explain the contents of the present invention by way of example, and are not intended to limit the present invention.

The chemicals used in the following examples were purchased from regular chemical suppliers and were chemically pure.

The media used in the following examples were prepared as follows:

YPD medium:

10g/1000mL of yeast extract, 20g/1000mL of glucose and 20g/1000mL of peptone. Weighing the reagent according to the above dosage, dissolving in deionized water, and obtaining the liquid YPD culture medium without adjusting pH. Agar is added into the liquid YPD medium according to the proportion of 1.5g/1000mL to obtain a solid YPD medium. The obtained culture medium is sterilized under high pressure at 121 deg.C for 15min, and cooled for use.

Cooling the sterilized solid YPD culture medium to a temperature of about 50 ℃, pouring the cooled solid YPD culture medium into a plate culture dish, and cooling and solidifying to obtain the YPD culture plate.

WL nutrient agar medium:

4g/1000mL of yeast extract powder, 0.0025g/1000mL of ferric chloride, 50g/1000mL of glucose, 0.0025g/1000mL of manganese sulfate, 0.425g/1000mL of potassium chloride, 0.022g/1000mL of bromocresol green, 0.125g/1000mL of calcium chloride, 0.125g/1000mL of magnesium sulfate, 5g/1000mL of acid hydrolyzed casein and 0.55g/1000mL of potassium dihydrogen phosphate. Weighing the reagent according to the dosage, dissolving in deionized water, and adjusting pH to 5.5 +/-0.2 to obtain the liquid WL culture medium. Agar is added into the liquid WL culture medium according to the proportion of 20g/1000mL to obtain the solid WL culture medium. The obtained culture medium is sterilized by autoclaving at 121 deg.C for 15min, and cooling for use.

And (3) cooling the sterilized solid WL culture medium to be not too hot (about 50 ℃), pouring the solid WL culture medium into a plate culture dish, and cooling and solidifying to obtain a WL culture plate.

Ethanol tolerance screening medium:

10g/1000mL of yeast extract, 20g/1000mL of glucose and 20g/1000mL of peptone. Weighing the reagents according to the above dosage, dissolving in deionized water, adding agar according to the proportion of 1.5g/100mL without adjusting pH, autoclaving at 121 ℃ for 15min, and cooling for later use.

And (3) cooling the sterilized culture medium to be not too hot to the hands (about 50 ℃), adding preheated ethanol to ensure that the concentration of the ethanol in the culture medium reaches 18 volume percent, uniformly mixing, pouring into a plate culture dish, and cooling and solidifying to obtain the ethanol tolerance screening culture plate.

High sugar (300 g/L) grape juice simulated medium:

150g/1000mL of glucose, 150g/L of fructose, 3 g/1000mL of tartaric acid, 0.3 g/1000mL of citric acid, 0.3 g/1000mL of L-malic acid, 0.3 g/1000mL of ammonium sulfate, 0.6 g/1000mL of asparagine, 4 mg/1000mL of manganese sulfate monohydrate, 4 mg/1000mL of zinc sulfate monohydrate, 2g/1000mL of potassium dihydrogen phosphate, 1 mg/1000mL of copper sulfate pentahydrate, 1 mg/1000mL of potassium iodide, 1 mg/1000mL of boric acid, (NH) ₄ ) ₆ MO ₇ O ₂₄ ·4H ₂ O 1 mg/1000mL，COCl ₂ ·6H ₂ O0.4 mg/1000mL, inositol 0.3 g/1000mL, biotin 0.04 mg/1000mL, vitamin B ₁ 1 mg/1000mL, vitamin B ₆ 1 mg/1000mL, 1 mg/1000mL nicotinic acid, 1 mg/1000mL pantothenic acid, 1 mg/1000mL para aminobenzoic acid. Weighing the reagents according to the above dosage, dissolving in deionized water, adjusting pH to 5.8, filtering and sterilizing to obtain the high-glucose grape juice simulated culture medium.

Example 1

This example illustrates the acquisition and preservation of Saccharomyces cerevisiae CGMCC No.24017.

(I) mutagenesis of strains

A strain of saccharomyces cerevisiae ZL03 (stored in a microbial strain bank of the technical center of wine industry of the Chinese grain nutrition and health research institute) is obtained by purifying and culturing wine grape mash naturally fermented in the eastern foot production area of the Helland, and is carried on a new generation mannequin test boat which is launched first through a carrier rocket of No. five of Long Zheng, and space mutagenesis is carried out to obtain space breeding saccharomyces cerevisiae HTZL03.

(II) purifying and screening bacterial strain

Slowly freezing a glycerol storage tube of a space mutagenic strain HTZL03 frozen at-80 ℃ at 4 ℃, inoculating the glycerol storage tube into a WL culture plate in an aseptic workbench in an inoculation amount of 2 volume percent, culturing for 24h at 30 ℃, randomly picking single bacterial colonies, streaking, separating and purifying for 3 times to obtain a plurality of single pure bacterial colonies.

Primary screening: and (3) adopting a point inoculation method, sequentially inoculating the bacterial colonies obtained by separation and purification into an ethanol tolerance screening culture plate by using a sterile inoculating needle, wherein three bacterial colonies are arranged in parallel. Culturing at 30 ℃ for 48h, observing the growth condition of bacterial colonies, and screening 8 bacterial strains with uniform size and good growth state, wherein the bacterial strains are numbered as HTZL03-1 to HTZL03-8 in sequence.

Re-screening: the strains after the passages are respectively subcultured on the YPD liquid culture medium by adopting HTZL03-1 to HTZL03-8, and then cultured by adopting a YPD culture plate, the strains are picked from the YPD culture plate and respectively inoculated into the YPD liquid culture medium (100 mL) in a 250mL conical flask, and the strains are cultured for 24 hours by a shaker (180rpm, 30 ℃) to be used as seed liquid.

Seed solutions were prepared in the same manner using the ground starter strain ZL03 and a commonly used imported commercial Saccharomyces cerevisiae XR (available from Lamoth-Abiet, france) as controls.

500mL Erlenmeyer flask according to 10 ⁶ CFU/mL inoculation amount seed liquid of HTZL03-1 to HTZL03-8, ZL03 and XR is inoculated into 300mL grape juice simulation culture medium, initial total reducing sugar concentration is 300g/L, fermentation plug liquid sealing is carried out, and standing culture is carried out for 10-14 days at 25 ℃ (the fermentation can be stopped when the reducing sugar content is less than 4g/L, and the fermentation can be stopped no matter the reducing sugar content at 14 days). The content of residual sugar and ethanol in the fermentation product was measured by liquid chromatography (Agilent HPLC 1200 series) and the results are detailed in Table 1.

TABLE 1 fermentation Performance and ethanol tolerance of space-mutagenized Saccharomyces cerevisiae

As can be seen from the data in Table 1, HTZL03-5 exhibited the best fermentability and the highest tolerance to ethanol (comparable to current commercial strain levels).

(III) identification of the Strain

Morphological identification: and (3) streaking the HTZL03-5 seed solution obtained in the test (II) on an YDP culture plate and a WL culture plate, culturing at 30 ℃ for 48h, and observing the obtained colony characteristics.

YPD culture plate culture results: the colony is milky white, round, shiny, neat in edge, sticky and easy to pick up (as shown in fig. 1).

WL culture plate culture results: the colonies were cream-colored, spherical, smooth-surfaced, opaque, creamy (see FIG. 2).

Molecular biology identification: the total DNA of the strain HTZL03-5 (extracted by adopting a kit DP6201 of the Baitek company according to the content of the instruction) is taken as a template, and ITS rDNA amplification is carried out by using fungus ITS rDNA universal primers according to a PCR system and an amplification program shown in a table 2.

TABLE 2 PCR systems and procedures for ITS identification

Sending the amplification product to biological engineering (Shanghai) limited for sequencing, and comparing the ITS rDNA sequence of the obtained strain HTZL03-5 with the data in NCBI to find that the strain HTZL03-5 and the strain NCBISaccharomyces cerevisiaeHas 100 percent of homology, combines the morphological identification result and the physiological and biochemical identification result, and comprehensively identifies the saccharomyces cerevisiae(s) ((Saccharomyces cerevisiae）。

(IV) preservation of the strains

In 2021, 12 and 02 days, saccharomyces cerevisiae(s) (Saccharomyces cerevisiae) Is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the address of No. 3 of Xilu No. 1 of Beijing province of rising Yang and the preservation number of CGMCC No.24017. BC 60685 in the deposit certificate is the strain HTZL03-5 screened in the example.

Example 2

This example is used to illustrate the osmotic stress resistance of Saccharomyces cerevisiae CGMCC No.24017.

HTZL03-5 seed liquid was prepared as described in example 1. The seed solution is inoculated into YPD liquid culture media with glucose contents of 200g/L, 300g/L, 400g/L, 500g/L and 600g/L respectively according to the inoculation amount of 3 volume percent by adopting a 96-pore plate, and the osmotic stress resistance of the strain is detected.

3 parallel replicates (total volume per well approximately 1 mL) were made for each concentration. 30 ℃ overnight (about 18 h) with shaking at 200rpm, and OD was measured with a microplate reader _600nm Values, glucose tolerance of Saccharomyces cerevisiae HTZL03-5 was calculated according to the following formula and a glucose tolerance curve was plotted (see FIG. 3 for details).

Glucose tolerance (%) = [ (OD) _{After cultivation} -OD _{Blank (same culture time)} )/(OD _{Before culturing} -OD _{Blank space} )-1]×100%

As can be seen from the figure, HTZL03-5 can normally grow and ferment under the condition of 500g/L glucose, and can ensure the normal brewing production of the wine. The strain is proved to have good osmotic stress resistance.

Example 3

This example is used to illustrate the fermentation effect and flavor-producing property of Saccharomyces cerevisiae CGMCC No.24017.

Seed liquor preparation for Saccharomyces cerevisiae HTZL03-5 and imported commercial Saccharomyces cerevisiae XR was performed as described in example 1.

500mL Erlenmeyer flask according to 10 ⁶ And (3) inoculating the inoculation amount of CFU/mL, respectively inoculating Saccharomyces cerevisiae HTZL03-5 and XR seed liquid into 300mL high-glucose grape juice simulation culture medium (500 mL conical flask), sealing with a fermentation plug liquid, standing at 25 ℃, fermenting and culturing for 10-14 days (stopping fermentation when the content of reducing sugar is less than 4 g/L), and obtaining a wine sample.

The contents of glucose, fructose, glycerol, ethanol, acetic acid, citric acid, malic acid and succinic acid in the wine samples were determined by liquid chromatography (GB/T15038-2006), and the results are detailed in Table 3.

TABLE 3 wine sample physical and chemical index test results

The volatile aroma content of the wine samples was determined using an Agilent 6890 Gas Chromatography (GC) and Agilent 5975 Mass Spectrometry (MS) combination (Agilent, USA). The specific conditions are as follows: capillary column HP-INNOWAX Polyethylene Glycol 60 m × 0.25 mm × 0.25 μm (J & W scientific, USA) carrier gas is high purity helium gas, and flow rate is 1 mL/min; manually injecting sample for headspace solid phase microextraction, adopting non-flow-dividing mode, inserting into sample inlet of gas chromatography, and thermally desorbing at 250 deg.C for 25 min. The temperature rising program of the column oven is as follows: keeping the temperature at 40 ℃ for 5min, then heating to 200 ℃ at the speed of 3 ℃/min, and keeping the temperature for 2 min. The interface temperature of the mass spectrum is 280 ℃, the temperature of the ion source is 230 ℃, the ionization mode EI is 70 ev, and the mass scanning range is 20-450 amu. The results are detailed in Table 4.

TABLE 4 comparison of aroma content in wine samples

* The higher alcohol content in table 4 means the total amount of alcohols shown in table 4, the ester content means the total amount of esters shown in table 4, and the acid content means the total amount of acids shown in table 4.

As can be seen from the contents of tables 3 and 4, the HTZL03-5 wine has a final sugar content of 2.88 g/L at the end of fermentation, and the fermentation can be completed (< 4 g/L). The alcohol content was 15.84 vol%, which is close to that of commercial yeast. The yeast can complete the wine fermentation under the stress of high osmotic pressure. As can be seen from the wine aroma brewed from HTZL03-5 in Table 4, the yeast had moderate higher alcohol yields, did not exceed the maximum off-flavor (400 mg/L), and had significantly higher total ester yields than commercial yeasts. The total amount of ester substances generated by the yeast is 2.95 mg/L, which is 1.4 times of that of a contrast, wherein the isoamyl acetate, ethyl hexanoate, ethyl octanoate and ethyl decanoate are obviously higher than those of a commercial contrast, so that the flavor characteristics of the wine, such as fruit aroma, flower aroma and the like, can be improved, and the aroma quality of the wine is improved.

Therefore, the saccharomyces cerevisiae HTZL03-5 provided by the invention not only can smoothly complete brewing of the dry red wine with high-sugar raw materials and enable the dry red wine to meet the national standard, but also can increase the complexity of the aroma of the wine and improve the aroma quality of the wine.

Example 4

This example illustrates the sensory evaluation of wine brewed with Saccharomyces cerevisiae CGMCC No.24017.

The wine brewing is carried out by using mashelan grape juice produced in western producing areas as a raw material. The preparation method of the masher orchid grape juice comprises the following steps: mechanically removing stalks of fructus Vitis Viniferae, crushing, juicing, pasteurizing (65 deg.C, 30 min), clarifying, collecting clear juice, sampling, coating, and detecting to confirm that the grape juice is sterile. The content of reducing sugar (calculated by glucose) in the grape juice is 300.81 plus or minus 10.25g/L, and the total acid content (calculated by tartaric acid) is 5.51 plus or minus 0.23g/L.

Respectively mixing HTZL03-5 and XR seed solutions according to the ratio of 10 ⁶ Inoculating the inoculation amount of CFU/mL into 10L of Matheran grape juice, standing at 20 ℃ for 24h, and heating to 22 ℃ to obtain an activation solution of HTZL03-5 and XR.

Respectively transferring the activation solution of HTZL03-5 and XR into a 100L fermentation tank (Huaqiang Zhongtian fluid equipment (Beijing) Co., ltd., HCHT-100L × 8) for fermentation under the following conditions: fermenting at 27 + -1 deg.C, and pressing the cap 3 times a day in the morning, at noon and at night to ensure the skin cap is soaked. Ending the fermentation when the concentration of reducing sugar in the fermentation liquor is less than 4g/L to obtain the wine. The fermentation time for HTZL03-5 and XR was 6 days.

Ten evaluators were invited to perform sensory evaluation of the wines brewed by the above method, and the ten invited evaluators included national grade wine tasters (five), professors from years of wine research in colleges and universities in China, subsidiary professors (two), professional masters of wines, and doctor researchers (three). The sensory evaluation items included: clarity, color, purity, richness, elegance, acidity, sweetness, balance, sustainability, etc., and comprehensive evaluation according to the overall feeling of the wine, the total score corresponding to each sensory evaluation item is detailed in table 5. The average value of each rater score was taken, and the results are shown in detail in FIG. 4 (note: the score in FIG. 4 is a value normalized by 10 points as the total score).

TABLE 5

According to the comparison of sensory evaluation scores in fig. 4, the mashelan dry red wine brewed by using the HTZL03-5 has higher elegance and richness, better fragrance property, obviously improved acidity of the wine, improved balance of the wine, better improved current situation of insufficient acidity of the dry red wine in western production areas, improved quality of the wine in the western production areas and good wine brewing potential.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A strain of Saccharomyces cerevisiaeSaccharomyces cerevisiae) The method is characterized in that the saccharomyces cerevisiae is the saccharomyces cerevisiae with the preservation number of CGMCC No.24017.

2. Use of the saccharomyces cerevisiae according to claim 1 for brewing wine.

3. A method for brewing wine, comprising inoculating the Saccharomyces cerevisiae according to claim 1 into grape juice and/or grape fruit pulp, and fermenting under brewing conditions to obtain wine, wherein the content of reducing sugar in the grape juice and/or grape fruit pulp is 200-500g/L, and the total acid content is 1-10 g/L.

4. A process as claimed in claim 3, wherein the ratio between the reducing sugar content and the total acid content in the must and/or pulp is not lower than 50.

5. A process as claimed in claim 4, wherein the ratio between the reducing sugar content and the total acid content of the must and/or pulp is comprised between 50 and 100.

6. The method according to any one of claims 3-5, wherein the grape cultivar used for making the grape juice and/or grape pulp is selected from at least one of Matheran, cabernet Sauvignon, merlot, pinlizhu, sira, heibeno, danfiter, st.longissima, guirenxiang and Chardonnay.

7. The method of claim 3, wherein the saccharomyces cerevisiae is inoculated in an amount of 10 ⁵ -10 ¹⁰ CFU/mL。

8. The method of claim 3, wherein the brewing conditions comprise: the temperature is 10-30 ℃ and the time is 5-20 days.

9. The method of claim 3, wherein the grape juice and/or grape pulp further comprises at least one of pectinase, tannin, a nitrogen source, zymosan, and an oak product.